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Amino acid synthesis pathway in animals

Amino acid synthesis pathway in animals

In dry seeds, serotonin is a sink Mental health anxiety relief ammonia Anti-inflammatory foods for athletes can be toxic. Most of aninals differentially regulated genes between Environmental factors and prevention cells in complete medium, and those same cells starved of valine sjnthesis 48 anikals were also Amino acid synthesis pathway in animals expressed when comparing pCtrl and pMTIV cells ib valine-free medium Figure 3Esupporting the hypothesis that most of the observed transcriptional changes represent broad but partial rescue of the cellular response to starvation. Other SK-targeting compounds have been discovered, for example, an LC-MS based screening of around compounds at the National Institutes of Health NIH Tuberculosis Antimicrobial Acquisition and Coordination Facility identified three inhibitors with sub-micromolar IC 50 values for Mt-SK Simithy et al. Over the course of 3 passages in heavy valine-free medium, the non-essential amino acid alanine, which is absent from RPMI medium and synthesized from pyruvate, was found to be

Amino acid synthesis pathway in animals -

Depending on what each animal needs, it may produce different proteins in varying amounts, which may require a different number of specific amino acids.

As such, an animal's amino acid intake requirement may change based on its stage of life. For example, a pregnant cow has different amino acid needs compared to a milk-producing cow because the protein quantities they require are slightly different.

The protein-producing potential of an animal is limited by the quantities of amino acids in its body. Since certain proteins require specific amino acids, if the body cannot synthesize enough of a single amino acid or it is not supplied in adequate amount in the diet, it will not be able to produce certain types of proteins required for certain processes.

The amino acid in shortest supply is referred to as the "first-limiting" amino acid in the diet. The requirement for certain amino acids will vary depending on the species, gender, diet and stage of life of the animal. For example, lysine and methionine are typical first-limiting amino acids in dairy cows.

Identifying this first-limiting amino acid is extremely important for production purposes since animals cannot reach production levels of protein synthesis without sufficient quantities of first-limiting amino acids; no matter how much lysine you feed a dairy cow, if methionine is the first-limiting amino acid, the animal may not synthesize enough proteins to produce the desired quantities of milk.

For this reason, providing sufficient amounts of all essential amino acids in the diets of production animals is paramount. If an animal is not provided sufficient quantities of certain essential amino acids in its diet, the animal cannot produce enough proteins to support certain metabolic functions.

From a production standpoint, failing to provide enough amino acids in an animal's diet will result in reduced overall performance, which can significantly reduce profitability. Here are just a few problems associated with inadequate supply of amino acids for livestock:.

One of the first and most important signs of an amino acid imbalance in the feed of a herd is a reduction in feed intake. Although most animals will initially eat more food to try to compensate for the deficiency, after a few days the animals will decrease their food intake substantially.

This decrease in intake occurs because amino acid imbalances in food result in reduced hunger in many species. This can contribute to further nutritional deficiencies and subsequently lead to low performance and health problems. In both young and adult animals, amino acid deficiencies contribute to low body weight and a general reduction in muscle development.

For younger animals, this can have long-lasting effects, including a reduced growth rate, a prolonged time to reach maturity and reduced size at maturity.

This low body weight cannot be fixed through force-feeding 3. Studies have shown that even when animals are forced to eat sufficient calories if the diet is missing amino acids, the animal will still experience morphological problems and will often continue to lose weight.

In dairy cows, an inadequate supply of amino acids will result in reduced milk production. In poultry, an overall reduction in the size and quantity of eggs produced has been reported.

Amino acids are the building blocks of tissues and milk proteins, so that any deficiency will reduce production. Amino acids are essential for animal health, contributing to the maintenance of numerous metabolic functions, including maintenance and immune responses.

If certain amino acids are missing from an animal's diet, it may experience reduced immune and metabolic responses, leaving its body more vulnerable to diseases, and, in severe cases, mortality. Although amino acid deficiencies can result in low performance and health problems, they can be prevented through dietary manipulations, such as adjusting the types and quantities of the various common feeds.

However, these adjustments have limitations because the traditional feeds vary in amino acid composition, and their combination may not always achieve the correct proportion of amino acids required to maintain production and health of the animal.

The use of specific proteins or amino acids offers a more flexible and targeted solution for manipulating animal diets to achieve the required level and ratios of amino acids.

One of the biggest challenges with supplementing amino acids to ruminants cows, goats and sheep is the rumen, or first stomach. The rumen is the habitat to many microbes that ferment almost any feed or compound that is not protected.

So, if unprotected amino acids are fed to ruminants, they will be degraded by the rumen microbes, which may be a waste. To manage this problem and ensure that ruminants get the amino acids they need in adequate quantities, animal health and nutrition companies have found ways to feed amino acids directly to the small intestine.

This is often accomplished by employing two mechanisms:. Rumen Protection: Rumen-protected amino acids are protected from the environment of the rumen so that they can reach the small intestine more consistently while avoiding degradation.

Intestinal Availability: Amino acids are useless if the intestine can't absorb them after passing through the rumen. Some amino acid products fail to release the amino acid at this point, and the amino acid is excreted in the feces. To avoid this, feed producers have developed products that release the rumen-protected amino acid after passing through the rumen so that it can be absorbed in the intestine.

Historically, blood meal has been used to get lysine through the rumen and into the bloodstream, but this product tended to be unreliable and often resulted in the excretion of excess nitrogen into the environment. Numerous feeding trials over the past several decades have shown that protein supplements can increase production for milk and eggs in livestock and poultry, respectively.

While the exact mechanism and amino acid balances differ based on the species and type of feed being used, cows, sheep and chickens all exhibited increased production when fed increased amounts of amino acids in their respective diets. For cattle and sheep specifically, introducing more dietary protein and a better amino acid makeup to cows can increase milk production substantially.

Depending on diet, the limiting amino acids for milk production can be methionine, lysine or any other amino acid.

However, research suggests that increasing overall amino acid availability to the small intestine results in an increase in production attributed to the increased availability of disposable non-essential amino acids.

In cows specifically, the delivery of high-quality protein with a well-balanced spread of amino acids was seen to produce a curvilinear increase in milk production 4 , leveling out as the cows reached their genetic limits.

Studies of egg-laying hens found similar results when fed more amino acids. Hens consistently produced more eggs of larger sizes. Unlike cows and sheep, hens do not have a rumen to consider, so unprotected amino acids can be added directly to the diet.

Typically, methionine tends to be the limiting amino acid in the diets of laying hens, and ideally they each should be fed around mg of methionine per day 5 to achieve maximum production.

Lysine and arginine are also highly significant in their diet, though it is equally important for hens to be fed enough pure caloric energy to produce since egg-laying is energetically expensive.

During the early phases of growth, all animals need access to as many essential amino acids as possible, as they need to produce sufficient proteins to support their growing bodies. Studies have shown that an increase in protein intake directly corresponds to an increase in protein deposition 6 within the bodies of growing animals, resulting in stronger, healthier mature animals.

Some amino acids are slightly more important than others, however. Amino Acids for Ruminates: For calves, the most important amino acids are methionine, lysine, isoleucine, threonine and leucine.

A deficiency in any of these amino acids results in a slowing of growth and delayed onset of maturity. The most important of these, methionine, is an essential amino acid. Though used inefficiently from a biological standpoint, methionine is important in cattle and sheep as a methyl group donor and a precursor for cysteine synthesis.

Lysine is the second most limiting amino acid for growing calves, especially in maize-based diets because maize is relatively low in lysine. Amino Acids for Pigs: Pigs have similar needs to calves, with the notable exception being arginine. While arginine is not an essential amino acid since it can be synthesized from glutamate and glutamine, it is essential to younger piglets in the neonatal and immediate postweaning phases.

Forty percent of pigs' arginine requirements 7 must be supplied through their diet, primarily due to their rapid growth rates and the fact that most arginine is used in the urea cycle of the liver. Amino Acids for Poultry: Growing poultry require similar amino acid balances as other growing animals, but they require arginine in their diets because they do not have a urea cycle and therefore cannot synthesize it on their own.

A deficiency of arginine often results in feather deformation in chickens 8. Lysine deficiencies can negatively affect feather growth in turkeys as well. Nutrition has a significant effect on the quality of eggs in all animals.

From the emergence of ovarian follicles through embryonic development, undernutrition can have a devastating effect on reproductive health for farm animals. By feeding animals sufficient amounts of amino acids to support egg production and embryonic health, you can ensure that your animals are producing healthy offspring at an optimal rate.

In ruminants, under-nutrition of amino acids can have a negative effect on fertility, especially during early ovulation. Most prominently, the intake of methionine and lysine have a strong effect throughout the fertility cycle.

These two amino acids are particularly important for embryonic development and consuming too little of either nutrient can negatively impact fertility. In one study, feeding rumen-protected methionine during the peripartum period of a cow's cycle significantly improved postpartum performance.

Additionally, studies have found that pregnancies are healthier when cows are fed sufficient amounts of methionine and lysine through the pregnancy, especially on days nine through 19, during which the cow's body determines whether to continue with a pregnancy. Pigs require a balanced diet that contains plenty of essential and non-essential amino acids.

While essential amino acids are important to support a pregnancy, sows also require dietary glutamine and arginine 9 to support mucosal integrity and neonatal growth, respectively. In summary, amino acids play varying importance roles based on the species of farm animal, its age and its production purpose.

Across all factors, however, protein supplements for cattle, pigs and poultry can deliver promising results and improve the performance and profitability of an animal.

Here are just a few ways that essential amino acids for animal health can benefit your bottom line:. When you raise the protein level in farm animal feed, farm animals will eat more food and digest it more efficiently, in turn increasing the amounts of amino acids and nutrients available to the animal.

This also improves feed efficiency, so there is less waste. Appropriate amino acid balances support improved growth rate so that animals will wean and reach mature weight early.

Tandem MS spectra for both positive and negative mode used a resolution of 15,, AGC target of 1e5, maximum IT of 50ms, isolation window of 0. The minimum AGC target was 1e4 with an intensity threshold of 2e5.

All data were acquired in profile mode. All valine data were processed using Thermo XCalibur Qualbrowser for manual inspection and annotation of the resulting spectra and peak heights referring to authentic valine standards and labeled internal standards as described.

QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates. Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina New England Biolabs, E , and sequenced on a NextSeq single-end 75 cycles high output with v2.

Differential gene enrichment analysis was performed with in R with DESeq2 and GO enrichment performed and visualized with clusterProfiler against the org. db database, with further visualization with the pathview, GoSemSim, eulerr packages.

Target plasmid was maintained in and purified from NEB beta electrocompetent E. coli New England Biolabs, CK. Lentivirus was packaged by plating 4×10 6 HEKT cells on 10 cm 2 and incubating cells overnight at 37°C. Cells were transfected with a plasmid mix consisting of 3. Transfected HEKT cells were incubated for 48 hr, before medium was collected, and centrifuged at ×g for 5 mins.

The resulting supernatant was filtered using a 0. The packaged virus was applied to cells for 24 hr before the medium was exchanged for fresh medium.

For RNA extraction, QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates. cDNA was generated from RNA using Invitrogen SuperScript IV Reverse Transcriptase Invitrogen, and oligo dT primers.

Each qPCR reaction was performed using SYBR Green Master I Roche, on a Light Cycler Roche, using the recommended cycling conditions. Primers were designed to amplify amplicons — bp in size. Sequencing data generated for this study is deposited in the NCBI SRA at accession number PRJNA Source data files have been provided for Figure 1 - figure supplement 1, Figure 1 - figure supplement 2D, Figure 2, Figure 2 - figure supplement 3, Figure 2 - figure supplement 4B, Figure 2 - figure supplement 5, Figure 2 - figure supplement 6, Figure 3, and Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, and Figure 5 - figure supplement 1.

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Thank you for submitting your article "Resurrecting essential amino acid biosynthesis in a mammalian cell" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Ivan Topisirovic as Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Philip Cole as the Senior Editor.

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Based on this, it was thought that more evidence is required to demonstrate that the introduction of valine biosynthetic pathway into CHO cells results in sustained proliferation and survival in the absence of valine supplementation.

Accordingly, it was deemed that the authors should monitor long-term ability of engineered CHO cells to sustain valine production and proliferate in valine-free media. To this end, monitoring flux via valine biosynthetic and degradation pathways, transcriptome and mTOR signaling at early and late time points was thought to be warranted.

These include lack of clarity pertinent to the rationale behind using "conditioned-medium" in the experiments. Moreover, potential utilization of other sources of valine e.

It was appreciated that the latter cells survive in valine-free media, but it seems that their proliferation is significantly lower than in valine containing media.

Moreover, it seems that after 6 passages only a fraction of the detected valine is synthesized de novo. Would this fraction further decrease in subsequent passages?

Related to this, it is not clear what is the efficiency of valine biosynthesis in CHO cells vs. a prototrophic organism. Perhaps comparing the rates of valine synthesis in cell free extracts of CHO cells vs. those derived from a prototrophic organism may be helpful to address this.

This in particular relates to amino-acid sensing pathways e. Were the enzymes mislocalized? Are there other regulatory factors involved? Moreover, considering that the overarching tenet is that metazoans lost the ability to produce essential amino acids due to energetic restraints, it may be worthwhile noting that culturing conditions and potential differences in energy resources may impact on functionalization of essential amino acid biosynthetic pathways.

The results put forth in this manuscript suggest the authors were marginally successful in introducing a valine biosynthetic pathway into CHO cells, but fall short of demonstrating a robust, self-sustaining engineered cell line under reasonable culture conditions.

This milestone should be met prior to final acceptance at eLife. Additionally, the following revisions should be carried out prior to acceptance. The authors should identify the timepoint at which pCTRL cells are no longer viable in dropout medium.

The authors should then compare transcriptional profiles of the pMTIV cells at that timepoint to the that of pMTIV cells harvested at 4hr and 48hr. Doing so may help identify key bottlenecks in the pathway.

If a bottleneck can be identified, authors should attempt to make the pathway more efficient, either by modifying expression strategy of that enzyme or testing homologs from other hosts. The pathway should be optimized until the major revision 1 above is achieved.

For clarity, these sections should be de-emphasized in writing and figures for clarity. The task that Wang et al. We thank the reviewers for this feedback. We understand the core issue to be the reduction in doubling time shown for later time points in Figure 2F and the suggestion that this represents a time-dependent lag in growth rate due to cumulative insufficient valine production.

In response to this feedback, we set out to attain a consistent doubling time in the valine-free condition. Importantly, this dihydroxy-acid dehydratase overexpressing cell line was passaged 10 times in the absence of valine with a consistent average doubling time of 3.

Doubling time remained consistent across the 39 days of culture and no medium conditioning was required Figure 5. Nonetheless, to alleviate concerns that the original prototrophic pMTIV cells were not able to sustain proliferation long-term in the absence of valine, we have also added additional evidence indicating that these cells retained valine prototrophy long-term:.

Given the rapid death phenotype experienced by pCtrl cells, continued survival of pMTIV cells at late passages should instead be considered an indicator of sustained prototrophy. Late time point transcriptomic data Figure 3 —figure supplement 5 for pMTIV cells demonstrating partial rescue of nutritional starvation at day 29 in conditioned valine-free FK medium.

We thank the reviewers for these comments. We have added a figure highlighting mTOR signaling differences in pMTIV and pCtrl cells at 48 h valine starvation, even though no clear signatures of mTOR activation could be detected Figure 3 —figure supplement 4. We have also added a new supplemental figure showing transcriptomic analysis of cells grown long-term 5 passages, 29 days in conditioned valine-free FK medium Figure 3 —figure supplement 5.

Additionally, we were able to gain insight into flux through the pathway with 13 C-tracing. No signal could be detected for pyruvate, 2-acetolactate or 2-oxoisovalerate; however we were able to specifically detect pathway intermediate 2,3-dihydroxy-isoverate and have added a panel to reflect this Figure 3 —figure supplement 1F.

It was unclear whether the detected 2,3-dihydroxy-isoverate represented a true pathway bottleneck. In order to test whether this was the case, we introduced extra copies of the downstream ilvD gene encoding the dihydroxy-acid dehydratase enzyme, by lentiviral transduction.

We apologize for the lack of clarity surrounding the use of conditioned medium and thank the reviewers for bringing this to our attention. We have added a panel demonstrating the utility of using conditioned medium in culturing pMTIV cells in the absence of valine Figure 2 —figure supplement 5B.

When culturing prototrophic cells in valine-free medium conditions, extracellular valine concentrations will be minimal, forcing cells to secrete valine until the appropriate equilibrium has been met. Examples supporting this rationale can be found in the literature.

For example, in a publication by Eagle and Piez 1 it was demonstrated that there is a population-dependent requirement of cultured cells for metabolites that are otherwise considered non-essential.

For instance, serine was required for growth when cells were cultured at low cell densities. Figure 2 —figure supplement 5B further supports this explanation by illustrating that the positive effect from medium conditioning cannot be recapitulated if the medium is conditioned with pCtrl cells, which excludes the possibility of cell debris or other effects from medium conditioning conferring the positive benefit.

It would therefore indicate that the benefit to cells that is derived from pMTIV medium conditioning is likely specifically caused by the valine synthesized and secreted by these cells. The serum was analyzed for the presence of 15 amino acids including valine, which was found to be present at 9.

Regarding autophagy, if such an effect would significantly alter the outcome of cells, this would not be specific to our engineered cells and any rescue effects thereof should be apparent for pCtrl cells as well, which was shown not to be the case Figure 2C, Figure 2E, Figure 2 —figure supplement 5.

Given the success with valine, we feel it appropriate to outline these results on their own terms. However, we agree that it would be beneficial to additionally discuss other efforts. We initially began our experimentation by designing an all-in-one construct that would introduce a isoleucine and valine biosynthesis using a shared 4-gene pathway b threonine biosynthesis by driving a typically degradative enzyme in reverse, and c rescue of methionine auxotrophy by bridging a gap in the sulfur shuttle.

The all-in-one format using 2A ribosome-skipping peptide sequences served to free up the limited number of available mammalian regulatory elements for potential addition of other pathway functionalities as well as to minimize the number of genes introduced and by extension the cost of DNA synthesis.

In particular, the gene choices made in the attempts to achieve b and c were optimistic and made in the interest of optimizing pathway number per DNA length. While the valine pathway in theory is able to conduct isoleucine biosynthesis activity as well, the choice of an E.

This may be necessary for meaningful isoleucine biosynthetic functionality but in addition, isoleucine biosynthesis additionally requires the presence of 2-oxobutanoate, which is not as involved in core metabolism as pyruvate and therefore is presumably found at much lower concentrations in cells.

We have added a panel Figure 4 —figure supplement 1B demonstrating increased proliferative ability of pMTIV cells in valine-free RPMI medium at a reduced 0.

In the case of threonine, we attempted to opportunistically take advantage of the bidirectionality of a typically degradative enzyme, ltaE.

However, this failed to rescue threonine auxotrophy, presumably because the mammalian metabolic equilibrium did not favor the reverse enzymatic reaction as intended.

In the case of methionine, rescue of biosynthesis was attempted by allowing for interconversion of cystathionine and homocysteine. Methionine is synthesized in mammalian cells from homocysteine, and we reasoned that increasing levels of cystathionine by introduction of E.

coli -derived metC would increase levels of homocysteine, which might increase cell viability in methionine-free conditions. However, cystathionine biosynthesis in E. coli and mammalian cells are divergent processes requiring different starting substrates. Whereas E. coli synthesizes cystathionine from cysteine and succinyl-homoserine, mammalian cells synthesize cystathionine from serine and homocysteine.

Introducing metC into a mammalian metabolic context therefore bridges a gap that is incompatible with the evolutionary developments of the past hundreds of millions of years, resulting in a circular pathway unlikely to produce significant quantities of methionine, which was confirmed empirically in our functional assay.

We would like to highlight to the reviewers that additional work is ongoing to rescue yet other essential amino acids, as well as our call for a wider community focus on such projects. We would like to clarify that the metabolomics data presented in the manuscript describes a separate experiment from the long-term culture experiments, and were collected after 3 passages or 12 days in unconditioned valine-free RPMI medium containing 13 C-glucose and 13 C-sodium pyruvate Figure 3 —figure supplement 1A.

To measure valine biosynthesis past the 3 rd passage as suggested, we set out to perform an additional metabolomics analysis looking at 13 C-valine levels — this time over a longer time period. In this time course, 13 C-glucose replaced its 12 C counterpart in the valine-free RPMI medium formulation as before; however the spiked in sodium pyruvate was not 13 C-labeled in this follow-up experiment due to limited reagent availability during the COVID pandemic.

This is important to note as it follows that the expected 13 C-labeling outcome is different. This is in contrast to the original experiment in which only 13 C sources of glucose and pyruvate were spiked in. In anticipation that cells might not perform well in unconditioned medium and in the new RPMI context, we therefore attempted to take measures to lose fewer cells to the harsh effects of passaging by culturing cells on plates coated with 0.

While it in theory was possible that cells were consuming valine derived from the 0. Furthermore, we later cultured cells long-term in unconditioned valine-free RPMI on plates not coated with 0.

In pMTIV cells, 13 C-valine content was lower than 12 C-valine content on days 14 and 24 while the opposite was true on days 2, 4, 12, and 18, demonstrating that the 13 C content of the cells was not on a downward trend but rather fluctuated up and down.

This may reflect an inability to adequately respond to valine demands given inefficient flux through the pathway. We thank the reviewer for these insights. We address this point above in our response to Essential Revision 4.

We agree with the reviewer on this point and have already begun efforts to test our pathways in other cell lines, such as HEK cells, but we believe these data are too preliminary to be included at this time, and are beyond the scope of this contribution.

We undertook a painstaking and extensive effort to demonstrate robust and self-sustaining valine-free growth over 39 days. This was achieved by increasing ilvD encoding a dihydroxy-acid dehydratase enzyme copy number in response to detecting a potential pathway bottleneck in 2,3-dihydroxy-isovalerate.

By increasing the efficiency of the final step in the introduced biosynthetic pathway, doubling time was reduced to a relatively consistent 3.

o It is possible residual valine from complete medium may help pCTRL and pMTIV cells survive at early timepoints. While it was possible to include an 8 day timepoint for collecting transcriptomic pMTIV samples we originally did not do so as there is no suitable control for analyzing such samples.

Nonetheless, we had previously collected RNA in duplicate samples from a late time point and as such have included transcriptomic data in a new figure for samples cultured in conditioned valine-free FK medium over 29 days or 5 passages i.

well past the point of pCtrl inviability Figure 3 —figure supplement 5. Evidently, these cells more closely resemble healthy cells cultured in complete medium than do pCtrl cells cultured in valine-free FK for just 48 h.

This excellent reviewer suggestion led to an important new finding — the specific accumulation of an intermediate suggesting a pathway bottleneck. We explored the presence of pathway intermediates in our original 13 C-tracing experiment.

The only pathway intermediate detected by metabolomics analysis was 2,3-dihydroxy-isovalerate, suggesting potential bottlenecking at this step.

This resulted in cells that double on average every 3. This data demonstrates long-term homeostasis and robust growth under reasonable culturing conditions.

We believed it would be misleading to describe our efforts to rescue valine biosynthesis alone. On the suggestion of Reviewer 1 above as well as the suggestion outlined in Essential Revision 4 sections have been adjusted but not removed.

The potential toxicity of valine pathway intermediates or perhaps toxic products from non-specific enzymatic activity is certainly an interesting question, given the introduction of a pathway sourced from a distantly removed species. However, we saw no signs of metabolic stress in cells harboring the pathway when grown on complete media, either by growth rate Figure 2D or in the transcriptomic data Figure 3D, Figure 3 —figure supplement 2.

The introduction of the pathway therefore does not appear to be a significant stressor to the cells. However, it remains to be seen if this will true for all essential amino acids, particularly as we look to introduce the more complex pathways.

We thank the reviewer for the vote of confidence, and share their excitement for the insights this work might enable. Eagle, H. and Piez, K. The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize.

Journal of Experimental Medicine , 29—43 Barak Z, Chipman D M, and Gollop N. Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria.

Journal of Bacteriology , — Mitchell Leslie A. et al. Science , eaaf Richardson Sarah M. Science , — The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank the members of the Boeke and Wang labs for comments and discussion on the work and manuscript. RMM additionally thanks personal support from Xiaoyu Weng. Defense Advanced Research Projects Agency HR HHW, JDB. National Science Foundation MCB HHW. Burroughs Wellcome Fund PATH HHW.

Irma T Hirschl Trust HHW. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion.

After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site s of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii , small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown.

Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis.

Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired.

The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine O-GlcNAc signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration.

Here, we define the specific metabolic pathway by which O-GlcNAc transferase ogt-1 loss of function mediates increased regenerative outgrowth.

Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans , we find that glycolysis, serine synthesis pathway SSP , one-carbon metabolism OCM , and the downstream transsulfuration metabolic pathway TSP are all essential in this process.

Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway.

These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals.

Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health.

Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response.

Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Phodependent phosphate sensing pathway.

We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila.

These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria. Share this article Doi.

Cite this article Julie Trolle Ross M McBee Andrew Kaufman Sudarshan Pinglay Henri Berger Sergei German Liyuan Liu Michael J Shen Xinyi Guo J Andrew Martin Michael E Pacold Drew R Jones Jef D Boeke Harris H Wang Resurrecting essential amino acid biosynthesis in mammalian cells.

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Figure 1 with 2 supplements see all. Download asset Open asset. Figure 2 with 6 supplements see all. Figure 2—source data 1 Raw cell count data for pMTIV valine-free and complete FK medium tests.

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zip Download elifefig5-data1-v2. Key resources table. Reagent type species or resource Designation Source or reference Identifiers Additional information Strain, strain background E.

coli TransforMax EPI Lucigen CC Chemically competent Strain, strain background E. coli NEB beta New England Biolabs CK Electrocompetent Cell line C. griseus CHO Flp-In ThermoFisher R Cell line C. griseus CHO pMTIV This paper Cell line maintained in J.

Boeke lab Cell line C. griseus CHO pCtrl This paper Cell line maintained in J. griseus CHO pPro This paper Cell line maintained in H. Wang lab Cell line C. griseus CHO pCtrl-mCh This paper Cell line maintained in H.

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BioEssays 39 :1— Dancis J Levitz M Westall RG Maple syrup urine disease: branched-chain keto-aciduria Pediatrics 25 — Eagle H Piez K The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize The Journal of Experimental Medicine — Fischer S Handrick R Otte K The art of CHO cell engineering: a comprehensive retrospect and future perspectives Biotechnology Advances 33 — Fredens J Wang K de la Torre D Funke LFH Robertson WE Christova Y Chia T Schmied WH Dunkelmann DL Beránek V Uttamapinant C Llamazares AG Elliott TS Chin JW Total synthesis of Escherichia coli with a recoded genome Nature — Gilbert LA Horlbeck MA Adamson B Villalta JE Chen Y Whitehead EH Guimaraes C Panning B Ploegh HL Bassik MC Qi LS Kampmann M Weissman JS Genome-Scale CRISPR-mediated control of gene repression and activation Cell — Guedes RLM Prosdocimi F Fernandes GR Moura LK Ribeiro HAL Ortega JM Amino acids biosynthesis and nitrogen assimilation pathways: a great genomic deletion during eukaryotes evolution BMC Genomics 12 Suppl 4 :S2.

Hefzi H Ang KS Hanscho M Bordbar A Ruckerbauer D Lakshmanan M Orellana CA Baycin-Hizal D Huang Y Ley D Martinez VS Kyriakopoulos S Jiménez NE Zielinski DC Quek L-E Wulff T Arnsdorf J Li S Lee JS Paglia G Loira N Spahn PN Pedersen LE Gutierrez JM King ZA Lund AM Nagarajan H Thomas A Abdel-Haleem AM Zanghellini J Kildegaard HF Voldborg BG Gerdtzen ZP Betenbaugh MJ Palsson BO Andersen MR Nielsen LK Borth N Lee D-Y Lewis NE A consensus genome-scale reconstruction of Chinese hamster ovary cell metabolism Cell Systems 3 — Heng BC Aubel D Fussenegger M Prosthetic gene networks as an alternative to standard pharmacotherapies for metabolic disorders Current Opinion in Biotechnology 35 — Isaacs FJ Carr PA Wang HH Lajoie MJ Sterling B Kraal L Tolonen AC Gianoulis TA Goodman DB Reppas NB Emig CJ Bang D Hwang SJ Jewett MC Jacobson JM Church GM Precise manipulation of chromosomes in vivo enables genome-wide codon replacement Science — Kemmer C Gitzinger M Daoud-El Baba M Djonov V Stelling J Fussenegger M Self-Sufficient control of urate homeostasis in mice by a synthetic circuit Nature Biotechnology 28 — Kitada T DiAndreth B Teague B Weiss R Programming gene and engineered-cell therapies with synthetic biology Science :eaad

All amino acids pathsay derived from intermediates in Amino acid synthesis pathway in animals, the citric Amin cycle, or the pentose phosphate pathway. Nitrogen enters these biosynthetic pathways by way of glutamate and glutamine. Some pathways are simple, others are not. Ten of the amino acids are just one or several steps removed from the common metabolite from which they are derived. The biosynthetic pathways for others, such as the aromatic amino acids, are more complex.

Amino acids are the structural units that make up proteins. They join synthesie to form short polymer chains called peptides or Sodium intake and bone health chains called either polypeptides or proteins. These polymers are linear and unbranched, Anti-inflammatory foods for athletes, with each amino acid Caloric balance the chain attached to Sodium intake and bone health neighboring Sodium intake and bone health acids.

The Sodium intake and bone health of synthesid proteins Manage cravings for salty foods called translation and involves qnimals step-by-step addition Superfood detox diets amino acids to adid growing protein chain by a ribozyme synhtesis is called a ribosome.

Twenty-two ahimals acids are naturally Preventing dental cavities into polypeptides syntheeis are naimals proteinogenic or natural Amino acid synthesis pathway in animals acids.

Of these, 20 are animaps by the universal genetic code. The remaining patjway, selenocysteine and pyrrolysine, are incorporated into proteins by unique synthetic mechanisms.

Synthesiz is incorporated when patway mRNA being translated includes a SECIS Cognitive function training, which causes the UGA codon to synthdsis selenocysteine pathwxy of a stop African Mango Elite. Pyrrolysine patthway used by Aminno methanogenic archaea in enzymes that they use to produce methane.

It is animalz with the codon UAG, payhway is pahway a stop codon in pathhway Sodium intake and bone health. Pyrrolysine abbreviated as ;athway or O is a naturally occurring amino acid similar to lysine, but with an added pyrroline Amino acid synthesis pathway in animals linked to the end accid the lysine side chain.

Produced by a specific tRNA and aminoacyl Sodium intake and bone health anjmals, it forms part of an animala genetic code syhthesis these Boost your thermogenic rate. It is anjmals the 22 nd proteinogenic amino acid.

This UAG Aacid is followed Amuno a PYLIS pathsay sequence. Organisms vary in their ability to synthesize the 20 common amino acids.

Most bacteria and plants can synthesize all Some simple parasites, such as the bacteria Mycoplasma pneumoniaelack all amino acid synthesis and take their amino acids directly from their hosts. All amino acids are synthesized from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway.

Nitrogen is provided by glutamate and glutamine. Amino acid synthesis depends on the formation of the appropriate alpha-keto acid, which is then transaminated to form an amino acid.

Amino acids are made into proteins by being joined together in a chain by peptide bonds. Each different protein has a unique sequence of amino acid residues: this is its primary structure.

Just as the letters of the alphabet can be combined to form an almost endless variety of words, amino acids can be linked in varying sequences to form a huge variety of proteins.

Proteins are made from amino acids that have been activated by attachment to a transfer RNA molecule through an ester bond. Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard.

Those either are not found in proteins for example carnitine, GABA or are not produced directly and in isolation by standard cellular machinery for example, hydroxyproline and selenomethionine.

Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein. For example, the carboxylation of glutamate allows for better binding of calcium cations.

The hydroxylation of proline is critical for maintaining connective tissues. Another example is the formation of hypusine in the translation initiation factor EIF5A, through modification of a lysine residue.

Such modifications can also determine the localization of the protein, e. Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids — for example, ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.

Search site Search Search. Go back to previous article. Sign in. Learning Objectives Recognize the factors involved in amino acid synthesis. Key Points All amino acids are synthesized from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway.

Of the 22 amino acids naturally incorporated into proteins, 20 are encoded by the universal genetic code and the remaining two, selenocysteine and pyrrolysine, are incorporated into proteins by unique synthetic mechanisms.

Key Terms pyrrolysine : An amino acid found in methanogenic bacteria. selenocysteine : A naturally-occurring amino acid, present in several enzymes, whose structure is that of cysteine but with the sulfur atom replaced by one of selenium.

genetic code : The set of rules by which the sequence of bases in DNA are translated into the amino acid sequence of proteins.

: Amino acid synthesis pathway in animals

Author Contributions

By comparison, pMTIV cells exhibited an average doubling time of 4. Plates were not coated with gelatin. In this work, we demonstrated the successful restoration of an EAA biosynthetic pathway in a metazoan cell.

Our results indicate that contemporary metazoan biochemistry can support complete biosynthesis of valine, despite millions of years of evolution from its initial loss from the ancestral lineage.

Interestingly, independent evidence for BCAA biosynthesis has also been obtained for sap-feeding whitefly bacteriocytes that host bacterial endosymbionts; metabolite sharing between these cells is predicted to lead to biosynthesis of BCAAs that are limiting in their restricted diet.

The malleability of mammalian metabolism to accept heterologous core pathways opens up the possibility of animals with designer metabolisms and enhanced capacities to thrive under environmental stress and nutritional starvation Zhang et al. Yet, our failure to functionalize designed methionine, threonine, and isoleucine pathways highlights outstanding challenges and future directions for synthetic metabolism engineering in animal cells and animals.

Other pathway components or alternative selections may be needed for different EAAs Rees and Hay, Studies to reincorporate EAAs into the core mammalian metabolism could provide greater understanding of nutrient-starvation in different physiological contexts including the tumor microenvironment Lim et al.

Emerging synthetic genomic efforts to build a prototrophic mammal may require reactivation of many more genes Supplementary files , iterations of the design, build, test DBT cycle, and a larger coordinated research effort to ultimately bring such a project to fruition. For pathway completeness analysis, the EC numbers of each enzyme in each amino acid biosynthesis pathway excluding pathways annotated as only occurring in prokaryotes were collected from the MetaCyc database Supplementary file 4.

Variant biosynthetic routes to the same amino acid were considered as separate pathways, generating distinct EC number lists. The resulting per-pathway EC number lists were checked against the KEGG, Entrez Gene, Entrez Nucleotide, and Uniprot databases using their respective web APIs for each listed organism.

CHO Flp-In cells ThermoFisher, R were used in all experiments. All cell lines tested negative for mycoplasma. Custom amino acid dropout medium was adjusted to a pH of 7. For metabolomics experiments, medium was prepared from an amino acid-free and glucose-free RPMI powder base US Biological, R , and custom combinations of amino acids and isotopically heavy glucose and sodium pyruvate were added in to match the standard amino acid concentrations for RPMI or as specified.

pH was adjusted to 7. Where specified, cells were cultured on plates coated with 0. Plates were washed with PBS prior to use. For evaluating effects of amino acid dropout on cell growth curves, cells were seeded at 1×10 4 into 6-well plates into FK media with lowered amino acid concentrations relative to typical FK media and then allowed to grow for 5 days.

Media was then aspirated off and replaced with PBS with Hoechst live nuclear stain for automated imaging and counting using a DAPI filter set on an Eclipse Ti2 automated inverted microscope.

To count, an automated microscopy routine was used to Figure 5 random locations within each well at 10× magnification, and then the cells present in imaged frames counted using automatic cell segregation and counting software.

Given differences in cell response to starvation, segregation and counting parameters were tuned in each experiment, but kept constant between starvation conditions and cells with and without the pathway. Conditioned medium was generated by seeding 1×10 6 pMTIV cells into 10 mL complete FK medium on 10 cm plates and replacing the medium with 10 mL freshly prepared valine-free FK medium the next day following a PBS wash step.

Cells conditioned the medium for 2 days at which point the medium was collected, centrifuged at ×g for 3 mins to remove potential cell debris, sterile filtered, and collected in mL vats to reduce batch-to-batch variation. Integrated constructs were synthesized de novo in 3 kb DNA segments with each segment overlapping neighboring segments by 80 bp.

Assembly was conducted in yeasto by co-transformation of segments into S. cerevisiae strain BY made competent by the LiOAc method Pan et al.

After 2 days of selection at 30°C on SC—Ura medium, individual colonies were picked and cultured overnight. Glass beads were added to each resuspension and the mixture was vortexed for 10 mins to mechanically shear the cells. Next, cells were subject to alkaline lysis by adding µl of P2 lysis buffer Qiagen, for 5 mins and then neutralized by addition of Qiagen N3 neutralization buffer Qiagen, Plasmid DNA was eluted in Zyppy Elution buffer and subsequently transformed into TransforMax EPI chemically competent E.

Cell were lysed in SKL Triton lysis buffer 50 mM Hepes pH7. NuPAGE LDS sample buffer ThermoFisher, NP supplemented with 1. The membrane was incubated in the secondary antibody solution for 1.

Cell pellets were generated by trypsinization, followed by low speed centrifugation, and the pellet was frozen at —80°C until further processing.

The LC column was a Millipore ZIC-pHILIC 2. Injection volume was set to 1 μL for all analyses 42 min total run time per injection. MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HF mass spectrometer operating in heated electrospray ionization mode HESI.

Spray voltage for both positive and negative modes was 3. Tandem MS spectra for both positive and negative mode used a resolution of 15,, AGC target of 1e5, maximum IT of 50ms, isolation window of 0.

The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. All valine data were processed using Thermo XCalibur Qualbrowser for manual inspection and annotation of the resulting spectra and peak heights referring to authentic valine standards and labeled internal standards as described.

QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates. Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina New England Biolabs, E , and sequenced on a NextSeq single-end 75 cycles high output with v2. Differential gene enrichment analysis was performed with in R with DESeq2 and GO enrichment performed and visualized with clusterProfiler against the org.

db database, with further visualization with the pathview, GoSemSim, eulerr packages. Target plasmid was maintained in and purified from NEB beta electrocompetent E.

coli New England Biolabs, CK. Lentivirus was packaged by plating 4×10 6 HEKT cells on 10 cm 2 and incubating cells overnight at 37°C.

Cells were transfected with a plasmid mix consisting of 3. Transfected HEKT cells were incubated for 48 hr, before medium was collected, and centrifuged at ×g for 5 mins. The resulting supernatant was filtered using a 0.

The packaged virus was applied to cells for 24 hr before the medium was exchanged for fresh medium. For RNA extraction, QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates.

cDNA was generated from RNA using Invitrogen SuperScript IV Reverse Transcriptase Invitrogen, and oligo dT primers. Each qPCR reaction was performed using SYBR Green Master I Roche, on a Light Cycler Roche, using the recommended cycling conditions. Primers were designed to amplify amplicons — bp in size.

Sequencing data generated for this study is deposited in the NCBI SRA at accession number PRJNA Source data files have been provided for Figure 1 - figure supplement 1, Figure 1 - figure supplement 2D, Figure 2, Figure 2 - figure supplement 3, Figure 2 - figure supplement 4B, Figure 2 - figure supplement 5, Figure 2 - figure supplement 6, Figure 3, and Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, and Figure 5 - figure supplement 1.

Our editorial process produces two outputs: i public reviews designed to be posted alongside the preprint for the benefit of readers; ii feedback on the manuscript for the authors, including requests for revisions, shown below.

We also include an acceptance summary that explains what the editors found interesting or important about the work. Thank you for submitting your article "Resurrecting essential amino acid biosynthesis in a mammalian cell" for consideration by eLife.

Your article has been reviewed by 3 peer reviewers, including Ivan Topisirovic as Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Philip Cole as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ran Kafri Reviewer 3.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Based on this, it was thought that more evidence is required to demonstrate that the introduction of valine biosynthetic pathway into CHO cells results in sustained proliferation and survival in the absence of valine supplementation.

Accordingly, it was deemed that the authors should monitor long-term ability of engineered CHO cells to sustain valine production and proliferate in valine-free media. To this end, monitoring flux via valine biosynthetic and degradation pathways, transcriptome and mTOR signaling at early and late time points was thought to be warranted.

These include lack of clarity pertinent to the rationale behind using "conditioned-medium" in the experiments. Moreover, potential utilization of other sources of valine e. It was appreciated that the latter cells survive in valine-free media, but it seems that their proliferation is significantly lower than in valine containing media.

Moreover, it seems that after 6 passages only a fraction of the detected valine is synthesized de novo. Would this fraction further decrease in subsequent passages? Related to this, it is not clear what is the efficiency of valine biosynthesis in CHO cells vs.

a prototrophic organism. Perhaps comparing the rates of valine synthesis in cell free extracts of CHO cells vs. those derived from a prototrophic organism may be helpful to address this. This in particular relates to amino-acid sensing pathways e.

Were the enzymes mislocalized? Are there other regulatory factors involved? Moreover, considering that the overarching tenet is that metazoans lost the ability to produce essential amino acids due to energetic restraints, it may be worthwhile noting that culturing conditions and potential differences in energy resources may impact on functionalization of essential amino acid biosynthetic pathways.

The results put forth in this manuscript suggest the authors were marginally successful in introducing a valine biosynthetic pathway into CHO cells, but fall short of demonstrating a robust, self-sustaining engineered cell line under reasonable culture conditions.

This milestone should be met prior to final acceptance at eLife. Additionally, the following revisions should be carried out prior to acceptance.

The authors should identify the timepoint at which pCTRL cells are no longer viable in dropout medium. The authors should then compare transcriptional profiles of the pMTIV cells at that timepoint to the that of pMTIV cells harvested at 4hr and 48hr.

Doing so may help identify key bottlenecks in the pathway. If a bottleneck can be identified, authors should attempt to make the pathway more efficient, either by modifying expression strategy of that enzyme or testing homologs from other hosts.

The pathway should be optimized until the major revision 1 above is achieved. For clarity, these sections should be de-emphasized in writing and figures for clarity.

The task that Wang et al. We thank the reviewers for this feedback. We understand the core issue to be the reduction in doubling time shown for later time points in Figure 2F and the suggestion that this represents a time-dependent lag in growth rate due to cumulative insufficient valine production.

In response to this feedback, we set out to attain a consistent doubling time in the valine-free condition. Importantly, this dihydroxy-acid dehydratase overexpressing cell line was passaged 10 times in the absence of valine with a consistent average doubling time of 3.

Doubling time remained consistent across the 39 days of culture and no medium conditioning was required Figure 5. Nonetheless, to alleviate concerns that the original prototrophic pMTIV cells were not able to sustain proliferation long-term in the absence of valine, we have also added additional evidence indicating that these cells retained valine prototrophy long-term:.

Given the rapid death phenotype experienced by pCtrl cells, continued survival of pMTIV cells at late passages should instead be considered an indicator of sustained prototrophy.

Late time point transcriptomic data Figure 3 —figure supplement 5 for pMTIV cells demonstrating partial rescue of nutritional starvation at day 29 in conditioned valine-free FK medium. We thank the reviewers for these comments.

We have added a figure highlighting mTOR signaling differences in pMTIV and pCtrl cells at 48 h valine starvation, even though no clear signatures of mTOR activation could be detected Figure 3 —figure supplement 4. We have also added a new supplemental figure showing transcriptomic analysis of cells grown long-term 5 passages, 29 days in conditioned valine-free FK medium Figure 3 —figure supplement 5.

Additionally, we were able to gain insight into flux through the pathway with 13 C-tracing. No signal could be detected for pyruvate, 2-acetolactate or 2-oxoisovalerate; however we were able to specifically detect pathway intermediate 2,3-dihydroxy-isoverate and have added a panel to reflect this Figure 3 —figure supplement 1F.

It was unclear whether the detected 2,3-dihydroxy-isoverate represented a true pathway bottleneck. In order to test whether this was the case, we introduced extra copies of the downstream ilvD gene encoding the dihydroxy-acid dehydratase enzyme, by lentiviral transduction.

We apologize for the lack of clarity surrounding the use of conditioned medium and thank the reviewers for bringing this to our attention. We have added a panel demonstrating the utility of using conditioned medium in culturing pMTIV cells in the absence of valine Figure 2 —figure supplement 5B.

When culturing prototrophic cells in valine-free medium conditions, extracellular valine concentrations will be minimal, forcing cells to secrete valine until the appropriate equilibrium has been met. Examples supporting this rationale can be found in the literature. For example, in a publication by Eagle and Piez 1 it was demonstrated that there is a population-dependent requirement of cultured cells for metabolites that are otherwise considered non-essential.

For instance, serine was required for growth when cells were cultured at low cell densities. Figure 2 —figure supplement 5B further supports this explanation by illustrating that the positive effect from medium conditioning cannot be recapitulated if the medium is conditioned with pCtrl cells, which excludes the possibility of cell debris or other effects from medium conditioning conferring the positive benefit.

It would therefore indicate that the benefit to cells that is derived from pMTIV medium conditioning is likely specifically caused by the valine synthesized and secreted by these cells. The serum was analyzed for the presence of 15 amino acids including valine, which was found to be present at 9.

Regarding autophagy, if such an effect would significantly alter the outcome of cells, this would not be specific to our engineered cells and any rescue effects thereof should be apparent for pCtrl cells as well, which was shown not to be the case Figure 2C, Figure 2E, Figure 2 —figure supplement 5.

Given the success with valine, we feel it appropriate to outline these results on their own terms. However, we agree that it would be beneficial to additionally discuss other efforts.

We initially began our experimentation by designing an all-in-one construct that would introduce a isoleucine and valine biosynthesis using a shared 4-gene pathway b threonine biosynthesis by driving a typically degradative enzyme in reverse, and c rescue of methionine auxotrophy by bridging a gap in the sulfur shuttle.

The all-in-one format using 2A ribosome-skipping peptide sequences served to free up the limited number of available mammalian regulatory elements for potential addition of other pathway functionalities as well as to minimize the number of genes introduced and by extension the cost of DNA synthesis.

In particular, the gene choices made in the attempts to achieve b and c were optimistic and made in the interest of optimizing pathway number per DNA length. While the valine pathway in theory is able to conduct isoleucine biosynthesis activity as well, the choice of an E.

This may be necessary for meaningful isoleucine biosynthetic functionality but in addition, isoleucine biosynthesis additionally requires the presence of 2-oxobutanoate, which is not as involved in core metabolism as pyruvate and therefore is presumably found at much lower concentrations in cells.

We have added a panel Figure 4 —figure supplement 1B demonstrating increased proliferative ability of pMTIV cells in valine-free RPMI medium at a reduced 0. In the case of threonine, we attempted to opportunistically take advantage of the bidirectionality of a typically degradative enzyme, ltaE.

However, this failed to rescue threonine auxotrophy, presumably because the mammalian metabolic equilibrium did not favor the reverse enzymatic reaction as intended. In the case of methionine, rescue of biosynthesis was attempted by allowing for interconversion of cystathionine and homocysteine.

Methionine is synthesized in mammalian cells from homocysteine, and we reasoned that increasing levels of cystathionine by introduction of E. coli -derived metC would increase levels of homocysteine, which might increase cell viability in methionine-free conditions. However, cystathionine biosynthesis in E.

coli and mammalian cells are divergent processes requiring different starting substrates. Whereas E. coli synthesizes cystathionine from cysteine and succinyl-homoserine, mammalian cells synthesize cystathionine from serine and homocysteine.

Introducing metC into a mammalian metabolic context therefore bridges a gap that is incompatible with the evolutionary developments of the past hundreds of millions of years, resulting in a circular pathway unlikely to produce significant quantities of methionine, which was confirmed empirically in our functional assay.

We would like to highlight to the reviewers that additional work is ongoing to rescue yet other essential amino acids, as well as our call for a wider community focus on such projects. We would like to clarify that the metabolomics data presented in the manuscript describes a separate experiment from the long-term culture experiments, and were collected after 3 passages or 12 days in unconditioned valine-free RPMI medium containing 13 C-glucose and 13 C-sodium pyruvate Figure 3 —figure supplement 1A.

To measure valine biosynthesis past the 3 rd passage as suggested, we set out to perform an additional metabolomics analysis looking at 13 C-valine levels — this time over a longer time period.

In this time course, 13 C-glucose replaced its 12 C counterpart in the valine-free RPMI medium formulation as before; however the spiked in sodium pyruvate was not 13 C-labeled in this follow-up experiment due to limited reagent availability during the COVID pandemic.

This is important to note as it follows that the expected 13 C-labeling outcome is different. This is in contrast to the original experiment in which only 13 C sources of glucose and pyruvate were spiked in.

In anticipation that cells might not perform well in unconditioned medium and in the new RPMI context, we therefore attempted to take measures to lose fewer cells to the harsh effects of passaging by culturing cells on plates coated with 0.

While it in theory was possible that cells were consuming valine derived from the 0. Furthermore, we later cultured cells long-term in unconditioned valine-free RPMI on plates not coated with 0. In pMTIV cells, 13 C-valine content was lower than 12 C-valine content on days 14 and 24 while the opposite was true on days 2, 4, 12, and 18, demonstrating that the 13 C content of the cells was not on a downward trend but rather fluctuated up and down.

This may reflect an inability to adequately respond to valine demands given inefficient flux through the pathway. We thank the reviewer for these insights. We address this point above in our response to Essential Revision 4.

We agree with the reviewer on this point and have already begun efforts to test our pathways in other cell lines, such as HEK cells, but we believe these data are too preliminary to be included at this time, and are beyond the scope of this contribution. We undertook a painstaking and extensive effort to demonstrate robust and self-sustaining valine-free growth over 39 days.

This was achieved by increasing ilvD encoding a dihydroxy-acid dehydratase enzyme copy number in response to detecting a potential pathway bottleneck in 2,3-dihydroxy-isovalerate. By increasing the efficiency of the final step in the introduced biosynthetic pathway, doubling time was reduced to a relatively consistent 3.

o It is possible residual valine from complete medium may help pCTRL and pMTIV cells survive at early timepoints. While it was possible to include an 8 day timepoint for collecting transcriptomic pMTIV samples we originally did not do so as there is no suitable control for analyzing such samples.

Nonetheless, we had previously collected RNA in duplicate samples from a late time point and as such have included transcriptomic data in a new figure for samples cultured in conditioned valine-free FK medium over 29 days or 5 passages i. well past the point of pCtrl inviability Figure 3 —figure supplement 5.

Evidently, these cells more closely resemble healthy cells cultured in complete medium than do pCtrl cells cultured in valine-free FK for just 48 h. This excellent reviewer suggestion led to an important new finding — the specific accumulation of an intermediate suggesting a pathway bottleneck.

We explored the presence of pathway intermediates in our original 13 C-tracing experiment. The only pathway intermediate detected by metabolomics analysis was 2,3-dihydroxy-isovalerate, suggesting potential bottlenecking at this step.

This resulted in cells that double on average every 3. This data demonstrates long-term homeostasis and robust growth under reasonable culturing conditions.

We believed it would be misleading to describe our efforts to rescue valine biosynthesis alone. On the suggestion of Reviewer 1 above as well as the suggestion outlined in Essential Revision 4 sections have been adjusted but not removed.

The potential toxicity of valine pathway intermediates or perhaps toxic products from non-specific enzymatic activity is certainly an interesting question, given the introduction of a pathway sourced from a distantly removed species.

However, we saw no signs of metabolic stress in cells harboring the pathway when grown on complete media, either by growth rate Figure 2D or in the transcriptomic data Figure 3D, Figure 3 —figure supplement 2.

The introduction of the pathway therefore does not appear to be a significant stressor to the cells. However, it remains to be seen if this will true for all essential amino acids, particularly as we look to introduce the more complex pathways.

We thank the reviewer for the vote of confidence, and share their excitement for the insights this work might enable. Eagle, H. and Piez, K. The population-dependent requirement by cultured mammalian cells for metabolites which they can synthesize.

Journal of Experimental Medicine , 29—43 Barak Z, Chipman D M, and Gollop N. Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria.

Journal of Bacteriology , — Mitchell Leslie A. et al. Science , eaaf Richardson Sarah M. Science , — The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We would like to thank the members of the Boeke and Wang labs for comments and discussion on the work and manuscript.

RMM additionally thanks personal support from Xiaoyu Weng. Defense Advanced Research Projects Agency HR HHW, JDB. National Science Foundation MCB HHW. Burroughs Wellcome Fund PATH HHW. Irma T Hirschl Trust HHW. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus. Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation.

The secretory organelles micronemes and rhoptries mediate the first steps of invasion. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules.

The site s of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii , small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown.

Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis.

Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury.

Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine O-GlcNAc signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration.

Here, we define the specific metabolic pathway by which O-GlcNAc transferase ogt-1 loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C.

elegans , we find that glycolysis, serine synthesis pathway SSP , one-carbon metabolism OCM , and the downstream transsulfuration metabolic pathway TSP are all essential in this process. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway.

These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism.

Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport.

Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question.

We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response.

Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Phodependent phosphate sensing pathway. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila.

These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Share this article Doi. Cite this article Julie Trolle Ross M McBee Andrew Kaufman Sudarshan Pinglay Henri Berger Sergei German Liyuan Liu Michael J Shen Xinyi Guo J Andrew Martin Michael E Pacold Drew R Jones Jef D Boeke Harris H Wang Resurrecting essential amino acid biosynthesis in mammalian cells.

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Download asset Open asset. Most bacteria, some archaea , fungi, algae, and plants use the DAP pathways. On the other hand, the AAA pathways synthesize Lys from alpha-ketoglutarate and acetyl coenzyme A. Most fungi, some algae, and some archaea use this route.

Why do we observe this diversity, and why does it occur particularly for Lys synthesis? Interestingly, the DAP pathways retain duplicated genes from the biosynthesis of arginine, whereas the AAA pathways retain duplicated genes from leucine biosynthesis Figure 2 , indicating that each of the pathways experienced at least one duplication event during evolution Hernandez-Montes et al.

Fani and coworkers performed a comparative analysis of the synthesis enzyme sequences and their phylogenetic distribution that suggested that the synthesis of leucine, lysine, and arginine were initially carried out with the same set of versatile enzymes.

Over the course of time came a series of gene duplication events and enzyme specializations that gave rise to the unambiguous pathways we know today.

Which of the pathways appeared earlier is still a source of query and debate. To support this hypothesis, there is evidence from a fascinating archaea, Pyrococcus horikoshii. This organism can synthesize leucine, lysine, and arginine, yet its genome contains only genes for one pathway.

Such a gap indicates that P. horikoshii has a mechanism similar to the ancestral one: versatile enzymes.

Biochemical experiments are needed to further support the idea that these enzymes can use multiple substrates and to rule out the possibility that amino acid synthesis in this organism does not arise from enzymes yet unidentified.

Selenocysteine SeC Bock is a genetically encoded amino acid not present in all organisms. Scientists have identified SeC in several archaeal, bacterial, and eukaryotic species even mammals.

When present, SeC is usually confined to active sites of proteins involved in reduction-oxidation redox reactions. It is highly reactive and has catalytic advantages over cysteine, but this high reactivity is undermined by its potential to cause cell damage if free in the cytoplasm.

Hence, it is too dangerous, and no pool of free SeC is available. How, then, is this amino acid synthesized for use in protein synthesis? The answer demonstrates the versatility of synthesis strategies deployed by organisms forced to cope with singularities.

The synthesis of SeC is carried out directly on the tRNA substrate before being used in protein synthesis. First, SeC-specific tRNA tRNA sec is charged with serine via seril-tRNA synthetase, which acts in a somehow promiscuous fashion, serilating either tRNA ser or tRNA sec.

Then, another enzyme modifies Ser to SeC by substituting the OH radical with SeH, using selenophosphate as the selenium donor Figure 2, pink pathway. This synthesis is a form of a trick to avoid the existence of a free pool of SeC while still maintaining a source of SeC-tRNA sec needed for protein synthesis.

Strictly speaking, this mechanism is not an actual synthesis of amino acids, but rather a synthesis of aminoacetylated-tRNAs. However, this technique involving tRNA directly is not exclusive to SeC, and similar mechanisms dependent on tRNA have been described for asparagine, glutamine, and cysteine.

Owing to its appearance of SeC across all three domains of life, scientists wonder if it is an ancestral mechanism for amino acid biosynthesis or simply a coincidence of selection pressures. In , Horowitz proposed the first accepted model for metabolic pathway evolution Horowitz Called the retrograde model, it states that after an enzyme consumes all its substrate available, another enzyme capable of producing the aforementioned substrate is required, so the last enzyme evolved to the preceding one by a gene duplication and selection mechanism.

In other words, enzymes evolve from others with similar substrate specificity, and the substrate of the last enzyme is the product of the preceding one. Also, the active site must bind both the substrate and the product. This model became very popular, but as more genes have been sequenced and more phylogenetic analyses performed, this mechanism has become less seemingly plausible and therefore unpopular.

An alternative model, the patchwork assembly model, proposes that ancestral enzymes were generalists, so they could bind a number of substrates to carry out the same type of reaction. Gene duplication events followed by evolutionary divergence would result in enzymes with high affinity and specificity for a substrate.

In other words, enzymes are recruited from others with the same type of chemical reaction. Whole genome analysis of Escherichia coli supports the patchwork evolution model Teichmann et al.

Duplication of whole pathways does not occur very often; nevertheless, examples include tryptophan to synthesize paraminobenzoate and histidine to synthesize nucleotides biosynthesis, as well as lysine, arginine, and leucine biosynthesis see aforementioned example.

Amino acids are one of the first organic molecules to appear on Earth. As the building blocks of proteins, amino acids are linked to almost every life process, but they also have key roles as precursor compounds in many physiological processes.

These processes include intermediary metabolism connections between carbohydrates and lipids , signal transduction , and neurotransmission.

Recent years have seen great advances in understanding amino acid evolution, yet many questions on the subject of amino acid synthesis remain. What was the order of appearance of amino acids over evolutionary history? How many amino acids are used in protein synthesis today?

How many were present when life began? Were there initially more than twenty used for building blocks, but intense selective process streamlined them down to twenty? Conversely, was the initial set much less than twenty, and did new amino acids successively emerge over time to fit into the protein synthesis repertoire?

What are the tempo and mode of amino acid pathway evolution? These questions are waiting to be tackled — with old or new hypotheses, conceptual tools, and methodological tools — and are ripe for a new generation of scientists.

Scientists now recognize twenty-two amino acids as the building blocks of proteins: the twenty common ones and two more, selenocysteine and pyrrolysine. Amino acids have several functions. Their primary function is to act as the monomer unit in protein synthesis. They can also be used as substrates for biosynthetic reactions; the nucleotide bases and a number of hormones and neurotransmitters are derived from amino acids.

Amino acids can be synthesized from glycolytic or Krebs cycle intermediates. The essential amino acids, those that are needed in the diet, require more steps to be synthesized.

Some amino acids need to be synthesized when charged onto their corresponding tRNAs. We have discussed only two biosynthetic routes: the Trp pathway, which appears to have evolved only once, and the Lys pathway, which seems to have evolved independently in different lineages.

Prevailing evidence suggests that metabolic pathways themselves seem to be evolving following the patchwork assembly model, which proposes that pathways originated through the recruitment of generalist enzymes that could react with a wide range of substrates.

The study of the evolution of amino acid metabolism has helped us understand the evolution of metabolism in general. Baumann, P. Biology bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annual Review of Microbiology 59 , — doi Bock, A.

Biosynthesis of selenoproteins — an overview. Biofactors 11 , 77—78 Fani, R. et al. The role of gene fusions in the evolution of metabolic pathways: The histidine biosynthesis case. BMC Evolutionary Biology 7 Suppl 2 , S4 doi Gordon, A. Partition chromatography in the study of protein constituents.

Biochemical Journal 37 , 79—86 Hernandez-Montes, G. The hidden universal distribution of amino acid biosynthetic networks: A genomic perspective on their origins and evolution.

Genome Biology 9 , R95 doi Horowitz, N. On the evolution of biochemical syntheses. Proceedings of the National Academy of Sciences 31 , Merino, E. Evolution of bacterial trp operons and their regulation. Current Opinion in Microbiology 11 , 78—86 doi Miller, S.

A production of amino acids under possible primitive earth conditions. Science , — Pal, C. Chance and necessity in the evolution of minimal metabolic networks. Nature , — doi Reeds, P. Dispensable and indispensable amino acids for humans.

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The evolution and structural anatomy of the small molecule metabolic pathways in Escherichia coli. Journal of Molecular Biology , — doi Velasco, A. Molecular evolution of the lysine biosynthetic pathways. Journal of Molecular Evolution 55 , — doi Xie, G.

Ancient origin of the tryptophan operon and the dynamics of evolutionary change. Microbiology and Molecular Biology Reviews 67 , — doi What Is a Cell? Eukaryotic Cells. Cell Energy and Cell Functions. Photosynthetic Cells. Cell Metabolism.

The Two Empires and Three Domains of Life in the Postgenomic Age. Why Are Cells Powered by Proton Gradients? The Origin of Mitochondria. Mitochondrial Fusion and Division. Beyond Prokaryotes and Eukaryotes : Planctomycetes and Cell Organization.

The Origin of Plastids. The Apicoplast: An Organelle with a Green Past. The Origins of Viruses. Discovery of the Giant Mimivirus. Volvox, Chlamydomonas, and the Evolution of Multicellularity.

Yeast Fermentation and the Making of Beer and Wine. Dynamic Adaptation of Nutrient Utilization in Humans. Nutrient Utilization in Humans: Metabolism Pathways. An Evolutionary Perspective on Amino Acids. Fatty Acid Molecules: A Role in Cell Signaling. Mitochondria and the Immune Response.

Stem Cells in Plants and Animals. G-Protein-Coupled Receptors, Pancreatic Islets, and Diabetes. Promising Biofuel Resources: Lignocellulose and Algae. The Discovery of Lysosomes and Autophagy. The Mystery of Vitamin C. The Sliding Filament Theory of Muscle Contraction.

An Evolutionary Perspective on Amino Acids By: Ana Gutiérrez-Preciado, B. Departamento de Microbiologia Molecular, Universidad Nacional Autonoma de Mexico , Hector Romero, B.

Departamento de Ciencias Naturales, Universidad Autonoma Metropolitana © Nature Education. Citation: Gutiérrez-Preciado, A. Nature Education 3 9

Amino Acids for Animal Health PubMed Google Pqthway Kim SW, Hurley WL, Wu G, Ji Plyometric training adaptations Ideal amino acid balance for sows during pathday and lactation. J Anim Synthessi Biotechnol. Article Sodium intake and bone health Google Scholar Rogers QR, Wigle AR, Laufer A, Sybthesis VH, Morris JG. The pathay domains Ln also been synhtesis subject of structural synthssis the solution structure of the regulatory domain of TyrOH Amino acid synthesis pathway in animals a core ACT domain similar to that found in PheOH. While glutamate was used to prepare isonitrogenous diets in the previous studies, none of these investigators considered that animals have a dietary requirement of glutamate for optimal growth and production performance. Pacold ME Brimacombe KR Chan SH Rohde JM Lewis CA Swier LJYM Possemato R Chen WW Sullivan LB Fiske BP Cho S Freinkman E Birsoy K Abu-Remaileh M Shaul YD Liu CM Zhou M Koh MJ Chung H Davidson SM Luengo A Wang AQ Xu X Yasgar A Liu L Rai G Westover KD Vander Heiden MG Shen M Gray NS Boxer MB Sabatini DM A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate Nature Chemical Biology 12 — This type of regulatory scheme allows control over the total flux of the aspartate pathway in addition to the total flux of individual amino acids.
What Are Essential Amino Acids?

Not much is known about the regulation of alanine synthesis. The only definite method is the bacterium's ability to repress Transaminase C activity by either valine or leucine see ilvEDA operon.

Other than that, alanine biosynthesis does not seem to be regulated. Valine is produced by a four-enzyme pathway. It begins with the condensation of two equivalents of pyruvate catalyzed by acetohydroxy acid synthase yielding α-acetolactate.

This is catalyzed by acetohydroxy isomeroreductase. The third step is the dehydration of α, β-dihydroxyisovalerate catalyzed by dihydroxy acid dehydrase. In the fourth and final step, the resulting α-ketoisovalerate undergoes transamination catalyzed either by an alanine-valine transaminase or a glutamate-valine transaminase.

Valine biosynthesis is subject to feedback inhibition in the production of acetohydroxy acid synthase. The leucine synthesis pathway diverges from the valine pathway beginning with α-ketoisovalerate. α-Isopropylmalate synthase catalyzes this condensation with acetyl CoA to produce α-isopropylmalate.

An isomerase converts α-isopropylmalate to β-isopropylmalate. The final step is the transamination of the α-ketoisocaproate by the action of a glutamate-leucine transaminase. Leucine, like valine, regulates the first step of its pathway by inhibiting the action of the α-Isopropylmalate synthase.

The genes that encode both the dihydroxy acid dehydrase used in the creation of α-ketoisovalerate and Transaminase E, as well as other enzymes are encoded on the ilvEDA operon. This operon is bound and inactivated by valine , leucine , and isoleucine.

Isoleucine is not a direct derivative of pyruvate, but is produced by the use of many of the same enzymes used to produce valine and, indirectly, leucine. When one of these amino acids is limited, the gene furthest from the amino-acid binding site of this operon can be transcribed.

When a second of these amino acids is limited, the next-closest gene to the binding site can be transcribed, and so forth.

The commercial production of amino acids usually relies on mutant bacteria that overproduce individual amino acids using glucose as a carbon source. Some amino acids are produced by enzymatic conversions of synthetic intermediates.

Aspartic acid is produced by the addition of ammonia to fumarate using a lyase. See Template:Leucine metabolism in humans — this diagram does not include the pathway for β-leucine synthesis via leucine 2,3-aminomutase. Contents move to sidebar hide. Article Talk.

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The set of biochemical processes by which amino acids are produced. For the non-biological synthesis of amino acids, see Strecker amino acid synthesis.

Demand Media. Retrieved 28 July Annual Review of Microbiology. doi : PMID The physiology and biochemistry of prokaryotes 3rd ed. New York: Oxford Univ. ISBN Journal of Molecular Biology.

PMC Principles of Biochemistry 3rd ed. New York: W. Lehninger, Principles of Biochemistry 3rd ed. New York: Worth Publishing. Microbial Biotechnology. ISSN The Journal of Biological Chemistry. Amino Acids. S2CID The Biosynthesis of Histidine and Its Regulation. Archived from the original on 9 December Retrieved 29 April International Journal of Biochemistry.

In Wendisch VF ed. Amino acid biosynthesis: pathways, regulation, and metabolic engineering. Berlin: Springer. Annual Review of Biochemistry. Ullmann's Encyclopedia of Industrial Chemistry.

Weinheim: Wiley-VCH. Gene expression. Bacterial Archaeal Eukaryotic. Transcription factor RNA polymerase Promoter. Ribosome Transfer RNA tRNA Ribosome-nascent chain complex RNC Post-translational modification.

Epigenetic imprinting Transcriptional Gene regulatory network cis-regulatory element lac operon Post-transcriptional sequestration P-bodies alternative splicing microRNA Translational Post-translational reversible irreversible. François Jacob Jacques Monod.

Metabolism , catabolism , anabolism. Metabolic pathway Metabolic network Primary nutritional groups. Purine metabolism Nucleotide salvage Pyrimidine metabolism Purine nucleotide cycle. Pentose phosphate pathway Fructolysis Polyol pathway Galactolysis Leloir pathway. Glycosylation N-linked O-linked.

Photosynthesis Anoxygenic photosynthesis Chemosynthesis Carbon fixation DeLey-Doudoroff pathway Entner-Doudoroff pathway. Xylose metabolism Radiotrophism. Fatty acid degradation Beta oxidation Fatty acid synthesis.

Steroid metabolism Sphingolipid metabolism Eicosanoid metabolism Ketosis Reverse cholesterol transport. Metal metabolism Iron metabolism Ethanol metabolism Phospagen system ATP-PCr.

Metabolism : Protein metabolism , synthesis and catabolism enzymes. Essential amino acids are in Capitals. Saccharopine dehydrogenase Glutaryl-CoA dehydrogenase. Alanine transaminase. D-cysteine desulfhydrase. The α-helix that links the two domains is shown in yellow and the N-terminal arm in green.

Bi Anthranilate synthase from Serratia marcrescens PDB code 1I7S in the α 2 β 2 heterotetramer conformation. The α subunit is in blue and the β subunit in red. Bii Anthranilate synthase from M. tuberculosis in the homodimer conformation.

C Anthranilate phosphoribosyltransferase from M. tuberculosis PDB code 2BPQ. The two domains of each monomer, small and large, are colored red and blue, respectively, and the active site cleft is indicated by the bound benzamidine molecule, shown as spheres with carbon atoms colored yellow.

Di Bifunctional E. coli phosphoribosyl anthranilate isomerase enzyme colored in blue and the indoleglycerolphosphate synthase domain colored red PDB code 1PII. The phosphate, shown as pink spheres, identifies the position of the phosphoribosyl anthranilate isomerase enzyme active site.

Dii Dimeric monofunctional phosphoribosyl anthranilate isomerase from T. thermophilus PDB code 1V5X. E The heterotetrameric tryptophan synthase from M. tuberculosis PDB code 5TCF. The α subunits are colored in red, while the β subunits are colored in blue and cyan to highlight the subunit interface.

Tryptophan is the most chemically complex and is the least abundant of the AAA. The biosynthesis of tryptophan occurs only in plants and microbes, and therefore contains multiple attractive targets for the development of herbicides and antimicrobials.

Chorismate is the branching point from where the biosynthesis of tryptophan diverges from that of phenylalanine and tyrosine.

The pathway from chorismate to tryptophan is shown in Figure 4. Figure 4. Synthesis of tryptophan from chorismate.

After the seven-step pathway via shikimate generates chorismate, the biosynthesis of tryptophan diverges from those of the other two AAA. Tryptophan synthase A or α-subunit cleaves indole glycerolphosphate into indole and glyceraldehydephosphate, while tryptophan synthase B or β-subunit is responsible for the PLP-dependent condensation of the latter two compounds into tryptophan.

The enzyme catalyzing the committed step of tryptophan biosynthesis is anthranilate synthase AS EC 4. AS, also designated as TrpG or TrpE, belongs to the lyase family, in particular to the oxo-acid-lyases capable of cleaving carbon-carbon bonds.

It is allosterically inhibited by the final product of the pathway, tryptophan, and is an important player in partitioning in chorismate toward tryptophan biosynthesis. The formation of anthranilate involves the transfer of ammonia from the donor glutamine to chorismate, generating glutamate and pyruvate apart from anthranilate.

This reaction is classified as a 1,4-nucleophilic substitution by ammonia followed by the elimination of pyruvate. Anthranilate synthase is considered to share evolutionary origins with other chorismate-metabolizing enzymes such as salicylate synthase EC 4.

It has been shown that S. Another study demonstrated that only a few mutations were sufficient to induce AS activity in an aminodeoxychorismate synthase, which in its native form does not eliminate pyruvate Culbertson et al. Anthranilate synthase contains two components, AS-I TrpE , and AS-II TrpG , both EC 4.

AS-I synthesizes the intermediate 2-aminodeoxyisochorismate ADIC from chorismate and ammonia Morollo et al. The enzyme mechanism and active chemistry are described in detail in another review Romero et al. Briefly, the AS-I subunits bind to aminate chorismate, when high concentrations of ammonia are present.

The AS-II subunits releases ammonia for the amination of chorismate via the formation of a γ-glutamyl- S- cysteinyl enzyme intermediate. Magnesium is suggested to make the 4-hydroxyl group of chorismate a better leaving group.

Anthranilate synthase organization and size differs between bacteria and plants. Some bacterial AS contain the two subunits, α and β Figure 3B , in various oligomeric compositions such as αβ, α 2 β 2 , or α 3 β 3 , or with a fusion of the two subunits Romero et al. Other bacterial AS such as the fused Streptomyces venezuelae are not only monomeric, but also cannot use ammonia instead of glutamine to aminate chorismate Ashenafi et al.

Among pathogenic bacteria from which crystallographic structures of AS are available, the oligomeric organization differs substantially between Serratia marcrescens and S. typhimurium , where the α 2 β 2 tetramer associates via the AS-I subunits Spraggon et al.

tuberculosis enzyme, AS-I is a homodimer, even when AS-II is present Bashiri et al. In the last enzyme, the allosteric binding site for the inhibitor tryptophan is found near the interfacial region. thaliana Niyogi and Fink, ; Niyogi et al. It is noteworthy that in some eukaryotes such as fungi, the AS complex may contain enzyme subunits with other functions in the tryptophan biosynthesis, i.

In plants, such as potato and tobacco, as well as in bacteria, such as Pseudomonas putida , the presence of tryptophan-sensitive and -insensitive AS isozymes have been suggested as indicative of the existence of complete but distinct pathways for both primary and secondary metabolism of tryptophan Hrazdina and Jensen, It was reported that the second set of AS gene products in P.

putida and P. aeruginosa , which are tryptophan-insensitive, participate in the biosynthesis of the blue-green phenazine pigment, pyocyanin Essar et al. Also known as anthranilate phosphoribosyl synthase APR synthase or TrpD EC 2. AnPRT is a member of the phosphoribosyl transferase PRT involved in nucleotide biosynthesis and salvage apart from AAA biosynthesis Sinha and Smith, A number of microbial AnPRT have been studied in detail, with the M.

tuberculosis enzyme showing some interesting structural features Castell et al. In this case, an unusual channel, which could deliver anthranilate to the active site, has been observed. Multiple anthranilate-binding sites have been reported within this channel and may account for the substrate inhibition caused by anthranilate.

The biochemical role of this substrate channeling may be to deliver the PRPP to anthranilate leading to phosphate attachment , instead of water which would lead to hydrolysis. AnPRT inhibitors based on these multiple binding sites have been explored Evans et al.

AnPRT enzymes are homodimeric, with each subunit constructed from two domains that interact via a hinge region that contains the active site Figure 3C.

Also known as PRA isomerase or TrpF EC 5. The enzyme architecture varies widely, with some bacteria such as E. coli containing TrpF fused to the C-terminus of the next enzyme in the pathway Wilmanns et al.

Due to the existence of an analogous Amadori rearrangement in the biosynthesis of histidine catalyzed by the ProFAR isomerase EC 5. Indeed, a dual-functional enzyme called PriA has been discovered in M. tuberculosis and S. coelicolor Due et al. The bifunctional enzyme is monomeric with the C-terminal end facilitating the phosphoribosyl anthranilate isomerase reaction Figure 3Di.

In contrast, the mono-functional enzyme is monomeric in mesophiles, whereas in thermophiles where increased structural stability is required, the phosphoribosyl anthranilate isomerase is observed primarily as a dimer Figure 3Dii.

Regardless whether this reaction is catalyzed by a mono- or bi-functional enzyme, the domain responsible for the isomerization adopts the same basic βα 8 fold. The active site is located at the C-terminal end of the barrel and in dimeric structures, the loops located at the N-terminal side of the barrel interlock the monomers together.

An increased helical content and increased numbers of charged residues observed in the thermophiles has also been proposed to contribute to enzyme stability. TrpC or indoleglycerol-phosphate synthase EC 4. In some bacteria such as E.

coli , the IGP synthase is fused with the previous enzyme in the pathway, leading to a bi-functional PRA synthase: IGP synthase Wilmanns et al. In this enzyme, a conserved glutamate and two conserved lysine residues have been identified as essential for catalysis.

A notable feature of the IGP synthase reaction is that it is a series of biochemical steps—condensation, decarboxylation and dehydration in that sequence, whose kinetic mechanisms have recently been elucidated Schlee et al.

The final step of tryptophan biosynthesis Figure 4 is catalyzed by tryptophan synthase or TrpAB EC 4. The enzyme consists of a α 2 β 2 tetramer Figure 3E , as demonstrated by the recent report of the M. tuberculosis crystal structure Wellington et al.

The α subunit generates indole from IGP by means of a retro-aldol type of reaction wherein glycerolphosphate is eliminated, and channels the indole into the second active site, which is present in the β subunit. The second active site chemistry involves a typical PLP Schiff base mechanism.

The activation of the substrate serine replaces an active site lysine attached to PLP. Indole acts as a nucleophile to displace a water molecule and the elimination-addition ends by indole condensing with a 3-carbon unit to form tryptophan.

The shielding of reaction intermediates from the bulk solution by tunneling between the active sites and the complex allosteric coupling of the bound subunits have been the subject of many structural and mutagenesis studies. NMR studies, in particular, have shed light on the intricate dance of the tryptophan synthase components Axe and Boehr, and an extensive account of the workings of tryptophan synthase nanomachine is available elsewhere Dunn et al.

Since animals lack the AAA biosynthetic pathways, pathogens that biosynthesize AAA are attractive targets for developing new anti-infective substances, especially since the rise of antibiotic resistance threatens the effectiveness of traditional antimicrobials.

An increase in the number of available structures of enzymes in protein structure databases solved by X-ray crystallography particularly enzymes from pathogenic microbes , combined with recent improvements in computational methods applied to elucidate and understand enzyme function as well as the increasing pace of genome sequencing and annotation of microbes in the last decade, have opened up enormous opportunities to develop new antimicrobial targets as well as to discover new antimicrobial molecules.

The shikimate pathway furnishes not only the AAA, but also molecules such as vitamins and cofactors, and therefore is attractive for the discovery and or development of new chemotherapeutic agents Lamichhane et al.

Enzymes involved in the shikimate pathway have been detected in the protists, Toxoplasma gondii which causes toxoplasmosis and one of the malarial parasites, Plasmodium falciparum Roberts et al.

However, only the last enzyme of the pathway, chorismate synthase has been identified conclusively on the basis of genome annotation, and the remaining enzymes seem to be missing. Six of the seven enzymes of the pathway were identified in M. tuberculosis Cole et al. For Helicobacter pylori , the causative agent of gastric ulcers and a type I carcinogen, four of the seven shikimate pathway genes, aroQ, aroE, aroK , and aroC , are essential.

pylori infections, and therapeutics based on inhibiting these two enzymes were reviewed extensively elsewhere González-Bello, SK phosphorylates shikimate at the 3-hydroxy group, at the expense of ATP. coli contains two types of enzymes SK1 AroK and SK II AroL , but most bacteria have only one SK variant.

SK inhibitors have been developed both by a substrate-mimetic strategy as well as by screening compound libraries. The P-loop is conserved in many ATP- and GTP-dependent proteins. Therefore, substrate mimics of shikimic acid were designed to bind the substrate-binding SB site of SK specifically Blanco et al.

Many of the compounds developed in that study for M. tuberculosis SK Mt-SK were reversible competitive inhibitors and some, such as the 3-aminoshikimates closely resembled the structure of shikimate. The inhibition kinetics and molecular modeling of these compounds showed that fixing the C4 and C5 hydroxyl groups in the diaxial conformation in the substrate mimic might be a good way to inhibit SK, due to the dramatic reduction in the flexibility of two domains— 1 the SB domain residues 9—17 in Mt-SK and 2 the LID domain residues — in Mt-SK.

Arg was identified as a key residue during ATP-binding, product release and also a Lewis acid during catalysis. Other SK-targeting compounds have been discovered, for example, an LC-MS based screening of around compounds at the National Institutes of Health NIH Tuberculosis Antimicrobial Acquisition and Coordination Facility identified three inhibitors with sub-micromolar IC 50 values for Mt-SK Simithy et al.

pylori SK Han et al. High-throughput virtual screening efforts have also been reported Segura-Cabrera and Rodríguez-Pérez, ; Coracini and de Azevedo, DHQ catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate, a reversible reaction.

Two forms of DHQ with no similarity at the level of primary sequences, and differing biochemical and biophysical properties are found Kleanthous et al. DHQ1 AroD found in plants, fungi and some bacteria such as E.

coli and Salmonella typhi , catalyzes a syn dehydration based on a Schiff base mechanism. Apart from biosynthesis, DHQ1 may also be required for virulence in some bacteria Racz et al.

It is essential in prominent pathogenic species such as M. tuberculosis and H. pylori Database for essential genes. In Mt-DHQ2 M. tuberculosis DHQ2 , a conserved aspartate Asp 88 residue from an adjacent enzyme subunit triggers the conversion of an essential tyrosine Tyr24 into tyrosinate, while the positive charge of Arg19 stabilizes the enolate intermediate generated from dehydroquinate.

The reaction was suggested to go through an enolate instead of an enol intermediate, due to the significantly lower energy of the enol intermediate Blomberg et al. Since the formation of the enolate involves abstraction of the C2 axial hydrogen by Tyr24, replacement of this hydrogen by another group is expected to inhibit the enzyme.

However, substitution of the C2 equatorial hydrogen by benzyl groups also generates effective inhibitors as the methylene group permits close stacking of the benzene ring close to the aromatic ring of the Tyr 24 residue González-Bello et al.

The reaction mechanism also entails a ring-flattening between the C2 and C3 positions during the elimination. Therefore, compounds mimicking the enolate transition state could be good competitive inhibitors and the first inhibitor synthesized and tested using this approach was 2,3-dehydroquinic acid Frederickson et al.

It was shown to be a reversible competitive inhibitor of Mt-DHQ2 and Sc-DHQ2 S. coelicolor- DHQ2. The solution of the X-ray diffraction structure of Sc-DHQ2 with an inhibitor containing a C2-C3 double bond bound at the active site PDB entry 1GU1, 1. An important feature of DHQ2 substrate recognition is the carboxylate binding pocket which has been a challenge for in vitro optimization of anti-tuberculosis drugs, but an ester prodrug approach was demonstrated to improve antibacterial activity by increasing the cell permeability of mycobacteria Tizón et al.

Several drug-like aromatic Mt-DHQ2 inhibitors with anti-tubercular activity in the micromolar range have been described, including some nitrobenzyl-gallate analogs González-Bello et al. TB is one of the great public health challenges and the need for new drugs, especially those with novel targets and modes of action Ma et al.

This has generated interest in the metabolism of the TB pathogen. Although the complete genome sequence of Mycobacterium tuberculosis has been published Cole et al. The genes already identified in this context are also not arranged in a single open reading frame ORF and were therefore identified only after genetic complementation and biochemical studies.

The work of the TB Structural Genomics Consortium TBSGC enabled the determination of crystal structures of many proteins from M. tuberculosis including those involved in tryptophan metabolism such as, TrpB, TrpC, and TrpE Lee et al.

TrpD was shown to be necessary for colonization of the lungs Lee et al. Remarkably, a tryptophan auxotrophic strain was not virulent even in mice with severe combined immunodeficiency Smith et al.

A decrease in tryptophan concentration and an increase in the activity of indoleamine-2,3-dioxygenase IDO EC 1. Therefore, it could be inferred that tryptophan degradation was induced specifically in the lungs by the host as a tryptophan starvation response to counter the pathogen.

While plant tryptophan synthase inhibitors with sub-micromolar potency Sachpatzidis et al. A sub-micromolar inhibitor for TrpC was reported Shen et al. Curiously, inhibitors designed with one enzyme in the biosynthetic pathway as targets, often have activities against other enzymes in the pathway Kozlowski et al.

One of the two putative anthranilate synthase genes in TB was found to be a salicylate synthase involved in mycobactin synthesis Harrison et al. The same authors showed further that TrpE knockout mutants of the bacterium were killed both in vitro and inside macrophages.

Novel IGP synthase inhibitors that are effective against MDR TB have been reported Shen et al. Anthranilates fluorinated at the 5- or 6- position of the benzene ring were demonstrated to be antibacterial, with modest in vivo activity in mouse models Zhang et al. Since the fluoro-anthranilates were not shown to be TrpD inhibitors for the E.

coli enzyme, but alternate substrates leading to formation of fluorinated IGP in vitro Cookson et al. Either, the biosynthesis is inhibited downstream of TrpD, or the fluorinated tryptophan formed at the end of this pathway is toxic to the bacterium.

Incorporation of fluorine at the 4-, 5-, or 6- positions of tryptophan was shown to be toxic to E. coli , possibly due to detrimental effects on protein structure Brown et al. Complete phenylalanine degradation has not been reported in plants Mazelis, and phenylalanine hydroxylase homologs are absent in plants as far as the published data indicate, with no candidates found in the genome of the model plant Arabidopsis thaliana.

However, a unique phenylalanine hydroxylase dependent on folate was discovered in non-flowering plants, which is localized in the chloroplast Pribat et al. Complete degradation of tyrosine in A.

thaliana was shown to proceed by the same pathway as in mammals Dixon and Edwards, The transamination of tyrosine by TAT occurs yielding 4-hydroxyphenylpyruvate, which is then transformed by 4-hydroxyphenylpyruvate dioxygenase to homogentisate. Homogentisate in plants acts as the precursor for tocopherols such as vitamin E and plastoquinones.

Further catabolic steps convert homogentisate via ring cleavage ultimately into fumarylacetoacetate. Hydrolysis of fumarylacetoacetate yields fumarate and acetoacetate, thereby linking tyrosine and fumarate metabolism, potentially both inside and outside of mitochondria.

Although tryptophan has been shown to be a precursor for the synthesis of many secondary metabolites such as auxins, phytoalexins, glucosinolates, and alkaloids Radwanski and Last, , to date, the elucidation of the tryptophan degradation pathway s has not been reported in plants.

In plants, phenylalanine and tyrosine are catabolized to generate anabolic precursors for the phenylpropanoid pathway. Phenylalanine can be converted to cinnamate by the enzyme phenylalanine ammonia lyase PAL EC 4.

The expression of PAL-encoding genes are highly regulated by different biotic and abiotic stresses, and conditions which increases the requirement of the cell wall component lignin Anterola and Lewis, Cinnamate can be further metabolized to p -coumaroyl CoA, a central metabolite in the phenylpropanoid pathway, which are involved in mediating responses pertaining to biotic and abiotic stresses Dixon, ; Casati and Walbot, The phenylpropanoid pathway has been reviewed extensively elsewhere Boudet, ; Vogt, These compounds impart mechanical strength to plant cells, and also participate in pest deterrence, drought resistance, UV protection, disease resistance, pollen viability and so on Nair et al.

Compounds such as lignans, lignins, cutin, suberin, catechins, sporopolleins, flavonoids, isoflavonoids, proanthocyanidins, aurones, phenylpropenes, stilbenes, alkaloids, and acylated polyamines are derived from this pathway and involved in plant defense. In addition, some of these compounds are involved in the synthesis of colorful pigments that are present in flowers and fruits Fraser and Chapple, Newer genome-based approaches, such as the creation of an extensive database for P superfamily genes CYPedia based on the microarray analysis of A.

thaliana and the analysis of over 4, re-annotated genes predicted to be active in plant metabolism for co-expression with P genes, have been described recently Ehlting et al.

Phenylalanine-derived volatile compounds are involved in plant reproduction and defense Dudareva et al. Phenylpropanoids, benzenoids, phenylpropenes, and nitrogenous aromatics are the major classes of volatiles in this context.

Phenylalanine is converted to phenylacetaldehyde by oxidative decarboxylation Kaminaga et al. Apart from phenylacetaldehyde, other phenylalanine-based volatiles include phenylethylacetate, 2-phenylethanol, methylbenzoate, and isoeugenol Watanabe et al.

Phenylalanine is also the precursor for a class of sulfur-containing secondary metabolites called phenylalanine glucosinolates Reichelt et al. Tyrosine, instead of phenylalanine, is the direct precursor of coumarate in the phenylpropanoid pathway in some plants Neish, ; MacDonald and D'Cunha, , where the enzyme responsible for the transformation is tyrosine-ammonia-lyase TAL EC 4.

Tyrosine decarboxylase TyrDC EC 4. TyrDC is distributed across the plant kingdom and is involved in the biosynthesis of defense compounds such as glycosides Ellis, and alkaloids Leete and Marion, The induction of TyrDC was shown to be induced upon wounding or fungal elicitor treatment Kawalleck et al.

In addition, recent studies using A. thaliana have demonstrated that TyrDC is involved in abiotic stress response especially during drought and exposure to high salt concentrations Lehmann and Pollmann, TyrDC in A. thaliana also feeds into the production of alkaloids as well as cell-wall hydroxycinnamic acid amides Facchini et al.

Tyrosine also serves as the starting compound for the biosynthesis of tocochromanols DellaPenna and Pogson, ; Mène-Saffranè and Dellapenna, as well as plastoquinones Norris et al.

The committed step of tocochromanol biosynthesis involves TAT, which converts tyrosine into p -hydroxyphenylppyruvate Norris et al.

Tyrosine is the precursor for meta -tyrosine, a non-proteogenic amino acid found in fescue grasses. It has been hypothesized that meta -tyrosine can be incorporated into proteins instead of phenylalanine by eukaryotic phenylalanine-tRNA synthases Duchêne et al.

The incorporation of meta -tyrosine can lead to wide range of plant growth defects including growth retardation and inhibition of root development Bertin et al. Tryptophan is the precursor to the family of auxins hormones Gibson et al. While indoleacetic acid IAA is the most abundant auxin, other indole-containing auxins such as; 4-chloro-indoleacetic acid 4-Cl-IAA , indole butyric acid IBA and indole propionic acid IPA are also important and have integral roles in plants.

Asymmetric auxin distribution in response to environmental cues govern the form, shape, strength and direction of growth of all organs and the interactions between various organs Benkov et al.

At least four pathways have been proposed for the production of IAA from tryptophan. It should be noted that the complete pathways for the degradation of IAA are still not elucidated Strader and Bartel, The two-step auxin biosynthesis pathway via indolepyruvate IPy , which is highly conserved throughout the plant kingdom and has been characterized in several monocot and dicot plants, is well known.

The first step of this pathway is the elimination of the amino group from the AA by the tryptophan aminotransferase TAA EC 2.

The latter compound then undergoes oxidative decarboxylation catalyzed by the YUC family of flavin monooxygenases to produce IAA. The enzymes of the transaminase-dependent pathway for IAA biosynthesis were characterized in vitro in the recombinant enzyme Stepanova et al. Recombinant TAA1 catalyzes the PLP-dependent transfer of an amino group from tryptophan to 2-oxoglutarate, yielding IPy and glutamate.

Disruption of TAA genes not only abolishes IPA production, but also affects the metabolism of other α-ketoacids and amino acids.

Recombinant YUC6 from A. thaliana was purified and shown to be a FAD-containing enzyme, wherein NADH reduces the bound FAD to FADH 2 , which then reacts with molecular oxygen to form the C4α- hydro peroxyflavin intermediate that is the actual oxidizing species Dai et al.

Other routes that generate IAA from tryptophan include 1 the indoleacetaldoxime IAOx pathway, which contains the two cytochrome P enzymes CYP79B2 and CYP79B3 EC 1.

In addition, a putative tryptophan-independent pathway of IAA biosynthesis directly from indole has been proposed Normanly et al. Enzymatic decarboxylation of tryptophan by the PLP-dependent TDC produces the indole alkaloid tryptamine, which is found in small amounts in many plants. It is deemed to be a feedstock compound for pathways involved in synthesis of terpenoid indole alkaloids TIA and those that influence growth and the microbiome.

Expression of TDC and TYDC in transgenic tobacco depleted the pools of tryptophan and tyrosine respectively, but in addition also perturbed pathways not directly involving AAA, such as methionine, valine, and leucine biosynthesis Guillet et al.

Tryptophan is also converted to compounds associated with plant-insect and plant-pathogen interactions known as the indole glucosinolates Halkier, , which are natural products containing thioglucose and sulfonate bound to the oxime derived from of the amino acid bound to an oxime function Halkier and Gershenzon, IAOx also feeds into the indole glucosinolate pathway via an oxime-metabolizing enzyme CYP83B1 Naur et al.

Another major category of tryptophan-derived secondary metabolites are the phytoalexins Pedras et al. The major indolic phytoalexin is camalexin, which accumulates upon infection with pathogens or the action of abiotic elicitors Zhao and Last, ; Böttcher et al.

In plants, chorismate is not only a precursor of the three AAA, but also the initial compound for the biosynthesis of folates, such as tetrahydrofolate or vitamin B9 Basset et al.

et al. Therefore, the shikimate pathway could potentially be engineered to augment the synthesis of folates or vitamin K in crop plants. Another enzyme in the shikimate pathway, 5-enolpyruvylshikimatephosphate synthase EPSPS , is the target of the well-known herbicide, N -phosphonomethylglycine or glyphosate commonly referred to as Roundup ® , which is a mimic of PEP and competitively inhibits EPSPS, thereby reducing the carbon flux through the pathway Healy-Fried et al.

Non-plant EPSPS are used to provide herbicide resistance in transgenic crops Duke and Powles, Being the basis for Roundup-Ready transgenic crops, EPSPS has received much research attention Singer and McDaniel, ; Smart et al.

The biosynthesis of AAA and secondary metabolites derived from them are often elevated in infection responses Ferrari et al. Manipulation of these responses could help improve plant protection against bacterial disease.

Invading bacteria trigger the transcription of pathways including AAA metabolism and pigment biosynthesis within 12 h of infection Truman et al. Salicylic acid SA is a plant defense compound that accumulates in leaves in response to local and systemic acquired resistance against phytopathogens Malamy et al.

SA applied externally on plant surfaces alone is able to trigger enhanced resistance to pathogens in A.

Although, SA was initially shown to be synthesized from phenylalanine via the PAL pathway, inhibition of this pathway did not prevent the synthesis of SA Mauch-Mani and Slusarenko, ; Coquoz et al.

It was shown in further studies that chorismate was converted into isochorismate by isochorismate synthase ICS EC 5.

Tryptamine production in transgenic tobacco was shown to severely inhibit the reproduction of whiteflies Thomas et al. Since this work was done with transgenic plants, the possibility exists for a generic TDC-based plant protection strategy against whiteflies.

The role of meta -tyrosine in inhibiting the growth of competing plants by fescue grasses Bertin et al. Tryptophan is the precursor of serotonin, which has multiple functions in plants. Tryptophan is decarboxylated to tryptamine by TDC, which then undergoes hydroxylation by a cytochrome P monooxygenase, forming serotonin Schröder et al.

In dry seeds, serotonin is a sink for ammonia which can be toxic. Serotonin is present in plant spines, such as those of stinging nettles and the pain caused as a result of contact with them Chen and Larivier, , may deter browsing animals from consuming the plants.

Since serotonin also affects the gut of animals, plants produce it in seeds and fruits as a way to promote the passage of seeds through the animal digestive tract in order to aid seed dispersal Feldman and Lee, Serotonin is further metabolized into the growth regulator melatonin, which is also synthesized in response to various biotic and abiotic stresses, such as pathogenic fungi, toxins, soil salinity, drought and extreme temperature Arnao and Hernández-Ruiz, Tryptophan is a precursor of thioquinolobactin, an antifungal agent that protects plants against the pathogen Pythium debaryanum Matthijs et al.

AAA obtained by animals from the diet can be broken down or converted into other necessary compounds, such as neurotransmitters see Figure 1. Phenylalanine is often converted into tyrosine in animals and both these AAA feed into the biosynthesis of neurotransmitters, such as L-3,4-dihydroxyphenylalanine L-DOPA , dopamine, epinephrine, and norepinephrine Figure 1.

It is not surprising, therefore, that metabolic defects in animals genes related to AAA catabolism have significant effects on their health. We will limit this review to a description of key AAA catabolic pathways in animals, along with a brief general discussion of pathologies related to each AAA catabolic pathway.

Complete AAA degradation pathways described for plants also occur in animals, whereby they break down phenylalanine and tyrosine from proteins for recycling.

Alkaptonuria is an inherited disorder affecting this function, caused by non-functional and or suboptimal activity of the enzyme homogentisate 1,2-dioxygenase dioxygenase HGD EC 1. Tyrosinemia is an inherited disorder in a single pathway involving mutations in one of three distinct enzymes involved in tyrosine degradation—fumaroylacetoacetate hydrolase EC 3.

This is a major pathway for the catabolism of phenylalanine and tyrosine catabolism in animals. Here, many enzymes have additional roles in the synthesis of multiple neuroactive substances.

The trace amines include all the neurotransmitters and neuroactive intermediates in this pathway except for L-DOPA, dopamine, epinephrine adrenaline and norepinephrin noradrenaline. It enables the biosynthesis of the neurotransmitters phenylethylamine and N -methylphenylethylamine directly from phenylalanine, in addition to dopamine, octapamine, tyramine, N -methyltyramine, syneprhine, 3-methoxytyramine, epinephrine and norepinephrine either directly from tyrosine or from phenylalanine, which is hydroxylated to tyrosine.

The physiological effects of these monoamine neurotransmitters are reviewed elsewhere Broadley, Phenylalanine is converted into tyrosine by phenylalanine 4-hydroxylase PheOH or PAH EC 1. Tyrosine hydroxylase converts tyrosine to L-DOPA, which is rate limiting for the synthesis of the catecholamines dopamine, epinephrine and norepinephrine.

Tryptophan hydroxylase converts tryptophan into 5-hydroxy-L-tryptophan en route to serotonin. All the AAAH enzymes contain iron and catalyze AAA hydroxylation using tetrahydrobiopterin.

They act as rate-limiting enzymes in their respective pathways Grenett et al. Detailed reviews of AAAH structural biology Flatmark and Stevens, , regulation Fitzpatrick, and AAAH-based therapeutic targets Waløen et al.

PheOH deficiency causes phenylketonuria PKU in humans, which is an inborn error of metabolism attributed to a single gene defect Erlandsen et al. PKU leads to a deficiency of tyrosine, which is continuously produced from phenylalanine in many animals for the synthesis of the catecholamine and trace amine neurotransmitters.

Untreated PKU can lead to seizures, intellectual disability, behavioral problems, and mental disorders Al Hafid and Christodoulou, Most of the more than mutations in this enzyme PAH DB 2 are linked to PKU, while a few different mutations have been identified among patients suffering from non-PKU hyperphenylalaninemia HPA.

In humans, knockout mutations in PheOH are not lethal, but the loss of TyrOH is, with the victims dying at a late embronic stage or briefly after birth Flatmark et al. Only two TyrOH mutations were so far associated with disorders of the basal ganglia Knappskog et al.

In later studies, the human TyrOH locus has also been linked to bipolar disorder Smyth et al. Even though their biochemical reaction mechanisms are the same and their substrate specificities are similar, they have different expression and regulation patterns, as well as different physiological roles McKinney et al.

TPH1 synthesizes most of the serotonin in circulation and is expressed chiefly in the gastrointestinal tract, adrenal glands, kidneys, and the pineal gland. TPH2 however, occurs in the serotonergic neurons with wide distribution in various cortices in the brain Amireault et al.

Each AAAH contains a non-heme iron center and a 6 R -L-erythro-5,6,7,8-tetrahydrobiopterin BH4 cofactor, and requires a dioxygen molecule during catalysis. The cofactor is oxidized to quinonoid dihydrobiopterin qBH2 , which is regenerated to BH4 by the NAD P H-dependent dihydropteridin reductase.

The structure of human PheOH hPheOH has been solved and shows an active site which is very open to the solvent and to the binding of exogenous ligands Kappock and Caradonna, ; Fusetti et al.

The negative potential and hydrophobic nature of the active site is considered to promote the binding of positively charged amphipathic molecules such as the actual substrates, pterin cofactors, and inhibitors such as catecholamines Hufton et al. The catalytic iron is situated at the entrance of the pocket containing the active site, with space enough for both the pterin cofactor and the substrate Hufton et al.

A highly conserved motif 27 amino acids long has been proposed to govern the binding of the cofactor tetrahydrobiopterin Jennings et al. The competitive inhibition of PheOH and TyrOH by catecholamines has been investigated using binary complexes of the dimeric proteins with various catecholamines.

The molecular basis for the inhibition has been proposed to be the binding of the inhibitors directly to the catalytic iron center via the bidentate coordination of the two hydroxyl groups Erlandsen et al. The regulatory domains have also been the subject of structural studies; the solution structure of the regulatory domain of TyrOH shows a core ACT domain similar to that found in PheOH.

When isolated, this domain of TyrOH forms a stable dimer, whereas the corresponding domain in PheOH exhibits an equilibrium between the monomer and dimer, with dimer stabilization afforded by the substrate phenylalanine.

This correlates well with the fact that TyrOH is regulated by the binding of catecholamines, while PheOH is regulated by the substrate binding to an allosteric site Fitzpatrick, Phenylalanine is converted to the neurotransmitter phenylethylamine by the PLP-dependent enzyme aromatic L-amino acid decarboxylase AADC or AAAD EC 4.

Phenylethylamine undergoes N -methylation catalyzed by phenylethanolamine N -methyltransferase PMNT EC 2. After the conversion of phenylalanine to tyrosine by AAAH, the latter is decarboxylated by AADC to form tyramine.

If instead of decarboxylation, tyrosine is rerouted via a second AAAH reaction, the product is L-DOPA. When L-DOPA is decarboxylated by AADC, dopamine is formed. A minor pathway leads from tyramine to dopamine, with the enzyme catalyzing the hydroxylation being brain CYP2D in humans Wang et al. Dopamine is methylated to 3-methoxytyramine by the action of catechol- O -methyltransferase COMT EC 2.

Dopamine is hydroxylated at the aminoethyl side chain in an R -specific manner by DBH to yield the major neurotransmitter norepinephrine. After tryptophan is hydroxylated by AAAH to 5-hydroxy-tryptophan 5-HTP , following which AADC catalyzes the decarboxylation of 5-HTP into serotonin.

In most animals, serotonin is found in the gastrointestinal tract gut , blood platelets as well as in the central nervous system. It has a variety of functions in the gastrointestinal and nervous systems, a detailed description of which can be found elsewhere Berger et al. Altered serotonin levels are involved in many diseases and disorders.

In the liver, serotonin is oxidized by monomine oxidase to the corresponding aldehyde, which is further oxidized by aldehyde dehydrogenase to 5-hydroxyindoelacetic acid 5-HIAA , which is eliminated via urine.

The amounts of serotonin and 5-HIAA are elevated in certain tumors and cancers. Serotonin is present in insect venoms, where it is the component responsible for causing pain to animals upon injection of these venoms Chen and Larivier, Pathogenic amoebae produce serotonin, which causes diarrhea in humans McGowan et al.

Serotonin is converted by serotonin N- acetyl transferase to N -acetyl serotonin; methylation of N -acetyl serotonin by S -adenosyl methionine SAM -dependent hydroxyindole O -methyl transferase yields melatonin. Melatonin is a neuro-hormone with many functions such as antioxidant, sleep-wake regulator and immune system regulator.

Tryptophan is also the source of the trace neurotransmitter tryptamine via an AADC catalyzed decarboxylation. All three decarboxylation reactions, namely, the conversion of phenylalanine into phenylethylamine, tyrosine into tyramine and tryptophan to 5-HTP have been considered to be catalyzed by the same enzyme, at least in animals.

Species-specific differences between AADC produced by various organisms exist and studies in Drosophila demonstrated that different tissues may contain distinct AADC isoforms.

It was shown that alternative splicing patterns from transcripts of the same gene caused the expression of tissue-specific variants Morgan et al. Deficiency of pyridoxine decreases AADC stability. Since, PLP is required for AADC catalysis, this is not surprising. However, the apoenzyme was found to degrade to a 20 times faster than the holoenzyme due to the involvement of a flexible loop covering the active site Matsuda et al.

The loop is fixed to the active site in the holoenzyme with a PLP Schiff base ligand interaction and stabilized; it fits into the entrance of the active site, held by hydrophobic interactions with the substrate catechol ring.

The flexible loop is expected to be stabilized in vivo by adopting a closed structure binding the substrate aldimine, whereas the apoenzyme does not bind the substrate, leading to its preferential proteolysis Matsuda et al.

The catalytic mechanism of AADC has been postulated to involve two intermediates, a Michaelis complex followed by an external aldimine. A flexible region around the residue Arg is exposed before ligand binding and forms a Michaelis complex. This in turn causes a conformational change, and during the following transaldimination, a more dramatic conformational change occurs, forming an external aldimine Ishii et al.

AADC deficiency is an inherited neuromuscular disorder in humans caused by a deficit of this enzyme. Patients show reduced catecholamine levels and elevated 3-O-methyldopa levels and have symptoms such as hypotonia, hypokinesia, and signs of autonomic dysfunction from an early age Pons et al.

All these mutations reduce the turnover number of the enzyme and most also alter the tertiary structure, with several experimental approaches pointing to incorrect conversion of the apoenzyme to the holoenzyme as the cause of the pathogenicity in a majority of the cases Montioli et al.

The most striking results are observed upon mutation of the residues His70, His72, Tyr79, Phe80, Pro81, Arg, and Arg, which map to a key loop structure participating in the switch of the apoenzyme to the holoenzyme Montioli et al. Melanins are pigments that in animals are responsible for the coloration of eyes, hair, skin, fur, feathers and scales.

While higher animals use melanins mainly for protection from radiation and in the immune response, insects utilize them many purposes such as hardening the cuticle, pigmentation of the exoskeleton, wound healing and innate immune responses. Melanins are derived from L-DOPA, which as mentioned before is derived from tyrosine.

Tyrosinase is a rate-limiting oxidase containing copper, which catalyzes two separate steps in melanin biosynthesis, the hydroxylation of a monophenol is the first reaction, followed by conversion of the o-diphenol to the corresponding o-quinone. The o-quinone, for example dopaquinone, is further metabolized to eumelanins and pheomelanins Solano, Dopaquinone combines with cysteine forming either 2- S -cysteinyl-DOPA or 5-cysteinyl-DOPA, both of which form pheomelanins via benzothiazine intermediates.

In the eumelanin pathway, dopaquinone is converted to leucodopachrome, which is the parent compound for dopachrome.

The next intermediate is either 5,6-dihydroxyindole DHI or 5,6-dihydroxyindolecarboxylic acid DHICA , both derived from dopachrome. Both are converted into quinone which eventually form the eumelanins.

A comprehensive review of the biosynthesis of the melanin pigments in insects and higher animals has been published elsewhere Sugumaran and Barek, There are multiple types of albinism caused by melanin deficiency, linked to different genes, among which type 3 oculocutaneous albinism results from a single-gene inborn metabolic defect, in this case mutations in the tyrosinase enzyme.

Albinism entails a partial or complete lack of pigmentation in the skin, hair, and eyes. Albinism in humans is commonly connected with a number of vision defects and the lack of skin pigmentation may lead to heightened susceptibility to sunburn and skin cancers.

Melanin granules are essential in immune cells and therefore, albinism leads to lowered immune defense Kaplan et al. Albinism is also associated in some cases with deaf-mutism Tietz, All known mammalian dopachrome converting enzymes transform dopachrome into DHICA, whereas insect and invertebrate enzymes convert dopachrome into DHI.

A D-dopachrome converting enzyme was first discovered in mammals Orlow et al. It was separated from the complex, characterized Aroca et al.

DCT is known to contain zinc at its active site Solano et al. Studies have shown that an increase in protein intake directly corresponds to an increase in protein deposition 6 within the bodies of growing animals, resulting in stronger, healthier mature animals. Some amino acids are slightly more important than others, however.

Amino Acids for Ruminates: For calves, the most important amino acids are methionine, lysine, isoleucine, threonine and leucine. A deficiency in any of these amino acids results in a slowing of growth and delayed onset of maturity.

The most important of these, methionine, is an essential amino acid. Though used inefficiently from a biological standpoint, methionine is important in cattle and sheep as a methyl group donor and a precursor for cysteine synthesis. Lysine is the second most limiting amino acid for growing calves, especially in maize-based diets because maize is relatively low in lysine.

Amino Acids for Pigs: Pigs have similar needs to calves, with the notable exception being arginine. While arginine is not an essential amino acid since it can be synthesized from glutamate and glutamine, it is essential to younger piglets in the neonatal and immediate postweaning phases.

Forty percent of pigs' arginine requirements 7 must be supplied through their diet, primarily due to their rapid growth rates and the fact that most arginine is used in the urea cycle of the liver. Amino Acids for Poultry: Growing poultry require similar amino acid balances as other growing animals, but they require arginine in their diets because they do not have a urea cycle and therefore cannot synthesize it on their own.

A deficiency of arginine often results in feather deformation in chickens 8. Lysine deficiencies can negatively affect feather growth in turkeys as well.

Nutrition has a significant effect on the quality of eggs in all animals. From the emergence of ovarian follicles through embryonic development, undernutrition can have a devastating effect on reproductive health for farm animals.

By feeding animals sufficient amounts of amino acids to support egg production and embryonic health, you can ensure that your animals are producing healthy offspring at an optimal rate. In ruminants, under-nutrition of amino acids can have a negative effect on fertility, especially during early ovulation.

Most prominently, the intake of methionine and lysine have a strong effect throughout the fertility cycle. These two amino acids are particularly important for embryonic development and consuming too little of either nutrient can negatively impact fertility.

In one study, feeding rumen-protected methionine during the peripartum period of a cow's cycle significantly improved postpartum performance. Additionally, studies have found that pregnancies are healthier when cows are fed sufficient amounts of methionine and lysine through the pregnancy, especially on days nine through 19, during which the cow's body determines whether to continue with a pregnancy.

Pigs require a balanced diet that contains plenty of essential and non-essential amino acids. While essential amino acids are important to support a pregnancy, sows also require dietary glutamine and arginine 9 to support mucosal integrity and neonatal growth, respectively. In summary, amino acids play varying importance roles based on the species of farm animal, its age and its production purpose.

Across all factors, however, protein supplements for cattle, pigs and poultry can deliver promising results and improve the performance and profitability of an animal.

Here are just a few ways that essential amino acids for animal health can benefit your bottom line:. When you raise the protein level in farm animal feed, farm animals will eat more food and digest it more efficiently, in turn increasing the amounts of amino acids and nutrients available to the animal.

This also improves feed efficiency, so there is less waste. Appropriate amino acid balances support improved growth rate so that animals will wean and reach mature weight early.

Additionally, well-fed calves, piglets and chicks tend to be healthier and larger as adults, producing more and experiencing disease at a lower rate.

The most prominent reason for culling cows is reproduction — if a cow doesn't calve, it doesn't produce milk. Conversely, the higher an animal's production potential, the higher the value of the pregnancy.

By increasing the amount and the quality of amino acids in feed, especially methionine and lysine, studies have shown an improvement in pregnancy rates 10 , which not only contribute to herd numbers but also improve milk production, increasing profitability.

Regardless of how much a cow is producing, it costs the same to keep it in the herd due to operating costs, fixed overhead costs, maintenance requirements and dry matter. To make the most of that cow, it is important that she produces enough milk to offset any costs of increasing feed quality.

By improving the ratio of amino acids in the diet, you can increase cow's milk production cost-effectively and achieve a positive return on investment.

Higher incidence of disease leads to diminished production and higher maintenance costs, reducing the profitability of your farm. Additionally, a disease can impact the future production potential of a segment of your herd, negatively affecting production in the long-term.

Are you interested in learning more about amino acid products and how they can help you achieve more with your livestock? Learn about the products that might be best for you, as well as their benefits and potential for improving your animals' performance and profitability.

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What Are Essential Amino Acids? Problems Associated With Lack of Amino Acids in Farm Animal Diets If an animal is not provided sufficient quantities of certain essential amino acids in its diet, the animal cannot produce enough proteins to support certain metabolic functions.

Here are just a few problems associated with inadequate supply of amino acids for livestock: 1. Changes in Intake One of the first and most important signs of an amino acid imbalance in the feed of a herd is a reduction in feed intake.

Resurrecting essential amino acid biosynthesis in mammalian cells G-Protein-Coupled Receptors, Pancreatic Islets, and Diabetes. Dickert, S. Clapper GM, Grieshop CM, Merchen NR, Russet JC, Brent JL Jr, Fahey GC Jr. The initial two stages of the DAP pathway are catalyzed by aspartokinase and aspartate semialdehyde dehydrogenase. Indole acts as a nucleophile to displace a water molecule and the elimination-addition ends by indole condensing with a 3-carbon unit to form tryptophan. Chorismate mutase, also known as hydroxyphenylpyruvate synthase or chorismate pyruvatemutase, is the isomerase enzyme involved and catalyzes the committed step of phenylalanine and tyrosine, namely the formation of prephenate.
An Evolutionary Perspective on Amino Acids Collagen Lathway comprised of two-thirds of AAs as glycine, proline, and 4-hydroxyproline, whereas keratins present in feather and hair are also rich in these three AAs Amjno cysteine Thermogenic weight loss pills serine the immediate precursor of animal. Kemmer Anti-inflammatory foods for athletes Gitzinger M Daoud-El Baba M Djonov V Stelling J Fussenegger M Self-Sufficient control of urate homeostasis in mice by a synthetic circuit Nature Biotechnology 28 — Properties of tyrosinase and dopa quinone imine conversion factor from pharate pupal cuticle of Manduca sexta. Efficient Intervention of Broiler Gut Health Issues Learn More. Importantly, this dihydroxy-acid dehydratase overexpressing cell line was passaged 10 times in the absence of valine with a consistent average doubling time of 3.
Major genomic deletions in independent eukaryotic Blueberry salad dressing recipe Sodium intake and bone health led to pathwat Anti-inflammatory foods for athletes loss synnthesis biosynthesis pathways for nine of the twenty acir amino acids. While the ysnthesis forces driving these polyphyletic Sodium intake and bone health events aicd not well sgnthesis, the synhesis is that extant metazoans are unable to produce nine acir amino acids EAAs. Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary CHO cell line.

Amino acid synthesis pathway in animals -

For example, the carboxylation of glutamate allows for better binding of calcium cations. The hydroxylation of proline is critical for maintaining connective tissues. Another example is the formation of hypusine in the translation initiation factor EIF5A, through modification of a lysine residue.

Such modifications can also determine the localization of the protein, e. Some nonstandard amino acids are not found in proteins.

Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids — for example, ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.

Search site Search Search. Go back to previous article. Sign in. Learning Objectives Recognize the factors involved in amino acid synthesis. Key Points All amino acids are synthesized from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway.

Of the 22 amino acids naturally incorporated into proteins, 20 are encoded by the universal genetic code and the remaining two, selenocysteine and pyrrolysine, are incorporated into proteins by unique synthetic mechanisms.

Key Terms pyrrolysine : An amino acid found in methanogenic bacteria. B Schematic of pMTIV construct after genomic integration and RNA-seq read coverage showing successful incorporation and active transcription. C Microscopy images of CHO-K1 cells with integrated pCtrl or pMTIV constructs in complete FK medium after 2 days or valine-free FK medium after 6 days.

Scale bar represents µm. D Growth curve of CHO-K1 cells with pCtrl or pMTIV in complete FK medium Figure 2—source data 1. Day-0 indicates number of seeded cells. Error bars represent data from three replicates. E Growth curve of CHO-K1 cells with pCtrl or pMTIV in valine-free FK medium Figure 2—source data 1.

Raw cell count data for pMTIV valine-free and complete FK medium tests. To test the biosynthetic capacity of pMTIV, we first introduced the construct into CHO cells.

Flp-In integration was used to stably insert either pMTIV, or a control vector pCtrl into the CHO genome. Successful generation of each cell line was confirmed by PCR amplification of junction regions formed during vector integration Figure 2—figure supplement 2A-B.

RNA-seq of cells containing the pMTIV construct confirmed transcription of the entire ORF Figure 2B. Western blotting of pMTIV cells using antibodies against the P2A peptide yielded bands at the expected masses of P2A-tagged proteins, confirming the production of separate distinct enzymes Figure 2—figure supplement 2C.

In reconstituted methionine-free, threonine-free, or isoleucine-free FK medium supplemented with dialyzed FBS to reduce FBS-derived AA content Figure 2—figure supplement 3 , cells containing the pMTIV construct did not show viability over 7 days, similar to cells containing the pCtrl control vector Figure 2—figure supplement 4.

In striking contrast, however, cells containing the integrated pMTIV showed relatively healthy cell morphology and viability in valine-free FK medium Figure 2C , whereas cells containing pCtrl exhibited substantial loss of viability over 6 days.

In complete FK medium, cells carrying the integrated pMTIV construct showed no growth defects compared to control cells Figure 2D. When cultured in valine-free FK medium over multiple passages with medium changes every 2 days, pMTIV cell proliferation was substantially reduced by the 3rd passage.

We hypothesized that frequent passaging might over-dilute the medium and prevent sufficient accumulation of biosynthesized valine necessary for continued proliferation as has been demonstrated for certain non-essential metabolites which become essential when cells are cultured at low cell densities Eagle and Piez, While use of pMTIV-conditioned medium improved the survival of cells harboring the pathway, it did not completely rescue valine auxotrophy in control cells, which exhibited substantial loss of cell viability over 8 days Figure 2—figure supplement 5A.

As a control, we generated pCtrl-conditioned valine-free FK medium using the same medium conditioning regimen, which failed to enable cells to grow to the same degree as that of pMTIV-conditioned medium, suggesting that the benefit conferred by medium conditioning is valine-specific Figure 2—figure supplement 5B.

Using this regimen, we were able to culture pMTIV cells for 9 passages without addition of exogenous valine Figure 2F. The doubling time was inconsistent across the 49 days of experimentation with cells exhibiting a mean doubling time of 5. Despite the slowed growth seen in later passages, cells exhibited healthy morphology and continued viability at day, suggesting that the cells could have been passaged even further.

The pIV construct similarly supported cell growth in valine-free FK medium, and exhibited similar growth dynamics to the pMTIV construct in complete medium Figure 2—figure supplement 6.

To confirm endogenous biosynthesis of valine, we cultured pCtrl and pMTIV cells in RPMI medium containing 13 C 6 -glucose in the place of its 12 C equivalent together with 13 C 3 -sodium pyruvate spiked in at 2 mM over three passages Figure 3—figure supplement 1A. High-resolution MS1 of MTIV cell lysates revealed a peak at The resulting fragmentation patterns for each peak Figure 3B matched theoretical expectations for each isotopic version of valine Figure 3—figure supplement 1B.

Taken together, this demonstrates that pMTIV cells are capable of biosynthesizing valine from core metabolites glucose and pyruvate, thereby proving successful metazoan biosynthesis of valine.

Over the course of 3 passages in heavy valine-free medium, the non-essential amino acid alanine, which is absent from RPMI medium and synthesized from pyruvate, was found to be Assuming similar turnover rates for alanine and valine within the CHO proteome, we expected to see similar percentages of 13 C-labeled valine.

However, just For pMTIV cells cultured in heavy but valine-replete medium, just 6. Together with the observed slow proliferation of pMTIV cells in valine-free medium, our data suggests that valine complementation is sufficient but sub-optimal for cell growth.

MS2 fragmentation patterns for each of these metabolites matched expectations Figure 3—source data 1. C RNA-seq dendrogram of pCtrl cells and pMTIV cells grown on complete FK medium or starved of valine for 4 hr or 48 hr.

D Principal Component Analysis PCA space depiction of pCtrl cells and pMTIV cells grown on complete FK medium, or starved of valine for 4 hr or 48 hr. We performed RNA-seq to profile the transcriptional responses of cells containing pMTIV or pCtrl in complete harvested at 0 hr and valine-free FK medium harvested at 4 hr and 48 hr, respectively Figure 3C , Figure 3—figure supplement 2A.

The transcriptional impact of pathway integration is modest Figure 3D. Only 51 transcripts were differentially expressed between pCtrl and pMTIV cells grown in complete medium, and the fold changes between conditions were small Figure 3E , Figure 3—figure supplement 2B. While some gene ontology GO functional categories were enriched Figure 3—figure supplement 2C , they did not suggest dramatic cellular stress.

Rather, these transcriptional changes may reflect cellular response to BCAA dysregulation due to altered valine levels Zhenyukh et al. In contrast, comparison of 48 hr valine-starved pCtrl and pMTIV cells yielded ~ differentially expressed genes.

Transcriptomes of pMTIV cells in valine-free medium more closely resembled cells grown on complete medium than did pCtrl cells in valine-free medium Figure 3D , Figure 3—figure supplement 3A. Differentially expressed genes between pCtrl and pMTIV cells showed enrichment for hundreds of GO categories, including clear signatures of cellular stress such as autophagy, changes to endoplasmic reticulum trafficking, and ribosome regulation Figure 3—figure supplement 3B.

Most of the differentially regulated genes between pCtrl cells in complete medium, and those same cells starved of valine for 48 hr were also differentially expressed when comparing pCtrl and pMTIV cells in valine-free medium Figure 3E , supporting the hypothesis that most of the observed transcriptional changes represent broad but partial rescue of the cellular response to starvation.

We also examined the integrated stress response ISR and mTOR signaling pathways, both of which are known to modulate cellular responses to starvation Pakos-Zebrucka et al.

We observed no clear signatures of mTOR activation Figure 3—figure supplement 4A , although a number of individual genes related to the mTOR pathway were significantly differentially expressed compared to pCtrl cells valine-starved for 48 hr Figure 3—figure supplement 4B. A manually curated list of ISR genes showed signals of ISR gene activation, but showed few differences between pCtrl and pMTIV cells at 48 hr of starvation Figure 3—figure supplement 4C.

pMTIV cells grown for 5 passages over 29 days on conditioned valine-free FK medium were more similar to pMTIV cells starved for 48 hr than to pCtrl cells starved for 48 hr Figure 3—figure supplement 5A. To improve rescue of the valine starvation phenotype, we looked for valine biosynthetic pathway intermediates in our metabolomics data that might suggest that the pathway was bottlenecked at any stage.

While no signal could be detected for pyruvate, 2-acetolactate or 2-oxoisovalerate, a signal was detected for pathway intermediate 13 C 5 -2,3-dihydroxy-isovalerate, which was specific to pMTIV cells cultured in both complete and in valine-free medium Figure 3—figure supplement 1F.

To determine whether the downstream pathway gene, ilvD , which encodes the dihydroxy-acid dehydratase enzyme, might constitute a bottleneck, we generated a lentivirus encoding a puromycin resistance cassette in addition to ilvD under control of a viral MMLV promoter Figure 4A.

Both pCtrl and pMTIV cells were infected and integrants were selected for on puromycin, resulting in a population-averaged integration count of 5. This resulted in a 0.

B ilvD qPCR on gDNA and cDNA from each cell line Figure 4—source data 1. Fold change levels were relativized to pMTIV. cDNA was reverse transcribed using oligo dT primers from RNA templates collected from each cell line.

Error bars show SD of three technical replicates. Error bars represent data from two replicates. We also reduced the isoleucine content of this isotopically heavy valine-free RPMI medium to match the isoleucine content of FK medium from 0.

In pMTIV cells, presence of pathway intermediate 2,3-dihydroxy-isovalerate fluctuated throughout the 24 days of culture with cells exhibiting higher concentrations in earlier time points. pMTIV samples on average contained We quantified the functional impact of modifying flux at this pathway bottleneck by culturing both cell lines in unconditioned, reduced-isoleucine, valine-free RPMI medium containing 2 mM sodium pyruvate over 10 passages on plates not coated with gelatin.

By comparison, pMTIV cells exhibited an average doubling time of 4. Plates were not coated with gelatin. In this work, we demonstrated the successful restoration of an EAA biosynthetic pathway in a metazoan cell.

Our results indicate that contemporary metazoan biochemistry can support complete biosynthesis of valine, despite millions of years of evolution from its initial loss from the ancestral lineage.

Interestingly, independent evidence for BCAA biosynthesis has also been obtained for sap-feeding whitefly bacteriocytes that host bacterial endosymbionts; metabolite sharing between these cells is predicted to lead to biosynthesis of BCAAs that are limiting in their restricted diet.

The malleability of mammalian metabolism to accept heterologous core pathways opens up the possibility of animals with designer metabolisms and enhanced capacities to thrive under environmental stress and nutritional starvation Zhang et al.

Yet, our failure to functionalize designed methionine, threonine, and isoleucine pathways highlights outstanding challenges and future directions for synthetic metabolism engineering in animal cells and animals.

Other pathway components or alternative selections may be needed for different EAAs Rees and Hay, Studies to reincorporate EAAs into the core mammalian metabolism could provide greater understanding of nutrient-starvation in different physiological contexts including the tumor microenvironment Lim et al.

Emerging synthetic genomic efforts to build a prototrophic mammal may require reactivation of many more genes Supplementary files , iterations of the design, build, test DBT cycle, and a larger coordinated research effort to ultimately bring such a project to fruition.

For pathway completeness analysis, the EC numbers of each enzyme in each amino acid biosynthesis pathway excluding pathways annotated as only occurring in prokaryotes were collected from the MetaCyc database Supplementary file 4.

Variant biosynthetic routes to the same amino acid were considered as separate pathways, generating distinct EC number lists.

The resulting per-pathway EC number lists were checked against the KEGG, Entrez Gene, Entrez Nucleotide, and Uniprot databases using their respective web APIs for each listed organism. CHO Flp-In cells ThermoFisher, R were used in all experiments. All cell lines tested negative for mycoplasma.

Custom amino acid dropout medium was adjusted to a pH of 7. For metabolomics experiments, medium was prepared from an amino acid-free and glucose-free RPMI powder base US Biological, R , and custom combinations of amino acids and isotopically heavy glucose and sodium pyruvate were added in to match the standard amino acid concentrations for RPMI or as specified.

pH was adjusted to 7. Where specified, cells were cultured on plates coated with 0. Plates were washed with PBS prior to use. For evaluating effects of amino acid dropout on cell growth curves, cells were seeded at 1×10 4 into 6-well plates into FK media with lowered amino acid concentrations relative to typical FK media and then allowed to grow for 5 days.

Media was then aspirated off and replaced with PBS with Hoechst live nuclear stain for automated imaging and counting using a DAPI filter set on an Eclipse Ti2 automated inverted microscope.

To count, an automated microscopy routine was used to Figure 5 random locations within each well at 10× magnification, and then the cells present in imaged frames counted using automatic cell segregation and counting software. Given differences in cell response to starvation, segregation and counting parameters were tuned in each experiment, but kept constant between starvation conditions and cells with and without the pathway.

Conditioned medium was generated by seeding 1×10 6 pMTIV cells into 10 mL complete FK medium on 10 cm plates and replacing the medium with 10 mL freshly prepared valine-free FK medium the next day following a PBS wash step. Cells conditioned the medium for 2 days at which point the medium was collected, centrifuged at ×g for 3 mins to remove potential cell debris, sterile filtered, and collected in mL vats to reduce batch-to-batch variation.

Integrated constructs were synthesized de novo in 3 kb DNA segments with each segment overlapping neighboring segments by 80 bp. Assembly was conducted in yeasto by co-transformation of segments into S.

cerevisiae strain BY made competent by the LiOAc method Pan et al. After 2 days of selection at 30°C on SC—Ura medium, individual colonies were picked and cultured overnight. Glass beads were added to each resuspension and the mixture was vortexed for 10 mins to mechanically shear the cells.

Next, cells were subject to alkaline lysis by adding µl of P2 lysis buffer Qiagen, for 5 mins and then neutralized by addition of Qiagen N3 neutralization buffer Qiagen, Plasmid DNA was eluted in Zyppy Elution buffer and subsequently transformed into TransforMax EPI chemically competent E.

Cell were lysed in SKL Triton lysis buffer 50 mM Hepes pH7. NuPAGE LDS sample buffer ThermoFisher, NP supplemented with 1. The membrane was incubated in the secondary antibody solution for 1.

Cell pellets were generated by trypsinization, followed by low speed centrifugation, and the pellet was frozen at —80°C until further processing. The LC column was a Millipore ZIC-pHILIC 2. Injection volume was set to 1 μL for all analyses 42 min total run time per injection. MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HF mass spectrometer operating in heated electrospray ionization mode HESI.

Spray voltage for both positive and negative modes was 3. Tandem MS spectra for both positive and negative mode used a resolution of 15,, AGC target of 1e5, maximum IT of 50ms, isolation window of 0.

The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. All valine data were processed using Thermo XCalibur Qualbrowser for manual inspection and annotation of the resulting spectra and peak heights referring to authentic valine standards and labeled internal standards as described.

QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates. Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina New England Biolabs, E , and sequenced on a NextSeq single-end 75 cycles high output with v2. Differential gene enrichment analysis was performed with in R with DESeq2 and GO enrichment performed and visualized with clusterProfiler against the org.

db database, with further visualization with the pathview, GoSemSim, eulerr packages. Target plasmid was maintained in and purified from NEB beta electrocompetent E.

coli New England Biolabs, CK. Lentivirus was packaged by plating 4×10 6 HEKT cells on 10 cm 2 and incubating cells overnight at 37°C. Cells were transfected with a plasmid mix consisting of 3. Transfected HEKT cells were incubated for 48 hr, before medium was collected, and centrifuged at ×g for 5 mins.

The resulting supernatant was filtered using a 0. The packaged virus was applied to cells for 24 hr before the medium was exchanged for fresh medium. For RNA extraction, QIAshredder homogenizer columns Qiagen, were used to disrupt the cell lysates.

cDNA was generated from RNA using Invitrogen SuperScript IV Reverse Transcriptase Invitrogen, and oligo dT primers. Each qPCR reaction was performed using SYBR Green Master I Roche, on a Light Cycler Roche, using the recommended cycling conditions.

Primers were designed to amplify amplicons — bp in size. Sequencing data generated for this study is deposited in the NCBI SRA at accession number PRJNA Source data files have been provided for Figure 1 - figure supplement 1, Figure 1 - figure supplement 2D, Figure 2, Figure 2 - figure supplement 3, Figure 2 - figure supplement 4B, Figure 2 - figure supplement 5, Figure 2 - figure supplement 6, Figure 3, and Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, and Figure 5 - figure supplement 1.

Our editorial process produces two outputs: i public reviews designed to be posted alongside the preprint for the benefit of readers; ii feedback on the manuscript for the authors, including requests for revisions, shown below.

We also include an acceptance summary that explains what the editors found interesting or important about the work. Thank you for submitting your article "Resurrecting essential amino acid biosynthesis in a mammalian cell" for consideration by eLife.

Your article has been reviewed by 3 peer reviewers, including Ivan Topisirovic as Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Philip Cole as the Senior Editor.

The following individual involved in review of your submission has agreed to reveal their identity: Ran Kafri Reviewer 3. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Based on this, it was thought that more evidence is required to demonstrate that the introduction of valine biosynthetic pathway into CHO cells results in sustained proliferation and survival in the absence of valine supplementation.

Accordingly, it was deemed that the authors should monitor long-term ability of engineered CHO cells to sustain valine production and proliferate in valine-free media. To this end, monitoring flux via valine biosynthetic and degradation pathways, transcriptome and mTOR signaling at early and late time points was thought to be warranted.

These include lack of clarity pertinent to the rationale behind using "conditioned-medium" in the experiments. Moreover, potential utilization of other sources of valine e. It was appreciated that the latter cells survive in valine-free media, but it seems that their proliferation is significantly lower than in valine containing media.

Moreover, it seems that after 6 passages only a fraction of the detected valine is synthesized de novo. Would this fraction further decrease in subsequent passages? Related to this, it is not clear what is the efficiency of valine biosynthesis in CHO cells vs.

a prototrophic organism. Perhaps comparing the rates of valine synthesis in cell free extracts of CHO cells vs. those derived from a prototrophic organism may be helpful to address this.

This in particular relates to amino-acid sensing pathways e. Were the enzymes mislocalized? Are there other regulatory factors involved? Moreover, considering that the overarching tenet is that metazoans lost the ability to produce essential amino acids due to energetic restraints, it may be worthwhile noting that culturing conditions and potential differences in energy resources may impact on functionalization of essential amino acid biosynthetic pathways.

The results put forth in this manuscript suggest the authors were marginally successful in introducing a valine biosynthetic pathway into CHO cells, but fall short of demonstrating a robust, self-sustaining engineered cell line under reasonable culture conditions.

This milestone should be met prior to final acceptance at eLife. Additionally, the following revisions should be carried out prior to acceptance. When cats are fed diets e. Finally, when the content of one of 10 EAAs Arg, Lys, His, Ile, Leu, Met, Phe, Thr, Trp, and Val; provided at 1.

Cats respond to nitrogen-free intake by decreasing whole-body AA oxidation, leading to reduced excretion of urinary total nitrogen, urea, and ammonia [ 22 ]. Interestingly, regarding AA metabolism, cats differ from omnivores e.

It is likely that 1 tissues of cats express high basal levels of enzymes for AA catabolism and urea-cycle enzymes; 2 when fed a low-protein diet, cats are unable to downregulate these enzymes, but actual metabolic fluxes through the enzymes in vivo are decreased under conditions of such a nutritional state e.

In support of this view, Russell et al. Likewise, increasing the dietary CP level from 9. Thus, cats are metabolically more capable of adapting to high protein intake than previously realized based on enzyme activity data [ ].

In animals including cats , AA oxidation increases to remove excess AAs [ 61 ]. This can be illustrated by studies with Labrador retrievers males plus females that were fed a growth diet containing Protein restriction for healthy older dogs can be detrimental to their health particularly regarding the mass and function of skeletal muscles and bones and, therefore, should be avoided in feeding practice.

To meet the dietary requirements of dogs for high-quality protein, animal-sourced foodstuffs [which provide proper ratios and adequate amounts of proteinogenic AAs as well as functional nutrients e. Like dogs, cats lose lean body mass with age. As for older dogs, older cats need adequate high-quality protein i.

Such diets should also help to improve the anti-oxidative and immune functions of senior cats. Consistent with this notion, adult cats neutered males needed 1. Protein-restricted diets for healthy adult cats must be avoided in feeding practice. To meet the dietary requirements of adult cats and dogs for high-quality protein, animal-sourced foodstuffs can be used to manufacture diets for these animals [ 54 ].

Dietary AA intake by cats and dogs, like other animals, depends on dietary AA content and food consumption [ 61 ]. Multiple factors should be considered in formulating diets, including endogenous AA synthesis, the digestibility and bioavailability of dietary nutrients, the presence of antinutritive factors in foodstuffs, the fermentability and quantity of dietary fiber, and interactions among food constituents [ 33 , 61 , ].

Furthermore, requirements of cats and dogs for dietary AAs including sulfur AAs may critically depend on the catabolism of these nutrients by the intestinal microbiota [ 75 , 99 ].

Average pregnancy length in cats and dogs appears to similar 65 and 63 d, respectively , but there are differences in the patterns of both maternal and fetal weight gains between these two species [ , ]. At present, little is known about the dietary requirements of pregnant dogs for taurine.

There are differences in maternal weight change after parturition between cats and dogs. Specifically, after giving births, the bitch generally returns to her pre-breeding BW immediately after delivery [ ]. As in other mammals, dietary deficiencies of AAs impair milk production by lactating cats and dogs [ 16 ].

To date, dietary requirements of pregnant and lactating cats and dogs for proteinogenic AAs have not been well defined. It has been assumed that dietary CP requirements for the growth of young cats and dogs would meet their requirements for gestation and lactation [ 16 ].

However, embryos of mammals, including cats and dogs, are highly sensitive to ammonia toxicity and, therefore, maternal intakes of dietary CP and AAs should not be excessive [ , ]. The NRC [ 16 ] recommends that the dietary requirements of dogs or cats for CP and AAs be the same during late pregnancy and peak lactation, but it is likely that such estimates do not reflect the true requirements of the animals because of marked differences in physiological states pregnancy versus lactation and products conceptus versus milk.

In addition, the NRC [ 16 ] recommends substantial increases in the dietary contents of CP and most proteinogenic AAs except methionine, cysteine and tryptophan for adult dogs during late gestation and peak lactation compared with non-pregnant and non-lactating counterparts Table 7.

Such recommendations do not appear to have physiological bases and should be revised in the future.

Interestingly, glutamine is the only AA in the arterial blood that is taken up by the small intestine for citrulline and arginine production; therefore, it is of nutritional and physiological importance to convert BCAAs the source of the amino group and amide nitrogen into glutamine in extra-hepatic tissues primarily skeletal muscle [ 61 ].

Because BCAAs are not formed de novo in all animals, these EAAs must be provided from high-quality and high-quantity protein. Even the same ingredient may not supply the same amount of nutrients depending on the method of food processing, and some of diet-derived small peptides can exert signaling and regulatory functions in the intestine and extraintestinal tissues [ ].

Despite an endogenous synthesis of arginine, both young and adult dogs must ingest adequate arginine in diets to maintain its vital physiological functions beyond nitrogen balance [ 43 ], as noted previously.

Interestingly, hydrolyzed feather is a rich source of arginine 5. Inclusion of hydrolyzed feather meal in dry or wet foods for dogs can meet their high requirements for arginine.

In addition, even when fed a diet containing sufficient methionine and cysteine, some breeds of dogs have a limited ability to synthesize taurine due to genetic mutations as noted previously and, therefore, must be provided with adequate dietary taurine e.

As noted previously, cats have a very limited ability to synthesize both arginine and taurine and, therefore, must consume diets containing these two AAs to ensure normal blood flow, the proper digestion of dietary lipids and fat-soluble vitamins, and maintain health particularly retinal, cardiac, skeletal, reproductive, and metabolic health [ 14 , 43 ].

These animals do not have preference for plant products that generally contain high amounts of carbohydrates including sweet sugars [ 14 ]. In recent years, much work has shown that animal-derived ingredients are abundant sources of both arginine and taurine for the diets of animals [ 54 ].

These foodstuffs also contain creatine that is essential for energy metabolism and anti-oxidative reactions in excitable tissues brain and skeletal muscle [ 61 ]. In contrast, all plant-sourced foodstuffs lack taurine and creatine [ 54 ] and, therefore, should not be fed solely to either cats or some breeds of dogs.

As for dogs, hydrolyzed feather meal can be included in diets as an abundant source of both arginine and taurine for cats. Another unique feature of animal-derived products is that they contain high amounts of either collagens e. Collagen is comprised of two-thirds of AAs as glycine, proline, and 4-hydroxyproline, whereas keratins present in feather and hair are also rich in these three AAs plus cysteine and serine the immediate precursor of glycine.

After feather and hair are properly hydrolyzed, their AAs are nutritionally available for both cats and dogs to use [ 54 ].

Thus, hydrolyzed feather meal contains high amounts of glycine, proline, 4-hydroxyproline, cysteine, and serine 8. Dietary provision of hydrolyzed feather meal can spare energy and materials that would be needed for de novo syntheses of these AAs in animals, thereby reducing energy expenditure as well as the associated production of oxidants e.

Of note, cysteine, glycine, and proline are crucial for the synthesis of hair proteins e. These unique proteins maintain the normal structures and integrity of hair and connective tissue while preventing its abnormalities particularly in association with aging.

Indeed, hair quality is considered by pet owners as a very important indicator of the nutritional adequacy of commercially manufactured pet foods or home-made meals [ ].

Thus, hydrolyzed feather meal may be a desirable pet-food ingredient to provide nutritionally and physiologically significant AAs including arginine, cysteine, glycine, proline, 4-hydroxyproline, and serine [ 54 ]. This new knowledge can help to dispel the unfounded myth that poultry-sourced hydrolyzed feather meal is of little nutritive value in feeding companion animals.

Glucosamine is a normal metabolite of glutamine and fructosephosphate in animals [ 61 ]. Poultry meal is manufactured from raw materials containing chicken cartilage, which consists of glycosaminoglycans including chondroitin and proteoglycans formed from glycosaminoglycans and protein backbones in addition to collagens and elastins.

Glycosaminoglycans are composed of N-acetylglucosamine and N-acetylgalactosamine, as well as their sulfate derivatives [ 61 ]. In the small intestine, proteoglycans are hydrolyzed to proteases e.

The aminosugars are absorbed into enterocytes and then the portal vein. Within cells, 4-epimerase converts galactosamine into glucosamine. Thus, poultry meal is a source of glucosamine for animals, including cats and dogs. Of particular note, glucosamine has anti-inflammatory and anti-oxidative effects in immunologically challenged mammalian cells by inhibiting the expression of inducible NO synthase and excessive NO production [ ].

This may explain why glucosamine plus chondroitin has been used to effectively treat dogs particularly elderly dogs and working dogs [ ] and cats [ ] with joint pain or osteoarthritis. Research is warranted to define the efficacy of dietary supplementation with poultry meal in improving the health of cat and dog joints.

AAs are essential for immune responses in all animals including cats and dogs through a plethora of mechanisms, such as the syntheses of proteins including antibodies and cytokines and glutathione a potent anti-oxidative tripeptide consisting of glycine, cysteine, and glutamate , as well as the killing of pathogens via production of NO from arginine and of chlorotaurine and bromotaurine from taurine [ 61 ].

In addition, intravenous administration of alanyl-glutamine to dogs undergoing a treatment with methylprednisolone sodium succinate enhanced phagocytic capacity and respiratory burst activity of leukocytes [ ].

Currently, there is a global pandemic of COVID caused by the severe acute respiratory syndrome coronavirus 2 that can also infect dogs [ ] and cats [ ]. Adequate AA nutrition [e. In addition, some animal-sourced foods, such as spray-dried animal plasma [ ] and spray-dried egg products [ ] contain a large amount of immunoglobulins and directly contribute to neutralizing the pathogens that invade the body.

Furthermore, spray-dried animal plasma provides other functional molecules, such as albumin, essential fatty acids, B-complex vitamins, and minerals such as calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, zinc, copper, manganese, and selenium [ , ].

This animal-sourced foodstuff is also a useful binder in canned pet food products due to its high content of globulins and fibrinogen as well as its desired physicochemical properties [ ]. Finally, animal plasma products are highly palatable to both cats and dogs [ ].

Thus, animal-derived ingredients used in dry or wet pet foods can help to improve the immune responses and health of all companion animals by providing not only AAs but also other essential nutrients e.

Both cats and dogs are carnivores from the taxonomical order Carnivora. During evolution, domestic dogs have adapted to omnivorous diets that contain both taurine-rich meat and starch-rich plant ingredients, while domestic cats remain obligate carnivores.

Thus, dogs differ from cats in many aspects of AA nutrition and metabolism, and dogs can thrive on taurine-free vegetarian diets supplemented with non-taurine nutrients that are inadequately synthesized de novo or absent from plants.

Much evidence shows that there are marked differences in both qualitative i. In comparison with swine [ 53 , ], recommended minimum requirements and allowances of dietary EAAs for growing and adult cats and dogs are summarized in Table 7. We suggest that companion animals have dietary requirements for NEAAs as do other mammals due to insufficient synthesis de novo.

Cats have greater endogenous nitrogen losses, as well as higher requirements for dietary protein including arginine and taurine than do dogs and, therefore, should not be fed dog foods. Because the composition of the milk of both cats and dogs differ from that of farm mammals, young pets should not be fed replacer diets formulated based on goat or cow milk.

As companion animals lose tremendous amounts of lean body mass with aging, their diets should contain adequate levels of high-quality protein, which may be much greater than the current AAFCO [ ] and NRC [ 16 ] recommendations to support muscle protein synthesis and mitigate muscle loss.

Effects of an excessive intake of a single AA in cats [ ] and dogs [ ] may be different, depending on dietary intakes of other AAs. We are not aware that comparisons of the metabolism or dietary requirements of any nutrients between modern breeds of cats and dogs were made in the same experiment.

Nonetheless, the fundamental knowledge of nutrient metabolism in cats and dogs is essential for guiding their feeding, as well as food manufacturing. In practice, animal-sourced foodstuffs provide proper ratios and amounts of all proteinogenic AAs as well as large amounts of functional nutrients e.

In addition, feather meal can be used as an ingredient or supplement in dry or wet foods for cats and dogs to meet their high requirements for both arginine and taurine [ 54 ]. Animal-derived ingredients alone or in combination are abundant sources of both proteinogenic AAs and taurine for adequate nutrition and metabolism in cats and dogs to optimize their growth, development, health, and well-being.

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Amino acid synthesis pathway in animals on the Research Topic Aromatic Amino Acid Anti-inflammatory foods for athletes. Aromatic amino acids, pathwa other proteinogenic amino pqthway, are the building blocks of proteins Sodium intake and bone health include phenylalanine, tryptophan, and tyrosine. Ackd plants and micro-organisms synthesize their own Body shape determination amino acids to make proteins Aniamls, ; Tzin and Galili, However, animals have lost these costly metabolic pathways for aromatic amino acids synthesis and must instead obtain the amino acids through their diet. Herbicides take advantage of this by inhibiting enzymes involved in aromatic amino acid synthesis, thereby making them toxic to plants but not to animals Healy-Fried et al. Tyrosine is the initial precursor for the biosynthesis of dopa, dopamine, octopamine, norepinephrine, and epinephrine, etc. In addition, tyrosine is the precursor for melanin synthesis in most organisms including humans and animals, and is particularly important in insects for protection Whitten and Coates, Amino acid synthesis pathway in animals

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