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Created by Tracy Kim Kovach. Want to join the conversation? Log in. Sort by: Top Voted. Aditya Bhattad. Posted 10 years ago. Downvote Button navigates to signup page. Flag Button navigates to signup page. Show preview Show formatting options Post answer.

At biological PH 7 the amino acid ends are both charged. After the hydrolyzed step, why does the heat only remove one acid group as opposed to both? Cthulhu Mittens. Posted 9 years ago. you need the second carbonyl group to act as an electron sink.

Comment Button navigates to signup page. at It is a pretty long mechanism. Basically what happens is the nitrogen in the cyanide keeps getting protonated by the acid until it wants to leave the carbon. While this is happening the carbon from the cyanide is gaining water from the acid group being deprotonated by the nitrogen until it is a carboxylic acid.

Here is a link to a website that shows the steps with the mechanism. Posted a year ago. personally im into gabriel synthesis more. Do both reactions produce a racemic mixture of amino acids?

yes they both do since both synthesis start with a planar molecule! Eric Dornoff. It is interesting to note that the so-called essential amino acids that cannot be synthesized in human and other organisms generally appear in these extensions. Furthermore, the bottom extension of basic amino acids appears to be most divergent containing multiple pathways for lysine biosynthesis and multiple gene sets for arginine biosynthesis.

Image resolution: High. By the time many students get to the study of amino acid biosynthesis, they have seen so many pathways that learning new pathways for the amino acids seems daunting, even though they can be clustered into subpathways. Most know that from a nutrition perspective, amino acids can be divided into nonessential and essential need external dietary supplementation amino acids.

These are shown for humans below. Three of the essential amino acids can be made in humans but need significant supplementation. Arginine is depleted in processing through the urea cycle.

When cysteine is low, methionine is used to replace it so its levels fall. If tyrosine is low, phenylalanine is used to replace it. For this chapter subsection, we will provide only the basic synthetic pathways in abbreviated form without going into mechanistic or structural details.

Ala can easily be synthesized from the alpha-keto acid pyruvate by a transamination reaction, so we will focus our attention on the others, the branched-chain nonpolar amino acids Val, Leu, and Ile. Since amino acid metabolism is so complex, it's important to constantly review past learning.

These enzymes play a key role in the biosynthesis of lysine , threonine , and methionine. Transcription of aspartokinase genes is regulated by concentrations of the subsequently produced amino acids, lysine, threonine, and methionine.

The higher these amino acids concentrations, the less the gene is transcribed. ThrA and LysC are also feed-back inhibited by threonine and lysine.

Finally, DAP decarboxylase LysA mediates the last step of the lysine synthesis and is common for all studied bacterial species. The formation of aspartate kinase AK , which catalyzes the phosphorylation of aspartate and initiates its conversion into other amino acids, is also inhibited by both lysine and threonine , which prevents the formation of the amino acids derived from aspartate.

Additionally, high lysine concentrations inhibit the activity of dihydrodipicolinate synthase DHPS. So, in addition to inhibiting the first enzyme of the aspartate families biosynthetic pathway, lysine also inhibits the activity of the first enzyme after the branch point, i.

the enzyme that is specific for lysine's own synthesis. The biosynthesis of asparagine originates with aspartate using a transaminase enzyme. The enzyme asparagine synthetase produces asparagine, AMP , glutamate, and pyrophosphate from aspartate, glutamine , and ATP.

In the asparagine synthetase reaction, ATP is used to activate aspartate, forming β-aspartyl-AMP. Glutamine donates an ammonium group, which reacts with β-aspartyl-AMP to form asparagine and free AMP.

Two asparagine synthetases are found in bacteria. Both are referred to as the AsnC protein. They are coded for by the genes AsnA and AsnB. AsnC is autogenously regulated, which is where the product of a structural gene regulates the expression of the operon in which the genes reside.

The stimulating effect of AsnC on AsnA transcription is downregulated by asparagine. However, the autoregulation of AsnC is not affected by asparagine.

Biosynthesis by the transsulfuration pathway starts with aspartic acid. Relevant enzymes include aspartokinase , aspartate-semialdehyde dehydrogenase , homoserine dehydrogenase , homoserine O-transsuccinylase , cystathionine-γ-synthase , Cystathionine-β-lyase in mammals, this step is performed by homocysteine methyltransferase or betaine—homocysteine S-methyltransferase.

Methionine biosynthesis is subject to tight regulation. The repressor protein MetJ, in cooperation with the corepressor protein S-adenosyl-methionine, mediates the repression of methionine's biosynthesis.

The regulator MetR is required for MetE and MetH gene expression and functions as a transactivator of transcription for these genes. MetR transcriptional activity is regulated by homocystein, which is the metabolic precursor of methionine.

It is also known that vitamin B12 can repress MetE gene expression, which is mediated by the MetH holoenzyme. In plants and microorganisms, threonine is synthesized from aspartic acid via α-aspartyl-semialdehyde and homoserine. Homoserine undergoes O -phosphorylation; this phosphate ester undergoes hydrolysis concomitant with relocation of the OH group.

The biosynthesis of threonine is regulated via allosteric regulation of its precursor, homoserine , by structurally altering the enzyme homoserine dehydrogenase. This reaction occurs at a key branch point in the pathway, with the substrate homoserine serving as the precursor for the biosynthesis of lysine, methionine, threonin and isoleucine.

High levels of threonine result in low levels of homoserine synthesis. The synthesis of aspartate kinase AK , which catalyzes the phosphorylation of aspartate and initiates its conversion into other amino acids, is feed-back inhibited by lysine , isoleucine , and threonine , which prevents the synthesis of the amino acids derived from aspartate.

So, in addition to inhibiting the first enzyme of the aspartate families biosynthetic pathway, threonine also inhibits the activity of the first enzyme after the branch point, i.

the enzyme that is specific for threonine's own synthesis. In plants and microorganisms, isoleucine is biosynthesized from pyruvic acid and alpha-ketoglutarate.

Enzymes involved in this biosynthesis include acetolactate synthase also known as acetohydroxy acid synthase , acetohydroxy acid isomeroreductase , dihydroxyacid dehydratase , and valine aminotransferase.

In terms of regulation, the enzymes threonine deaminase, dihydroxy acid dehydrase, and transaminase are controlled by end-product regulation. the presence of isoleucine will downregulate threonine biosynthesis. High concentrations of isoleucine also result in the downregulation of aspartate's conversion into the aspartyl-phosphate intermediate, hence halting further biosynthesis of lysine , methionine , threonine , and isoleucine.

coli , the biosynthesis begins with phosphorylation of 5-phosphoribosyl-pyrophosphate PRPP , catalyzed by ATP-phosphoribosyl transferase. Phosphoribosyl-ATP converts to phosphoribosyl-AMP PRAMP. His4 then catalyzes the formation of phosphoribosylformiminoAICAR-phosphate, which is then converted to phosphoribulosylformimino-AICAR-P by the His6 gene product.

After, His3 forms imidazole acetol-phosphate releasing water. His5 then makes L -histidinol-phosphate, which is then hydrolyzed by His2 making histidinol. His4 catalyzes the oxidation of L -histidinol to form L -histidinal, an amino aldehyde. In the last step, L -histidinal is converted to L -histidine.

In general, the histidine biosynthesis is very similar in plants and microorganisms. The enzymes are coded for on the His operon. This operon has a distinct block of the leader sequence, called block This leader sequence is important for the regulation of histidine in E.

The His operon operates under a system of coordinated regulation where all the gene products will be repressed or depressed equally.

The main factor in the repression or derepression of histidine synthesis is the concentration of histidine charged tRNAs. The regulation of histidine is actually quite simple considering the complexity of its biosynthesis pathway and, it closely resembles regulation of tryptophan.

In this system the full leader sequence has 4 blocks of complementary strands that can form hairpin loops structures. When histidine charged tRNA levels are low in the cell the ribosome will stall at the string of His residues in block 1.

This stalling of the ribosome will allow complementary strands 2 and 3 to form a hairpin loop. The loop formed by strands 2 and 3 forms an anti-terminator and translation of the his genes will continue and histidine will be produced. However, when histidine charged tRNA levels are high the ribosome will not stall at block 1, this will not allow strands 2 and 3 to form a hairpin.

Instead strands 3 and 4 will form a hairpin loop further downstream of the ribosome. When the ribosome is removed the His genes will not be translated and histidine will not be produced by the cell. Serine is the first amino acid in this family to be produced; it is then modified to produce both glycine and cysteine and many other biologically important molecules.

Serine is formed from 3-phosphoglycerate in the following pathway:. The conversion from 3-phosphoglycerate to phosphohydroxyl-pyruvate is achieved by the enzyme phosphoglycerate dehydrogenase.

This enzyme is the key regulatory step in this pathway. Phosphoglycerate dehydrogenase is regulated by the concentration of serine in the cell. At high concentrations this enzyme will be inactive and serine will not be produced.

At low concentrations of serine the enzyme will be fully active and serine will be produced by the bacterium. Glycine is biosynthesized from serine, catalyzed by serine hydroxymethyltransferase SHMT. The enzyme effectively replaces a hydroxymethyl group with a hydrogen atom. SHMT is coded by the gene glyA.

The regulation of glyA is complex and is known to incorporate serine, glycine, methionine, purines, thymine, and folates, The full mechanism has yet to be elucidated.

Homocysteine is a coactivator of glyA and must act in concert with MetR. PurR binds directly to the control region of glyA and effectively turns the gene off so that glycine will not be produced by the bacterium.

The genes required for the synthesis of cysteine are coded for on the cys regulon. The integration of sulfur is positively regulated by CysB. Effective inducers of this regulon are N-acetyl-serine NAS and very small amounts of reduced sulfur.

CysB functions by binding to DNA half sites on the cys regulon. These half sites differ in quantity and arrangement depending on the promoter of interest. There is however one half site that is conserved. It lies just upstream of the site of the promoter.

There are also multiple accessory sites depending on the promoter. In the absence of the inducer, NAS, CysB will bind the DNA and cover many of the accessory half sites.

Without the accessory half sites the regulon cannot be transcribed and cysteine will not be produced. It is believed that the presence of NAS causes CysB to undergo a conformational change. This conformational change allows CysB to bind properly to all the half sites and causes the recruitment of the RNA polymerase.

The RNA polymerase will then transcribe the cys regulon and cysteine will be produced. Further regulation is required for this pathway, however. CysB can down regulate its own transcription by binding to its own DNA sequence and blocking the RNA polymerase. In this case NAS will act to disallow the binding of CysB to its own DNA sequence.

OAS is a precursor of NAS, cysteine itself can inhibit CysE which functions to create OAS. Without the necessary OAS, NAS will not be produced and cysteine will not be produced.

There are two other negative regulators of cysteine. These are the molecules sulfide and thiosulfate , they act to bind to CysB and they compete with NAS for the binding of CysB. Pyruvate, the result of glycolysis , can feed into both the TCA cycle and fermentation processes.

Reactions beginning with either one or two molecules of pyruvate lead to the synthesis of alanine, valine, and leucine. Feedback inhibition of final products is the main method of inhibition, and, in E.

coli , the ilvEDA operon also plays a part in this regulation. Alanine is produced by the transamination of one molecule of pyruvate using two alternate steps: 1 conversion of glutamate to α-ketoglutarate using a glutamate-alanine transaminase, and 2 conversion of valine to α-ketoisovalerate via Transaminase C.

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.

Furthermore, the bottom extension of basic amino acids appears to be most divergent containing multiple pathways for lysine biosynthesis and multiple gene sets for arginine biosynthesis.

Image resolution: High. Link: Normal Module. ID search. Reaction module Reaction modules Carboxylic acid metabolism 2-Oxocarboxylic acid chain extension RM 2-Oxocarboxylic acid chain extension by tricarboxylic acid pathway 2-Oxocarboxylic acid chain modification RM Carboxyl to amino conversion using protective N-acetyl group basic amino acid synthesis RM Carboxyl to amino conversion without using protective group RM Branched-chain addition branched-chain amino acid synthesis.

Figure 2—figure supplement 1. The two remaining genes were included to test potential routes to simultaneously rescue threonine and methionine auxotrophy by selectively supplementing individual missing metabolic steps, in addition to complete pathway reconstruction for valine and isoleucine.

To biosynthesize methionine, we chose the E. coli metC gene, which encodes cystathionine-ß-lyase and converts cystathionine to homocysteine, a missing step in CHO-K1 cells in a potential serine to methionine biosynthetic pathway.

Threonine production was tested using E. coli L-threonine aldolase ltaE , which converts glycine and acetaldehyde into threonine. For branched chain amino acids BCAAs valine and isoleucine, three additional biosynthetic enzymes and one regulatory subunit are needed in theory to convert pyruvate and 2-oxobutanoate into valine and isoleucine, respectively.

In the case of valine, pyruvate is converted to 2-acetolactate, then to 2,3-dihydroxy-isovalerate, then to 2-oxoisovalerate and finally to valine. For isoleucine, 2-oxobutanoate is converted to 2-acetohydroxybutanoate, then to 2,3-dihydroxymethylpentanoate, then to 3-methyloxopentanoate, and finally to isoleucine.

The final steps in the biosynthesis of both BCAAs can be performed by native CHO catabolic enzymes Bcat1 and Bcat2 Hefzi et al.

The final pMTIV construct comprises metC , itaE , ilvN, ilvB , ilvC, and ilvD organized as a single open reading frame ORF with a 2A sequence variant lying between each protein coding region Figure 2B , and driven by a single strong spleen focus-forming virus SFFV promoter.

A Three enzymatic steps encoded by E. coli genes ilvN regulatory subunit, acetolactate synthase , ilvB catalytic subunit, acetolactate synthase , ilvC ketol-acid reductoisomerase , and ilvD dihydroxy-acid dehydratase are required for valine biosynthesis in Chinese hamster ovary CHO -K1 cells.

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.

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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. If you are synthesizing an amino acid with a more reactive R group - say glutamate or arginine - how do you prevent the R group from participating in either synthesis Strecker or Gabriel?

Daniel Isaac. Posted 6 years ago. There are protecting groups available for protecting essentially every R group found on an amino acid. Such amino acid derivatives are typically used in solid phase peptide synthesis when a long peptide is desired.

Posted 7 years ago. yes, because both are starting with planar molecules, sorry for responding 6 years late hope you graduated as a doctor lol. is this is all what we have to know about Gabriel synthesis for the MCAT?

Aadim Npl. Posted 5 years ago. Where is n-phthalimidomalonic ester found? Is it found in food? Darren Savage. Is this a part of content category 1A? Video transcript Hey. So we're going to be talking about amino acid synthesis.

And we're just going to stick with two of the main methods for synthesizing amino acids. And they both just happened to be named after old German chemists because synthesizing amino acids was probably hot stuff back in the mid to late s, And the first method that we're going to be talking about is Gabriel synthesis, named after Siegmund Gabriel.

And the second method is called Strecker synthesis, which is named after Adolph Strecker. So let's start out with Gabriel synthesis first. In Gabriel synthesis, we begin with a molecule of what's called phthalimidomalonic ester. So n-phthalimidomalonic ester is what this molecule is called, and that's kind of a mouthful so I'm just going to call this "thad.

And so let me draw an alpha amino acid over here to kind of remind us what our end product or end goal is going to be. And so remember that an amino acid has, first, the amino group, and I'm going to draw it in the protonated form. And then we have our alpha carbon, and then the R group, or side chain, is over here.

And then bound to the alpha carbon is the hydrogen and a carobxylic acid group. So if we come back over to our molecule thad over here, we can see that the nitrogen atom is going to serve as our amino group.

And then we have our alpha carbon here in the center, and then our carboxylic acid, here, is on the bottom. And then we have this temporary ester group at the top. So I'm going to highlight those key atoms for you here, the nitrogen and the alpha carbon and the carbonyl carbon.

And the reason why we started out with all these other groups attached to our key atoms is for various reasons. For example, our amide is prevented or, quote, "protected" from acting as a nucleophile by having this phthalimide group attached to it. And then the carboxylic acid is protected with this ethyl ester that's attached, and the [INAUDIBLE] carbon is further activated by this additional ester group at the top.

Now in the presence of a base and having a source of an alkyl group, our molecule of thad will become alkylated to look like this. So now you can see that the alkyl group here has been substituted onto the carbon atom, and so this is known as the alkylated step.

And then the next step involves acid hydrolysis, which yields this molecule. And as you can see here, the phthalimide group was hydrolyzed along with the two esters. And this is the hydrolyzed step. And then finally, we can add a little heat to decarboxylate this molecule or remove its carboxyl group up top here.

And we get our final alpha amino acid. OK, so this is Gabriel synthesis in a nut shell. So you start out with an n-phthalimidomalonic ester, and then you add up a base and a source of an alkyl group. And you get an alkylated amide malonic acid here, and then you hydrolyze this to get your carboxylic acid group as well as your amino group.