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 α-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.     

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.     

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum.

Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.     

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.     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.     

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium hom ^dr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isoleucine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.     

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。     

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.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.     

Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum

Corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) as anaplerotic enzymes for growth on carbohydrates. To analyze the significance of PCx for the amino acid production by this organism, the wild-type pyc gene, encoding PCx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of C. glutamicum were tested in comparison to the respective host strains. No PCx activity was observed in the pyc-inactive mutants whereas the pyc-overexpressing strains showed eight-to elevenfold higher specific PCx activity when compared to the host strains. In a detergent-dependent glutamate production assay, the pyc-overexpressing strain showed more than sevenfold higher, the PCx-deficient strain about twofold lower glutamate production than the wild-type. Overexpression of the pyc gene and thus increasing the PCx activity in a lysine-producing strain of C. glutamicum resulted in approximately 50% higher lysine accumulation in the culture supernatant whereas inactivation of the pyc gene led to a decrease by 60%. In a threonine-producing strain of C. glutamicum, the overexpression of the pyc gene led to an only 10 to 20% increase in threonine production, however, to a more than 150% increase in the production of the threonine precursor homoserine. These results identify the anaplerotic PCx reaction as a major bottleneck for amino acid production by C. glutamicum and show that the enzyme is an important target for the molecular breeding of hyperproducing strains.     

Identification and Characterization of a Biodegradative Form of Threonine Dehydratase in Senescing Tomato (Lycopersicon esculentum) Leaf

Threonine dehydratase (TD; EC.4.2.1.16) is a key enzyme involved in the biosynthesis of isoleucine. Inhibition of TD by isoleucine regulates the flow of carbon to isoleucine. We have identified two different forms of TD in tomato (Lycopersicon esculentum) leaves. One form, present predominantly in younger leaves, is inhibited by isoleucine. The other form of TD, present primarily in older leaves, is insensitive to inhibition by isoleucine. Expression of the latter enzyme increases as the leaf ages and the highest enzyme activity is present in the old, chlorotic leaves. The specific activity of the enzyme present in older leaves is much higher than the one present in younger leaves. Both forms can use threonine and serine as substrates. Whereas TD from the older leaves had the same Km (0.25 mM) for both substrates, the enzyme from the young leaves preferred threonine (Km = 0.25 mM) over serine (Km = 1.7 mM). The molecular masses of TD from the young and the old leaves were 370,000 and 200,000 D, respectively. High levels of the isoleucine-insensitive form of threonine dehydratase in the older leaves suggests an important role of threonine dehydratase in nitrogen remobilization in senescing leaves.     

Threonine Synthetase-Catalyzed Conversion of Phosphohomoserine to α-Ketobutyrate in Bacillus subtilis

An enzyme activity of Bacillus subtilis has been found that catalyzes the dephosphorylation and deamination of phosphohomoserine to alpha-ketobutyrate, resulting in a bypass of threonine in isoleucine biosynthesis. In crude extracts of a strain deficient in the biosynthetic isoleucine-inhibitable threonine dehydratase, phosphohomoserine was converted to alpha-ketobutyrate. Phosphohomoserine conversion to alpha-ketobutyrate was shown not to involve a threonine intermediate. Single mutational events affecting threonine synthetase also affected the phosphohomoserine-deaminating activity, suggesting that the deamination of phosphohomoserine was catalyzed by the threonine synthetase enzyme. It was demonstrated in vivo, in a strain deficient in the biosynthetic threonine dehydratase, that isoleucine was synthesized from homoserine without intermediate formation of threonine.     

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。     

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     

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.     

Purification and properties of threonine deaminase from the X-1 isolate of the genus Thermus

Threonine deaminase (l-threonine dehydratase EC 4.2.1.16) has been partially purified from a new extreme thermophilic bacterium, Thermus X-1, which is similar to T. aquaticus YT-1. The threonine deaminase of strain X-1 has a maximal rate of reaction at 85 to 90 C and is more thermostable than the threonine deaminase from mesophilic bacteria. The enzyme has an apparent molecular weight of 100,000 to 115,000, a K(m) for l-threonine of 14 mM, a pH optimum of 8.0, and like other threonine deaminases also catalyzes the deamination of serine. However the Thermus X-1 threonine deaminase does not show a strong feedback inhibition by isoleucine. It is suggested that the regulation of the biosynthesis of isoleucine in this extreme theromophile may resemble that reported in Rodospirillum rubrum.     

Metabolic Engineering To Produce Tyrosine or Phenylalanine in a Tryptophan-Producing Corynebacterium glutamicum Strain

The aromatic amino acids are synthesized via a common biosynthetic pathway. A tryptophan-producing mutant of Corynebacterium glutamicum was genetically engineered to produce tyrosine or phenylalanine in abundance. To achieve this, three biosynthetic genes encoding the first enzyme in the common pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS), and the branch-point enzymes chorismate mutase and prephenate dehydratase were individually cloned from regulatory mutants of C. glutamicum which have either of the corresponding enzymes desensitized to end product inhibition. These cloned genes were assembled one after another onto a multicopy vector of C. glutamicum to yield two recombinant plasmids. One plasmid, designated pKY1, contains the DS and chorismate mutase genes, and the other, designated pKF1, contains all three biosynthetic genes. The enzymes specified by both plasmids were simultaneously overexpressed approximately sevenfold relative to the chromosomally encoded enzymes in a C. glutamicum strain. When transformed with pKY1 or pKF1, tryptophan-producing C. glutamicum KY10865, with the ability to produce 18 g of tryptophan per liter, was altered to produce a large amount of tyrosine (26 g/liter) or phenylalanine (28 g/liter), respectively, because the accelerated carbon flow through the common pathway was redirected to tyrosine or phenylalanine.