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.

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.

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

A 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.

Substitution of histone H3 arginine 72 to alanine leads to the deregulation of isoleucine biosynthesis in the budding yeast Saccharomyces cerevisiae.

Histone residues play an essential role in the regulation of various biological processes. In the present study, we utilized the H3/H4 histone mutant library to probe the functional aspects of histone residues in amino acid biosynthesis. We found that the histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of the arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in minimal medium. Histone mutant H3R72A requires external supplementation of either isoleucine, serine, or threonine for growth in minimal medium. We also observed that the H3R72 residue and leucine amino acid in synthetic complete medium might play a crucial role in determining the intake of isoleucine and threonine in yeast. Furthermore, gene deletion analysis of ILV1 and CHA1 in the H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in the budding yeast Saccharomyces cerevisiae.

Effect of sodium dodecyl sulfate on the production of L-isoleucine by the fermentation of Corynebacterium glutamicum.

Corynebacterium glutamicum is a safe and popular industrial microorganism that it is gram-positive bacteria with thick cell walls, which hinder the extracellular secretion of products. Surfactant has good surface or interface activity and can destroy the cell membrane of microorganisms. In this study, the surfactant SDS was used to artificially destroy the cell membrane of Corynebacterium glutamicum, increase the permeability of the cell membrane, and increase the ability of the strain to secrete L-isoleucine. This is the first time that surfactants have been applied to the fermentation of Corynebacterium glutamicum. Results indicated that after optimization, the output of L-isoleucine reached 43.67 g/L, which was 13.01% higher than that without sodium dodecyl sulfate. The yield of the by-products, such as valine, leucine, and alanine, was reduced by 72.30%, 64.30%, 71.70%, respectively. This method can promote the production of L-isoleucine while minimizing the damage of SDS to the strain.

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.

[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.

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, ß-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.

Increased productivity of L-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of L-threonine deaminase.

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae.

BACKGROUND: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. RESULTS: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. CONCLUSION: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

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 ß-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.

Biochemical and functional characterization of MRA_1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages.

Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).

Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae (baker's yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker's yeast. RESULTS: We first designed a two-step heterologous pathway, starting with the endogenous amino acid L-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional L-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional L-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol. CONCLUSIONS: Our results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology.

The dietary protein paradox and threonine 15 N-depletion: Pyridoxal-5'-phosphate enzyme activity as a mechanism for the δ15 N trophic level effect.

RATIONALE: Nitrogen stable isotope ratios (δ15 N values) are used to reconstruct dietary patterns, but the biochemical mechanism(s) responsible for the diet to tissue trophic level effect and its variability are not fully understood. Here δ15 N amino acid (AA) values and physiological measurements (nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates) are used to investigate increased dietary protein consumption and oxidative stress (vitamin E deficiency) in rat total plasma protein. METHODS: Using gas chromatography/combustion/isotope ratio mass spectrometry, the δ15 N values from N-pivaloyl-i-propyl esters of 15 AAs are reported for rats (n = 40) fed casein-based diets with: adequate protein (AP, 13.8%; n = 10), medium protein (MP, 25.7%; n = 10), high protein (HP, 51.3%; n = 10) or HP without vitamin E (HP-E; n = 10) for 18 weeks. RESULTS: Between the HP and AP groups, the δ15 NAA values of threonine (-4.0‰), serine (+1.4‰) and glycine (+1.2‰) display the largest differences and show significant correlations with: nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates. This indicates increased AA catabolism by the dietary induction of shared common metabolic pathways involving the enzymes threonine ammonia-lyase (EC 4.3.1.19), serine hydroxymethyltransferase (EC 2.1.2.1) and the glycine cleavage system (EC 2.1.2.10). The δ15 NAA values of the HP-E and HP groups were not found to be significantly different. CONCLUSIONS: The 15 N-depleted results of threonine are linked to increased activity of threonine ammonia-lyase, and show potential as a possible biomarker for protein intake and/or gluconeogenesis. We hypothesize that the inverse nitrogen equilibrium isotope effects of Schiff base formation, between AAs and pyridoxal-5'-phosphate cofactor enzymes, play a key role in the bioaccumulation and depletion of 15 N in the biomolecules of living organisms and contributes to the variability in the nitrogen trophic level effect. Copyright © 2017 John Wiley & Sons, Ltd.

Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly.

RATIONALE: The "Threonine Anomaly" relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in 15 N relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its 15 N depletion. Plots of delta 15 N against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in 15 N is clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright © 2016 John Wiley & Sons, Ltd.

MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress.

Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile.

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria.

An Unexpected Route to an Essential Cofactor: Escherichia coli Relies on Threonine for Thiamine Biosynthesis.

UNLABELLED: Metabolism consists of biochemical reactions that are combined to generate a robust metabolic network that can respond to perturbations and also adapt to changing environmental conditions. Escherichia coli and Salmonella enterica are closely related enterobacteria that share metabolic components, pathway structures, and regulatory strategies. The synthesis of thiamine in S. enterica has been used to define a node of the metabolic network by analyzing alternative inputs to thiamine synthesis from diverse metabolic pathways. To assess the conservation of metabolic networks in organisms with highly conserved components, metabolic contributions to thiamine synthesis in E. coli were investigated. Unexpectedly, we found that, unlike S. enterica, E. coli does not use the phosphoribosylpyrophosphate (PRPP) amidotransferase (PurF) as the primary enzyme for synthesis of phosphoribosylamine (PRA). In fact, our data showed that up to 50% of the PRA used by E. coli to make thiamine requires the activities of threonine dehydratase (IlvA) and anthranilate synthase component II (TrpD). Significantly, the IlvA- and TrpD-dependent pathway to PRA functions in S. enterica only in the absence of a functional reactive intermediate deaminase (RidA) enzyme, bringing into focus how these closely related bacteria have distinct metabolic networks. IMPORTANCE: In most bacteria, including Salmonella strains and Escherichia coli, synthesis of the pyrimidine moiety of the essential coenzyme, thiamine pyrophosphate (TPP), shares enzymes with the purine biosynthetic pathway. Phosphoribosylpyrophosphate amidotransferase, encoded by the purF gene, generates phosphoribosylamine (PRA) and is considered the first enzyme in the biosynthesis of purines and the pyrimidine moiety of TPP. We show here that, unlike Salmonella, E. coli synthesizes significant thiamine from PRA derived from threonine using enzymes from the isoleucine and tryptophan biosynthetic pathways. These data show that two closely related organisms can have distinct metabolic network structures despite having similar enzyme components, thus emphasizing caveats associated with predicting metabolic potential from genome content.

A recyclable biotransformation system for L-2-aminobutyric acid production based on immobilized enzyme technology.

OBJECTIVE: To make the previously developed biosynthesis of L-2-aminobutyric acid (L-ABA) more suitable for the industrial-scale production. RESULTS: A recyclable biotransformation system was developed based on immobilized enzyme technology. The conversion yield of L-threonine (at 90 g l(-1)) reached 99.9 % and the theoretical yield of L-ABA reached more than 90 % using the optimized biotransformation system by the individual immobilization of threonine deaminase and the co-immobilization of L leucine dehydrogenase and formate dehydrogenase. 90 g L-threonine l(-1) was converted to 73.9 g L-ABA l(-1) >95 % theoretical yield, within 120-145 min in 30 batch transformation experiments. CONCLUSION: The recyclable biotransformation system is promising to fulfill industrial requirements for L-ABA production.

Generation of mutant threonine dehydratase and its effects on isoleucine synthesis in Corynebacterium glutamicum.

Isoleucine synthesis is strongly regulated by its end product (isoleucine) in Corynebacterium glutamicum, especially at threonine dehydratase (TD) node. Multiple alignments of TD sequences of C. glutamicum and other sources were performed. According to the structural analysis, three TD variants were constructed by site-directed mutagenesis. These TD variants improved the performance of the holoenzyme. The specific activity of V140M variant was 1.5-fold higher than that of the wild-type TD, whereas F383A variant showed complete resistance to feedback inhibition by isoleucine. V140M-F383A variant had all the advantages of V140M and F383A variants and displayed 1.5-fold specific activity and complete resistance to isoleucine. In C. glutamicum, overexpression of V140M, F383A, and V140M-F383A variants accumulated 0.55, 0.63, and 0.73 g/l isoleucine, and overexpression of wild-type TD produced 0.47 g/l isoleucine. Thus, these novel TD variants, particularly V140M-F383A, showed great potential in isoleucine synthesis.