Threonine Deaminase Research Articles

Targeting threonine deaminase with chiral Au NPs: A novel strategy for E. coli inhibition

Bacterial infections are becoming a significant threat to global human health due to the growing prevalence of biofilm-related infections and the rise in antibiotic resistance. D/l-cysteine functionalized chiral gold nanoparticles (D/P-Au NPs or L/P-Au NPs) have demonstrated a potent antibacterial effect against E. coli, while the mechanism remains to be elucidated through additional research. Threonine deaminase (TD) is a crucial enzyme involved in branched-chain amino acid (BCAA) biosynthesis in E. coli and is involved in cysteine's antimicrobial effects. This study investigated the interaction between chiral Au NPs (D/P-Au NPs or L/P-Au NPs) and TD as well as its effect on enzyme activity. It demonstrates that chiral Au NPs interact with TD through hydrophobic forces, forming a ground state complex that induces changes in the secondary structure of TD and reduces enzyme activity in a concentration-dependent manner. We found that the exogenous supplementation of isoleucine and valine (2 mg/mL) significantly reduced the antibacterial activity of chiral Au NPs, especially for L/P-Au NPs. The proteomics results indicate that the expression of ilvA and ilvB was down-regulated after L/P-Au NPs treatment, which would interfere with the synthesis of BCAAs. These results demonstrate that chiral Au NPs cause cell death of E. coli partly due to inhibition of TD enzyme activity and the synthesis of branched-chain amino acids.

Structure and function of the pyridoxal 5′-phosphate-dependent (PLP) threonine deaminase IlvA1 from Pseudomonas aeruginosa PAO1

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Prolonging genetic circuit stability through adaptive evolution of overlapping genes.

The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

An unexpected role for tomato threonine deaminase 2 in host defense against bacterial infection.

The hormones salicylic acid (SA) and jasmonic acid (JA) often act antagonistically in controlling plant defense pathways in response to hemibiotrophs/biotrophs (hemi/biotroph) and herbivores/necrotrophs, respectively. Threonine deaminase (TD) converts threonine to α-ketobutyrate and ammonia as the committed step in isoleucine (Ile) biosynthesis and contributes to JA responses by producing the Ile needed to make the bioactive JA-Ile conjugate. Tomato (Solanum lycopersicum) plants have two TD genes: TD1 and TD2. A defensive role for TD2 against herbivores has been characterized in relation to JA-Ile production. However, it remains unknown whether TD2 is also involved in host defense against bacterial hemi/biotrophic and necrotrophic pathogens. Here, we show that in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin flg22 peptide, an activator of SA-based defense responses, TD2 activity is compromised, possibly through carboxy-terminal cleavage. TD2 knockdown (KD) plants showed increased resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae but were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, suggesting TD2 plays opposite roles in response to hemibiotrophic and necrotrophic pathogens. This TD2 KD plant differential response to different pathogens is consistent with SA- and JA-regulated defense gene expression. flg22-treated TD2 KD plants showed high expression levels of SA-responsive genes, whereas TD2 KD plants treated with the fungal PAMP chitin showed low expression levels of JA-responsive genes. This study indicates TD2 acts negatively in defense against hemibiotrophs and positively against necrotrophs and provides insight into a new TD2 function in the elaborate crosstalk between SA and JA signaling induced by pathogen infection.

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

Simultaneous Preparation of (S)-2-Aminobutane and d-Alanine or d-Homoalanine via Biocatalytic Transamination at High Substrate Concentration

(S)-2-Aminobutane, d-alanine, and d-homoalanine are important intermediates for the production of various active pharmaceutical ingredients and food additives. The preparation of these small chiral amine or amino acids with high water solubility still demands searching for efficient methods. In this work, we identified an ω-transaminase (ω-TA) from Sinirhodobacter hungdaonensis (ShdTA) that catalyzed the kinetic resolution of racemic 2-aminobutane at a concentration of 800 mM using pyruvate as the amino acceptor, leading to the simultaneous isolation of enantiopure (S)-2-aminobutane and d-alanine in 46% and 90% yield, respectively. In addition, (S)-2-aminobutane (98% ee) and d-homoalanine (99% ee) were isolated in 45% and 93% yield, respectively, in the kinetic resolution of racemic 2-aminobutane at a concentration of 400 mM coupled with deamination of l-threonine by threonine deaminase. We thus developed a biocatalytic process for the practical synthesis of these valuable small chiral amine and d-amino acids.

High-Level Production of Isoleucine and Fusel Alcohol by Expression of the Feedback Inhibition-Insensitive Threonine Deaminase in Saccharomyces cerevisiae.

ABSTRACTA variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols.IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Solar-Driven Producing of Value-Added Chemicals with Organic Semiconductor-Bacteria Biohybrid System.

Photosynthetic biohybrid systems exhibit promising performance in biosynthesis; however, these systems can only produce a single metabolite and cannot further transform carbon sources into highly valuable chemical production. Herein, a photosynthetic biohybrid system integrating biological and chemical cascade synthesis was developed for solar-driven conversion of glucose to value-added chemicals. A new ternary cooperative biohybrid system, namely bacterial factory, was constructed by self-assembling of enzyme-modified light-harvesting donor-acceptor conjugated polymer nanoparticles (D-A CPNs) and genetically engineered Escherichia coli (E. coli). The D-A CPNs coating on E. coli could effectively generate electrons under light irradiation, which were transferred into E. coli to promote the 37% increment of threonine production by increasing the ratio of nicotinamide adenine dinucleotide phosphate (NADPH). Subsequently, the metabolized threonine was catalyzed by threonine deaminase covalently linking with D-A CPNs to obtain 2-oxobutyrate, which is an important precursor of drugs and chemicals. The 2-oxobutyrate yield under light irradiation is increased by 58% in comparison to that in dark. This work provides a new organic semiconductor-microorganism photosynthetic biohybrid system for biological and chemical cascade synthesis of highly valuable chemicals by taking advantage of renewable carbon sources and solar energy.

Magnesium oxide induces immunity against Fusarium wilt by triggering the jasmonic acid signaling pathway in tomato

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a worldwide tomato disease. Although Fusarium wilt management remains unsuccessful, enhancing host FOL resistance using magnesium oxide to activate plant immunity may enable effective control. We demonstrated that MgO-pretreatment of roots induced FOL resistance in susceptible tomato plants. Resistance was not induced in tomato mutants deficient in the jasmonic acid (JA) signaling pathway, whereas the opposite trend was observed in mutants deficient in the salicylic acid and ethylene signaling pathways, suggesting that JA signaling activation is essential for MgO-induced FOL immunity. Quantitative real-time polymerase chain reaction analysis of MgO-pretreated tomato plants, and challenge-inoculated with FOL, revealed that MYELOCYTOMATOSIS ONCOGENE HOMOLOG 2 (MYC2), the master regulator of JA signaling, as well as MYC2-targeted transcription factors that directly regulate the JA-induced transcription of late defense genes and their downstream wound-responsive genes were preferentially upregulated in both roots and stems. Moreover, in MgO-pretreated tomato plants challenge-inoculated with FOL, the late wound-responsive THREONINE DEAMINASE 2 (TD) gene was expressed earlier than its upstream genes, including MYC2, suggesting that a primed state for defense was established in MgO-pretreated plants. We conclude that MgO is a promising agent for the control of Fusarium wilt.

Underground isoleucine biosynthesis pathways in E. coli.

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

Threonine deaminase (TD, EC 4.3.1.19) mediates α,β-elimination (deamination), which has untapped potential in the synthesis of unnatural α-keto acids. However, the narrow substrate scope of wild-ty...

Production of L-2-aminobutyric acid from L-threonine using a trienzyme cascade

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Defluorination of 4-fluorothreonine by threonine deaminase.

4-Fluorothreonine (4-FT) is the only naturally occurring fluorinated amino acid antibiotic. Although two conserved proteins in the 4-FT pathway have been found to be involved in self-detoxification mechanisms, the 4-FT-producing strains may also require an alternative pathway to degrade the intracellular 4-FT. In this study, we examined the possible degradation role of three enzymes involved in threonine metabolite pathways toward 4-FT as a possible degradation route to avoid in vivo 4-FT accumulation. Among these three enzymes, threonine deaminase was found to catalyse a defluorination reaction to generate 4-hydroxy-α-ketobutyrate, which is supposed to be further metabolised by an aldolase that likely is a unique occurrence in the 4-FT-producing strains. Our finding may constitute a 4-FT degradation pathway as a complementary resistance mechanism.

Trichoderma atroviride P1 Colonization of Tomato Plants Enhances Both Direct and Indirect Defense Barriers Against Insects.

Numerous microbial root symbionts are known to induce different levels of enhanced plant protection against a variety of pathogens. However, more recent studies have demonstrated that beneficial microbes are able to induce plant systemic resistance that confers some degree of protection against insects. Here, we report how treatments with the fungal biocontrol agent Trichoderma atroviride strain P1 in tomato plants induce responses that affect pest insects with different feeding habits: the noctuid moth Spodoptera littoralis (Boisduval) and the aphid Macrosiphum euphorbiae (Thomas). We observed that the tomato plant–Trichoderma P1 interaction had a negative impact on the development of moth larvae and on aphid longevity. These effects were attributed to a plant response induced by Trichoderma that was associated with transcriptional changes of a wide array of defense-related genes. While the impact on aphids could be related to the up-regulation of genes involved in the oxidative burst reaction, which occur early in the defense reaction, the negative performance of moth larvae was associated with the enhanced expression of genes encoding for protective enzymes (i.e., Proteinase inhibitor I (PI), Threonine deaminase, Leucine aminopeptidase A1, Arginase 2, and Polyphenol oxidase) that are activated downstream in the defense cascade. In addition, Trichoderma P1 produced alterations in plant metabolic pathways leading to the production and release of volatile organic compounds (VOCs) that are involved in the attraction of the aphid parasitoid Aphidius ervi, thus reinforcing the indirect plant defense barriers. Our findings, along with the evidence available in the literature, indicate that the outcome of the tripartite interaction among plant, Trichoderma, and pests is highly specific and only a comprehensive approach, integrating both insect phenotypic changes and plant transcriptomic alterations, can allow a reliable prediction of its potential for plant protection.

The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript

ABSTRACT The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (σB) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the σB regulon in facilitating this transition are understood the role of σB-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a σB-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in a deletion mutant lacking the rli47 gene. The Δrli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking σB. The impact of the Δrli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

Metabolic engineering of Escherichia coli for d-pantothenic acid production

Escherichia coli was engineered to produce d-pantothenic acid via systematic metabolic engineering. Firstly, genes of acetohydroxy acid synthase II, pantothenate synthetase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, 2-dehydropantoate 2-reductase and ketol-acid reductoisomerase were edited in E. coli W3110 with a resulting d-pantothenic acid yield of 0.49 g/L. Expressions of valine-pyruvate aminotransferase and branched-chain-amino-acid aminotransferase were then attenuated to decrease the carbon flux in l-valine biosynthetic pathway which is a competing pathway to the d-pantothenic acid biosynthetic pathway, and the yield increased to 1.48 g/L. Mutagenesis of pantothenate kinase and deletion of threonine deaminase further increased the production to 1.78 g/L. Overexpressions of panC and panB from Corynebacterium glutamicum enhanced the production by 29%. In fed-batch fermentations, strain DPA-9/pTrc99a-panBC(C.G) exhibited a highest d-pantothenic acid yield of 28.45 g/L. The findings in this study demonstrate the systematic metabolic engineering in Escherichia coli W3110 would be a promising strategy for industrial production of d-pantothenic acid.

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase.

The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, β-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs – but not SDHs – are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu fermentation.

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded β-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.

A Regulatory Hierarchy of the Arabidopsis Branched-Chain Amino Acid Metabolic Network.

The branched-chain amino acids (BCAAs) Ile, Val, and Leu are essential nutrients that humans and other animals obtain from plants. However, total and relative amounts of plant BCAAs rarely match animal nutritional needs, and improvement requires a better understanding of the mechanistic basis for BCAA homeostasis. We present an in vivo regulatory model of BCAA homeostasis derived from analysis of feedback-resistant Arabidopsis thaliana mutants for the three allosteric committed enzymes in the biosynthetic network: threonine deaminase (also named l-O-methylthreonine resistant 1 [OMR1]), acetohydroxyacid synthase small subunit 2 (AHASS2), and isopropylmalate synthase 1 (IPMS1). In this model, OMR1 exerts primary control on Ile accumulation and functions independently of AHAS and IPMS AHAS and IPMS regulate Val and Leu homeostasis, where AHAS affects total Val+Leu and IPMS controls partitioning between these amino acids. In addition, analysis of feedback-resistant and loss-of-function single and double mutants revealed that each AHAS and IPMS isoenzyme contributes to homeostasis rather than being functionally redundant. The characterized feedback resistance mutations caused increased free BCAA levels in both seedlings and seeds. These results add to our understanding of the basis of in vivo BCAA homeostasis and inform approaches to improve the amount and balance of these essential nutrients in crops.