The Comparative Genomics Analysis Of The L-serine-producing Corynebacterium Glutamicum Strains

As a nonessential amino acid, L-serine has been widely used in the pharmaceuticals, cosmetics and food industries. Corynebacterium glutamicum, as a food-safe bacterium, has been widely used to produce several animo acids. Previously, we isolated an L-serine-producing wild-type strain C. glutamicum SYPS-062. Then random mutagenesis was applied on it, resulting in the strain C. glutamicum SYPS-062-33 a. The strain showed a higher accumulation of L-serine, L-alanine and L-valine, and showed a higher growth and sugar consumption rate. The general genomic characterizations of SYPS-062 and the relationship of the genetic variations and phenotype between SYPS-062 and SYPS-062-33 a could provide theoretical guidance for the genetic modifications of C. glutamicum. In this study, the genome sequences of SYPS-062 and SYPS-062-33 a were obtained by the next generation-sequencing. The general genomic characterization of SYPS-062 was analyzed. The influences of the variant genes between SYPS-062 and SYPS-062-33 a to the phenotype of the strain were investigated. The main results were described as follows:(1) The genome sequences of SYPS-062 and SYPS-062-33 a were obtained by Illumina next generation-sequencing with the Genbank accession number of JXBH00000000 and JYEC00000000. The phylogenetic analysis of SYPS-062 and other Corynebacterium glutamicum strains showed that SYPS-062 was the most closely related to C. glutamicum ATCC14067. The COG classification analysis, core and special genes analysis, SNP and InDel analysis of C. glutamicum SYPS-062, ATCC14067 and ATCC13032 showed that the genome of SYPS-062 was more similar to ATCC14067, which analyzed the general characterization of the SYPS-062 genome.(2) Comparative genomics analysis was performed between SYPS-062 and SYPS-062-33 a by bioinformatics tools, and a total of 12 genes were found out to have amino acid mutation. Among the 12 genes, the three genes which were annotated as transporter proteins were deleted separately in the genome of SYPS-062-33 a. The two genes, which were related to the pyruvate metabolism: dxs(encoding the first enzyme in the MEP pathway) and aceE(encoding the E1 enzyme of PDHC), were reversely mutated in the genome of SYPS-062-33 a. We did not observe any phenotype variations of the strain through the deletions of the three transporters or dxs reverse mutation. The aceE reverse mutation strain C. glutamicum SYPS-062-33a(aceE594) showed a significant reduction in L-alanine and L-valine accumulation of 82.5% and 75.6% respectively, compared to SYPS-062-33 a, while its L-serine production, the growth and sugar consumption rate showed no obvious variations. The result showed that the aceE(His-594â†'Tyr) mutation was the reason for the higher L-alanine and L-valine accumulation of SYPS-062-33 a compared to SYPS-062.(3) The metabolic flux analysis and PDHC activity determinations were performed on SYPS-062-33 a and SYPS-062-33a(aceE594). In SYPS-062-33a(aceE594), the carbon flowed into the L-alanine and L-valine metabolic pathways form the pyruvate node were 39.9% and 7.9% less than that in SYPS-062-33 a, and the carbon flowed into the TCA cycle was 69.5% more than that in SYPS-062-33 a. The PDHC activity was 17.86 mU·mg-1 in SYPS-062-33a(aceE594), which was 28.3% higher than that in SYPS-062-33a(13.92 mU·mg-1). The results showed that the aceE(His-594â†'Tyr) mutation changed the carbon flux distribution at the pyruvate node which enabled SYPS-062-33 a to produce more L-alanine and L-valine.(4) The two strains C. glutamicum 33aΔSS(with 591 bp of the C-terminal domain of PGDH and L-serDH deletions in the genome of SYPS-062-33a) and C. glutamicum 33aΔSSAAI(with Ava A and AlaT deletions to block the L-alanine synthesis pathway and AHAS attenuation to attenuate the L-valine synthesis pathway in the genome of 33aΔSS) which were different in their phenotypes were selected to analyze the influence of the aceE mutation to the phenotype of the strain. After aceE was reversely mutated in the genome of 33aΔSS, the strain 33aΔSS(aceE594) showed a reduction in L-alanine and L-valine accumulation of 78.2% and 72.6% respectively, compared to 33aΔSS, while its L-serine production, the growth and sugar consumption rate showed no obvious variations. After aceE was reversely mutated in the genome of 33aΔSSAAI, the strain C. glutamicum 33aΔSSAAI(aceE594) produce no L-alanine and L-valine, and showed severe growth retardation. When 1 mM L-alanine was added to the medium, its growth rate was restored obviously. Those results further comfirmed that the aceE(His-594â†'Tyr) mutation could influence the accumulation of L-alanine and L-valine of the strain significantly.