The identification of enzyme targets for the optimization of a valine producing Corynebacterium glutamicum strain using a kinetic model

The enzyme targets for the rational optimization of a Corynebacterium glutamicum strain constructed for valine production are identified by analyzing the control of flux in the valine/leucine pathway. The control analysis is based on measurements of the intracellular metabolite concentrations and on a kinetic model of the reactions in the investigated pathway. Data‐driven and model‐based methods are used and evaluated against each other. The approach taken gives a quantitative evaluation of the flux control and it is demonstrated how the understanding of flux control is used to reach specific recommendations for strain optimization. The flux control coefficients (FCCs) with respect to the valine excretion rate were calculated, and it was found that the control is distributed mainly between the acetohydroxyacid synthase enzyme (FCC = 0.32), the branched chain amino acid transaminase (FCC = 0.27), and the exporting translocase (FCC = 0.43). The availability of the precursor pyruvate has substantial influence on the valine flux, whereas the cometabolites are less important as demonstrated by the calculation of the respective response coefficients. The model is further used to make in‐silico predictions of the change in valine flux following a change in enzyme level. A doubling of the enzyme level of valine translocase will result in an increase in valine flux of 31%. By optimizing the enzyme levels with respect to valine flux it was found that the valine flux can be increased by a factor 2.5 when the optimal enzyme levels are implemented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Monitoring and modeling of the reaction dynamics in the valine/leucine synthesis pathway in Corynebacterium glutamicum

The intracellular concentrations of the valine and leucine pathway intermediates in a Corynebacterium glutamicum strain were measured during a transient state. The data were obtained by performing a glucose stimulus-response experiment with the use of a rapid sampling device and advanced mass spectrometry. The glucose stimulus resulted in a 3-fold increase in the intracellular pyruvate concentration within less than a second, demonstrating the very fast interactions in metabolic networks. The samples were taken at subsecond intervals for a time period of 25 s. The time courses of the metabolite concentrations formed the experimental basis of a mathematical model simulating the fluxes and concentrations in the valine/leucine pathway. The implementation of a model selection criterion based on the second law of thermodynamics is demonstrated to be essential for the identification of realistic and unique models. Large differences between the enzyme properties determined in vitro and those determined in vivo by the model were observed with the in vivo maximal rates being almost an order of magnitude larger than the in vitro maximal rates. The transamination of ketoisovalerate (KIV) to valine is carried out mainly by the transaminase B enzyme, with the transaminase C enzyme playing a minor role. The availability of the cofactors NADP and NADPH only has modest influence on the flux through the valine pathway, while the influence of NAD and NADH on the flux through the leucine pathway is negligible.     

Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step.

Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mutagenized cultures. Single proC mutants are auxotrophic for proline and secrete delta1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes or proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass.     

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route

Amino Acids - 2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing...     

Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates

Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2'R,6'R)-decaprenoxanthin di-(beta-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-epsilon,psi-carotene, and sarcinene, 2,2'-bis(3-methyl-2-butenyl)-epsilon,epsilon-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C(50)-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-psi,psi-carotene, and flavuxanthin, 2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1',16'-tetradehydro-1,2,1',2'-tetrahydro-psi,psi-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.     

Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Engineering of a xylose metabolic pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Metabolic engineering Corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway

The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l^?1 h^?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum.     

Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation

The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of L-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants. Of nine candidate ORFs (four of which were known previously), seven ORFs (cg0754 (metX), cg0755 (metY), cg1290 (metE), cg1702 (metH), cg2383 (metF), cg2536 (aecD), and cg2687 (metB)) were demonstrated to be part of the pathway while two others (cg0961 and cg3086) could be excluded. C. glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine. C. glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction. For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine beta-lyase in C. glutamicum, while Cg3086 can act neither as cystathionine gamma-synthase nor as cystathionine beta-lyase. Finally, MetE and MetH, which catalyze the conversion of L-homocysteine to L-methionine, could be newly identified, together with MetF which provides the necessary N(5)-methyltetrahydrofolate.     

代谢木糖重组谷氨酸棒杆菌的构建

为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

Identification of a gene involved in plasmid structural instability in Corynebacterium glutamicum

Expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. In order to enhance plasmid stability in recombinant Corynebacterium glutamicum, an expression plasmid containing genes of the Clostridium acetobutylicum butyryl-CoA synthesis operon with high structural instability within wild-type C. glutamicum was employed. From a total of 133 mutants exhibiting disruptions in 265 suspect genes, only cgR_0322-deficient mutant was able to maintain the expression plasmid intact. The mutant exhibited normal growth under standard laboratory conditions but its transformation efficiency was about one order of magnitude lower than that of wild-type strain. The cgR_0322 gene encodes an endonuclease that is active against single- as well as double-stranded DNA substrates in the presence of Mg²⁺. The cgR_0322-deficient strain should therefore facilitate the development of more robust C. glutamicum strains to be used as microbial production hosts.     

Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.     

Analysis of Corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metB encoding cystathionine gamma-synthase.

The metB gene encoding cystathionine y-synthase, the second enzyme of methionine biosynthetic pathway, was isolated from a pSL109-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli metB mutant. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 1161 bp which encodes a protein with the molecular weight of 41,655 comprising of 386 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. Introduction of a plasmid carrying the cloned metB into the C. glutamicum resulted in a 10-fold increase in cystathionine gamma-synthase activities, demonstrating the identity of the cloned gene. The C. glutamicum metB mutant which was generated by the site-specific integration of the cloned DNA into its chromosome did not lose the ability to grow on glucose minimal medium lacking supplemental methionine. The growth rate of the mutant strain was also comparable to that of the parental strain. These data indicate that, in addition to the transsulfuration pathway, other methionine biosynthetic pathways may be present in C. glutamicum.     

Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro

Superoxide dismutase (SOD) of Corynebacterium glutamicum was purified and characterized. The enzyme had a native molecular weight of about 80kDa, whereas a monomer with molecular weight of 24kDa was found on SDS-PAGE suggesting it to be homotetramer. The native SOD activity stained gel revealed a unique cytosolic enzyme. Supplementing growth media with manganese increased the specific activity significantly, while adding iron did not result in significant difference. No growth perturbation was observed with the supplemented media. In vitro metal removal and replacement studies revealed conservation of about 85% of the specific activity by substitution with manganese, while substitution with copper, iron, nickel or zinc did not restore any significant specific activity. Manganese was identified by atomic absorption spectrometer, while no signals corresponding to fixing other metallic elements were detected. Thus, C. glutamicum SOD could be considered a strict (non-cambialistic) manganese superoxide dismutase (MnSOD).     

Molecular analysis of the Corynebacterium glutamicum gene involved in lysine uptake

Two Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S-(2-aminoethyl)-cysteine (AEC). A 5.6 kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2 kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine-ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.     

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.