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Xu J.,Jiangnan University | Zhang J.,OriGene Biotechnology Co. | Guo Y.,Jiangnan University | Zhang W.,Jiangnan University
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

The aspartate aminotransferase (AAT) and aspartate ammonia-lyase (AAL) catalyzes respectively the reversible reaction of oxaloacetate (OAA) and fumarate to form l-aspartate. However, the effects of AAT and AAL on metabolite variations have not been clearly elucidated as yet. Now, the effects of AAT and AAL on metabolite variations in genetically defined l-lysine producing strains were studied by genetically modifying AAT gene aspB and AAL gene aspA. AAL was not detected in Corynebacterium glutamicum Lys1 but increased in aspB-deleted strain. Inversely, AAT exhibited high activity in Lys1, but it was not detected in aspB-deleted strains. Moreover, the deletion of aspB was bad for cell growth and metabolites production. The expression of Escherichia coli aspA in aspB-deleted strain not only restored cell growth and l-lysine production, but also accumulated some metabolites. However, the over-expression of aspB or aspA in aspB-natural strain did not affect cell growth and metabolites production except l-aspartate production. Although E. coli AAL could used to restore cell growth and metabolites accumulation in aspB-deleted strain, the effect on l-lysine production was significantly worse than that of AAT. Results indicates that native AAT is both necessary and sufficient for cell growth and l-lysine production, and deepens our understanding of the role of native aspB and E. coli aspA on cell growth and metabolites productions. © 2015 Elsevier B.V. Source


Xu J.,Jiangnan University | Zhang J.,OriGene Biotechnology Co. | Guo Y.,Jiangnan University | Zai Y.,Jiangnan University | Zhang W.,Jiangnan University
Journal of Industrial Microbiology and Biotechnology | Year: 2013

Fructose-1,6-bisphosphatase (FBPase) and fructokinase (ScrK) have important roles in regenerating glucose-6-phosphate in the pentose phosphate pathway (PPP), and thus increasing L-lysine production. This article focuses on the development of L-lysine high-producing strains by heterologous expression of FBPase gene fbp and ScrK gene scrK in C. glutamicum lysCfbr with molasses as the sole carbon source. Heterologous expression of fbp and scrK lead to a decrease of residual sugar in fermentation broth, and heterologous expression of scrK prevents the fructose efflux. Heterologous expression of fbp and scrK not only increases significantly the activity of corresponding enzymes but also improves cell growth during growth on molasses. FBPase activities are increased tenfold by heterologous expression of fbp, whereas the FBPase activity is only increase fourfold during coexpression of scrK and fbp. Compared with glucose, the DCW of heterologous expression strains are higher on molasses except co-expression of fbp and scrK strain. In addition, heterologous expression of fbp and scrK can strongly increase the L-lysine production with molasses as the sole carbon source. The highest increase (88.4 %) was observed for C. glutamicum lysCfbr pDXW-8-fbp-scrK, but the increase was also significant for C. glutamicum lysCfbr pDXW-8-fbp (47.2 %) and C. glutamicum lysCfbr pDXW- 8-scrK (36.8 %). By-products, such as glycerol and dihydroxyacetone, are decreased by heterologous expression of fbp and scrK, whereas trehalose is only slightly increased. The strategy for enhancing L-lysine production by regeneration of glucose-6-phosphate in PPP may provide a reference to enhance the production of other amino acids during growth on molasses or starch. Source


Xu J.,Jiangnan University | Xia X.,Wuxi Institute of Commerce | Zhang J.,OriGene Biotechnology Co. | Guo Y.,Jiangnan University | And 2 more authors.
Plasmid | Year: 2014

A method for the simultaneous replacement of a given gene by a target gene, leaving no genetic markers, has been developed. The method is based on insertional inactivation and double-crossover homologous recombination. With this method, the lysCT311I, fbp and ddh genes were inserted into Corynebacterium glutamicum genome, and the pck, alaT and avtA genes were deleted. Mobilizable plasmids with lysCT311I, fbp and ddh cassettes and two homologous arms on the ends of pck, alaT and avtA were constructed, and then transformed into C. glutamicum. The target-expression cassettes were inserted in the genome via the first homologous recombination, and the genetic markers were removed via the second recombination. The target-transformants were sequentially screened from kanamycin-resistance and sucrose-resistance plates. The enzyme activities of transformants were stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated cassettes in host were successfully expressed and maintained as stable chromosomal insertions in C. glutamicum. The target-transformants were used to optimize the l-lysine production, showing that the productions were strongly increased because the selected genes were closely linked to l-lysine production. In short, this method can be used to construct amino acid high-producing strains with unmarked gene amplification and simultaneous deletion in genome. © 2014 Elsevier Inc. Source


Xu J.,Jiangnan University | Han M.,Jiangnan University | Han M.,State Key Laboratory of Dairy Biotechnology | Zhang J.,OriGene Biotechnology Co. | And 3 more authors.
Journal of Chemical Technology and Biotechnology | Year: 2014

BACKGROUND: Corynebacterium glutamicum was engineered for improvement of L-lysine production and minimization of by-products synthesis by genetically engineering. RESULTS: The most promising recombinant strain C. glutamicum Lys9 produced 62.1 mmol L-1 L-lysine with substrate-specific yield (YP/S) of 0.28 mol per mol of glucose in shake flasks fermentation, whereas parental strain showed more than four times lower L-lysine production and more than 10 times lower biomass-specific yield (YP/X) than that of C. glutamicum Lys9. L-lysine production and cell growth were drastically decreased by isocitrate dehydrogenase (ICD) attenuation in aceE deletion strains, indicating that down-regulation of ICD activity in aceE deletion strains adversely affects L-lysine production. In fed-batch fermentation, C. glutamicum Lys9 produced 526 mmol L-1 L-lysine, i.e. 96.8 g L-1 L-lysine-HCl with high yield of 0.422 mol per mol of glucose and productivity of 2.69 g L-1 h-1. Corynebacterium glutamicum Lys9 was devoid of any detectable L-alanine and L-lactate synthesis. CONCLUSION: Superior to classical producers to some degree, C. glutamicum Lys9 is more adaptable for industrial L-lysine production. In addition to L-lysine, pyruvate, oxaloacetate (OAA) and L-valine were produced by C. glutamicum Lys9, suggesting further optimization to improve L-lysine production by engineering the L-lysine and/or NADPH biosynthetic pathway. © 2013 Society of Chemical Industry. Source


Xu J.,Jiangnan University | Zhang J.,OriGene Biotechnology Co. | Han M.,Jiangnan University | Han M.,State Key Laboratory of Dairy Biotechnology | Zhang W.,Jiangnan University
Journal of Industrial Microbiology and Biotechnology | Year: 2016

The gene integration method is an important tool to stably express desirable genes in bacteria. To avoid heavy workload and cost, we constructed a rapid and efficient method for genome modification. This method depended on a mobilizable plasmid, which contains a Ptac promoter, an introduced multiple cloning site (iMCS), and rrnBT1T2 terminator. Briefly, the mobilizable plasmid pK18-MBPMT with the Ptac-iMCS-rrnBT1T2 cartridge derived from pK18mobsacB was prepared to directly integrate hetero-/homologous DNA into the Corynebacterium glutamicum genome. Like our previous method, this method was based on insertional inactivation and double-crossover homologous recombination, which simultaneously achieved gene overexpression and inactivation in the genome without the use of genetic markers. Compared to the previous method, this protocol omitted the construction of a recombinant expression plasmid and clone of the target gene(s) cassette, which significantly decreased the workload, cost, and operational time. Using this method, the heterologous gene amy and the homologous gene lysCT311I were successfully integrated into the C. glutamicum genome at alaT and avtA loci, respectively. Moreover, the operation time of this method was shorter than that of the previous method, especially for repeated integration. This method, which is based on the mobilizable plasmid pK18-MBPMT, thus represents a potentially attractive protocol for the integration of genes in the course of genetic modification of C. glutamicum. © 2016 Society for Industrial Microbiology and Biotechnology Source

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