Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were constructed and analyzed. The result was that unlike ilvA in vectors with low copy numbers, ilvA in high-copy-number vectors increased the final l-isoleucine yield by about 20%. An additional 40% increase in l-isoleucine yield was obtained by the use of ilvA alleles encoding feedback-resistant threonine dehydratases. The strain with the highest yield was characterized by three hom(Fbr) copies encoding feedback-resistant homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM l-isoleucine, without any l-threonine as a by-product. The highest specific productivity was 0.052 g of l-isoleucine per g of biomass per h. This comparative flux analysis of isogenic strains showed that high levels of l-isoleucine formation from glucose can be achieved by the appropriate balance of homoserine dehydrogenase and threonine dehydratase activities in a strain background with feedback-resistant aspartate kinase. However, still-unknown limitations are present within the entire reaction sequence.     

谷氨酸棒杆菌YILW苏氨酸脱水酶基因的克隆表达及酶学性质

将L-异亮氨酸生产菌谷氨酸棒杆菌(Corynebacterium glutamicum YILW)苏氨酸脱水酶(threonine de-hydratase,TD)的编码基因ilvA在大肠杆菌中进行异源表达及进行初步的酶学性质研究。分别以C.glutamicum ATCC13032、YILW的基因组DNA为模板,利用PCR技术扩增出苏氨酸脱水酶的编码基因ilvA,测序获得编码序列。利用质粒PET-His将该基因在大肠杆菌BL21(DE3)中进行重组表达、金属螯合纯化,对其酶学性质进行初步研究。结果显示C.glutamicum YILW编码基因序列与已报道的ilvA序列相差5个碱基,相似度为99.6%,第383位氨基酸由苯丙氨酸突变为缬氨酸。酶学性质研究表明:重组酶YilwTD最适反应温度为32℃,在20~55℃范围内该酶较稳定,最适pH为6.7,该酶底物专一性强,对最适底物苏氨酸的米氏常数Km=8.32 mmol/L,最大反应速度Vmax=3.18×104U/mg,与野生型酶相比,突变(F383V)后可显著降低终产物对酶的反馈抑制作用。为揭示突变对苏氨酸脱水酶活性的影响及进一步利用基因工程技术改造L-异亮氨酸生产菌,提高L-异亮氨酸产量奠定了基础。     

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

Increase in Isoleucine Accumulation by α-Aminobutyric Acid-Resistant Mutants of Serratia marcescens

Several alpha-aminobutyric acid-resistant (Abu-r) mutants of Serratia marcescens were found to be superior to the parent strain in converting d-threonine to l-isoleucine. One of them accumulated 1.5 times more l-isoleucine that the parent strain. The level of acetohydroxy acid (AHA) synthetase in this mutant increased twofold above that of the parent strain. In the parent strain, AHA synthetase was repressed and l-isoleucine accumulation was decreased by either l-valine or l-leucine, whereas in the mutant the AHA synthetase level and l-isoleucine accumulation were not affected by these amino acids. AHA synthetase of the Abu-r mutant was feedback-inhibited by l-valine to the same extent as that of the parent strain. The level of d-threonine dehydratase in both strains was only slightly affected by several amino acids tested. l-Threonine dehydratase of the parent strain and of the mutant was almost completely inhibited by l-isoleucine. These results indicate that the increase in l-isoleucine accumulation by Abu-r mutants is due to the genetic derepression of AHA synthetase.     

Inhibition of acetohydroxy acid synthase by leucine

The enzymatic reaction of acetohydroxy acid synthase in crude extracts of Escherichia coli K-12 is inhibited by leucine. Inhibition is most pronounced at low pH values and is low at pH values higher than 8.0. Both isoenzymes of acetohydroxy acid synthase present in E. coli K-12 (isoenzyme I and isoenzyme III) are inhibited by leucine. Isoenzyme I, which is responsible for the majority of acetohydroxy acid synthase activity in E. coli K-12 at physiological pH, is inhibited almost completely by 30 mM leucine at pH 6.25-7.0 and is not affected at all at pH values higher than 8.4. Inhibition of isoenzyme I by leucine is a mixed noncompetitive process. Leucine inhibition of isoenzyme III is pH-independent and reaches only 40% at 30 mM leucine. The inhibition of acetohydroxy acid synthase by leucine at physiological pH, observed in vitro in this study, correlates with the idea that acetohydroxy acid synthase is a target for the toxicity of the abnormally high concentrations of leucine in E. coli K-12.     

细菌苏氨酸脱水酶受L-异亮氨酸反馈抑制的关键在于其调控结构域

苏氨酸脱水酶是细菌L-异亮氨酸合成途径中的关键酶,酶活力受终产物L-异亮氨酸的反馈抑制。苏氨酸脱水酶包含催化结构域及调控结构域,其中调控结构域所起的作用有待进一步研究。本文在大肠杆菌中分别克隆表达了四种不同的苏氨酸脱水酶:来自谷氨酸棒杆菌的苏氨酸脱水酶CgIlvA及其不合调控结构域的突变体CgIlvA~M和来自大肠杆菌的苏氨酸脱水酶EcIlvA及其不合调控结构域的突变体EcIlvA~M。通过蛋白纯化和酶活分析发现,CgIlvA~M和EcIlvA~M的酶活力比CgIlvA和EcIlvA的酶活力有所降低,但它们不再受L-异亮氨酸的反馈抑制,说明L-异亮氨酸对苏氨酸脱水酶的反馈抑制是通过其调控结构域来实现的。     

Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards L-isoleucine in Corynebacterium glutamicum

 The synthesis of L-isoleucine by Corynebacterium glutamicum involves 11 reaction steps, with at least 5 of them regulated in activity or expression. Using gene replacement we constructed a vector-free C. glutamicum strain having feedback-resistant aspartate kinase and feedback-resistant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydratase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L-isoleucine was achieved, whereas with the parent strain only 4 mM L-isoleucine was obtained. Applying a closed-loop control fed-batch strategy to strain SM1 a final titre of 138 mM L-isoleucine was achieved with an integral molar yield of 0.11 mol/mol, and a maximal specific productivity of 0.28 mmol (g h)^-1. This shows that high L-isoleucine yields can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation strategy. In addition, the specific profiles of 2-oxoglutarate and pyruvate accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process. Received: 16 October 1995/Received revision: 21 December 1995/Accepted: 8 January 1996     

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.     

Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.     

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.     

Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate. When the resulting strain, C. glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.     

Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.

The pheA gene of Corynebacterium glutamicum encoding prephenate dehydratase was isolated from a gene bank constructed in C. glutamicum. The specific activity of prephenate dehydratase was increased six-fold in strains harboring the cloned gene. Genetic and structural evidence is presented which indicates that prephenate dehydratase and chorismate mutase were catalyzed by separate enzymes in this species. The C. glutamicum pheA gene, subcloned in both orientations with respect to the Escherichia coli vector pUC8, was able to complement an E. coli pheA auxotroph. The nucleotide sequence of the C. glutamicum pheA gene predicts a 315-residue protein product with a molecular weight of 33,740. The deduced protein product demonstrated sequence homology to the C-terminal two-thirds of the bifunctional E. coli enzyme chorismate mutase-P-prephenate dehydratase.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

谷氨酸棒杆菌NAD激酶的过表达对L-异亮氨酸合成的促进作用

NAD激酶催化辅酶Ⅰ[NAD(H)]发生磷酸化,转变成辅酶Ⅱ[NADP(H)],而还原态辅酶Ⅱ(NADPH)是L-异亮氨酸合成的必要辅因子。为了提高NADPH的供应,首先克隆了谷氨酸棒杆菌NAD激酶基因ppnK,并利用大肠杆菌-棒状杆菌诱导型穿梭表达载体pDXW-8和组成型穿梭表达载体pDXW-9在L-异亮氨酸合成菌——乳糖发酵短杆菌JHI3-156中进行表达。摇瓶发酵后,ppnK诱导表达菌JHI3-156/pDXW-8-ppnK的NAD激酶酶活(4.33±0.74 U/g)比pDXW-8空载菌提高了83.5%,辅酶Ⅱ与辅酶Ⅰ的比例提高了63.8%,L-异亮氨酸产量(3.86±0.12 g/L)提高了82.9%;ppnK组成表达菌JHI3-156/pDXW-9-ppnK的NAD激酶酶活(7.67±0.65 U/g)比pDXW-9空载菌提高了2.20倍,辅酶Ⅱ与辅酶Ⅰ的比例提高了1.34倍,NADPH含量提高了21.7%,L-异亮氨酸产量(2.99±0.18 g/L)提高了41.7%。这说明NAD激酶有助于辅酶Ⅱ的供应和L-异亮氨酸的生物合成,这对于其他氨基酸的生产也有一定的参考依据。