Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [¹³C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO₂, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.     

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.     

谷氨酸棒杆菌TL1105的L-组氨酸生物合成途径分析

目的：对谷氨酸棒杆菌TL1105由葡萄糖生物合成L-组氨酸的代谢途径进行分析,以确定L-组氨酸合成的最佳途径和最大理论产率。方法：运用METATOOL软件对谷氨酸棒杆菌TL1105合成L-组氨酸进行途径分析。结果：确定了L-组氨酸合成的最佳途径,并确定最大理论产率为1．2;通过比较途径分析所获得的基础反应模型,确定了5-磷酸核糖焦磷酸是L-组氨酸合成途径的关键节点,并且确定了谷氨酸的大量合成是L-组氨酸合成的重要前提;添加谷氨酸,L-组氨酸的产量提高了39．2％。结论：以途径分析为指导,改变外界环境因子,L-组氨酸的产量得到显著的提高。     

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.     

Effects of Carbon Dioxide on Metabolism by the Glycollate Pathway in Leaves

When solutions of [¹⁴C]glycollate, glycine, serine, glycerate,or glucose were supplied to segments of wheat leaves throughtheir cut bases in the light, most of the ¹⁴C was incorporatedinto sucrose in air but in CO₂-free air less sucrose was made.The synthesis of sucrose was decreased because metabolism ofserine was partly blocked. Sucrose synthesis from glucose andglycerate in CO₂-free air was decreased but to a smaller extent;relatively more CO₂ was evolved and serine accumulated. Theeffects of DCMU and light of different wavelengths on metabolismby leaves of L-[U-¹⁴C]serine confirmed that simultaneous photosyntheticassimilation of carbon was necessary for the conversion of serineto sucrose. Of various products of photosynthesis fed exogenouslyto the leaves -keto acids were the most effective in promotingphotosynthesis of sucrose and release of ¹⁴CO₂ from ¹⁴C-labelledserine. This suggests that in CO₂-free air the metabolism ofserine may be limited by a shortage of -keto acid acceptorsfor the amino group. In CO₂-free air added glucose stimulatedproduction of CO₂ and sucrose from D-[U-¹⁴C]- glycerate andno competitive effects were evident even though glucose is convertedrapidly to sucrose under these conditions. In addition to asupply of keto acid, photosynthesis may also provide substratesthat can be degraded and provide energy in the cytoplasm forthe conversion of glycerate to sugar and phosphates and sucrose.     

C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H¹³CO₃ ^–-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% ¹³C enrichment in C-4 and 3.5% ¹³C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C₃-carboxylation reaction that also takes place in the absence of PEPCx. Received: 27 December 1995 / Accepted: 20 March 1996     

Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.     

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.     

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II^D213G and thus by coupling to electron transport phosphorylation (ETP).     

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.     

Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum

The physiological behavior of Corynebacterium glutamicum in glucose-limited chemostat cultures was examined from both growth kinetics and enzymatic viewpoints. Metabolic fluxes within the central metabolism were calculated from growth kinetics and analyzed in relation to specific enzyme activities. At high growth rates, incomplete glucose removal was observed, and this was attributed to rate-limiting capacity of the phosphotransferase system transporter and the probable contribution of a low-affinity permease uptake mechanism. The improved biomass yield observed at high growth rates was related to a shift in the profile of anaplerotic carboxylation reactions, with pyruvate carboxylase replacing malic enzyme. Phosphoenolpyruvate carboxylase, an activity often assumed to be the major anaplerotic reaction during growth of C. glutamicum on glucose, was present at only low levels and is unlikely to contribute significantly to tricarboxylic acid cycle fuelling other than at low growth rates.     

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 RES167Δncg12918 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.     

Efflux permease CgAcr3-1 of Corynebacterium glutamicum is an arsenite-specific antiporter

Resistance to arsenite (As(III)) by cells is generally accomplished by arsenite efflux permeases from Acr3 or ArsB unrelated families. We analyzed the function of three Acr3 proteins from Corynebacterium glutamicum, CgAcr3-1, CgAcr3-2, and CgAcr3-3. CgAcr3-1 conferred the highest level of As(III) resistance and accumulation in vivo. CgAcr3-1 was also the most active when everted membranes vesicles from Escherichia coli or C. glutamicum mutants were assayed for efflux with different energy sources. As(III) and antimonite (Sb(III)) resistance and accumulation studies using E. coli or C. glutamicum arsenite permease mutants clearly show that CgAcr3-1 is specific for As(III). In everted membrane vesicles expressing CgAcr3-1, dissipation of either the membrane potential or the pH gradient of the proton motive force did not prevent As(III) uptake, whereas dissipation of both components eliminated uptake. Further, a mutagenesis study of CgAcr3-1 suggested that a conserved cysteine and glutamate are involved in active transport. Therefore, we propose that CgAcr3-1 is an antiporter that catalyzes arsenite-proton exchange with residues Cys129 and Glu305 involved in efflux.     

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD^Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.     

Analysis of cepA encoding an efflux pump-like protein in Corynebacterium glutamicum

A gene encoding a homolog of purine efflux proteins of Escherichia coli and Bacillus subtilis was identified in the genome of Corynebacterium glutamicum and designated as cepA. The gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. To elucidate the function of the gene, we constructed a cepA deletion mutant (ΔcepA) and a cepA-overexpressing strain and analyzed their physiological characteristics. The cepA gene could be deleted with no critical effect on cell growth. However, the cell yield of a ΔcepA strain was decreased by 10% as compared to that of a strain carrying a cepA-overexpression plasmid (P₁₈₀-cepA). Further analysis identified increased resistance of the P₁₈₀-cepA strain to the purine analogues 6-mercaptopurine and 6-mercaptoguanine, but not to 2-aminopurine and purine nucleoside analogues. Moreover, this strain showed increased resistance to the antibiotics nalidixic acid and ampicillin. Collectively, these data suggest that cepA is a novel multidrug resistance gene and probably functions in the efflux of toxic substances from the inside of cells to the environment, thus allowing cells to reach a higher cell yield.     

Respirometric 13C flux analysis--Part II: in vivo flux estimation of lysine-producing Corynebacterium glutamicum

A novel method for metabolic flux studies of central metabolism which is based on respirometric (13)C flux analysis, i.e., parallel (13)C tracer studies with online CO(2) labeling measurements is applied to flux quantification of a lysine-producing mutant of Corynebacterium glutamicum. For this purpose, 3 respirometric (13)C labeling experiments with [1-(13)C(1)], [6-(13)C(1)] and [1,6-(13)C(2)] glucose were carried out in parallel. All fluxes comprising the reactions of glycolysis, of TCA cycle, of C3- and C4-metabolite interconversion and of lysine biosynthesis as well as the net reactions in the pentose phosphate pathway could be quantified solely using experimental data obtained from CO(2) labeling and extracellular rate measurements. At key branch points, 68+/-5% of glucose 6-phosphate were observed to be metabolized into pentose phosphate pathway and 48+/-1% of pyruvate into TCA cycle via pyruvate dehydrogenase. The results showed a good agreement with the previous studies using (13)C tracer cultivation and GC/MS analysis of proteinogenic amino acids. Also, respiratory quotient calculated from flux estimates using redox balance showed a high accordance with the value determined directly from the measured specific rates of O(2) consumption and CO(2) production. The results strongly support that the respirometric (13)C metabolic flux analysis is suited as an alternative to the conventional methods to study functional and regulatory activities of cells. The developed method is applicable to study growing or non-growing cells, primary and secondary metabolism and immobilized cells. Due to the non-accumulating nature of CO(2) labeling and instantaneous nature of the resulting fluxes, the method can also be used for dynamic profiling of metabolic activities. Therefore, it is complementary to conventional methods for metabolic flux analysis.     

Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids

The hom-1-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. They showed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and of homoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy of hom-1-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of hom-1-thrB increased, increasing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threonine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.     

谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较

【背景】谷氨酸棒状杆菌的基因敲除系统较为匮乏且效率不高,难以对其进行代谢工程改造,不利于高性能工业菌株的构建及规模生产。【目的】分别采用CRISPR-Cpf1和Cre/loxP基因敲除系统对谷氨酸棒状杆菌ATCC13032(CorynebacteriumglutamicumATCC13032)基因组上的argR和argF基因进行敲除,比较两种敲除方法的优缺点,为合理选择敲除系统提供依据。【方法】特异性重组的Cre/loxP敲除系统是首先利用同源重组将基因组上的靶基因替换为两端带有重组位点loxP的kanR片段,然后由重组酶Cre识别loxP位点并发生重组反应,从而去除替换到基因组上的kanR片段,进一步利用质粒的温敏特性将其消除,从而实现靶基因的敲除。CRISPR-Cpf1敲除系统是利用Cpf1对pre-crRNA进行加工,形成的成熟crRNA引导Cpf1识别和结合到靶DNA的特定序列上并切割双链DNA分子,通过同源重组作用去除靶基因,基于质粒自身的温敏特性将其消除,从而完成基因敲除的整个过程。【结果】Cre/lox P系统可在8N+2 d内完成N轮迭代基因敲除,而CRISPR-Cpf1系统可在5N+2d内完成N轮迭代基因无痕敲除,理论上还可以一次对多个靶位点进行编辑,效率更高,但存在同源重组效率较低、假阳性率高等缺点。【结论】与Cre/loxP系统相比,CRISPR-Cpf1辅助的同源重组基因敲除方法可省时、省力地实现基因的无痕敲除,理论上还可实现多个基因的同时敲除、总体效率更高,然而编辑效率还有提高的空间。