Systems Pathway Engineering Of L-arginine High Producing Corynebacterium Crenatum

L-arginine is a semi-essential amino acid that is widely involved in biological functions such as stimulating secretion of hormones, enhancing wound healing and nitric oxide formation, and has numerous applications in food, pharmaceutical and feed industries. In recent years, due to the increasing of L-arginine demand, its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum(subspecies of Corynebacterium glutamicum) SYPA5-5 through mutation breeding. In this work, we performed systems pathway engineering of C. crenatum SYPA5-5 for improved L-arginine production.(1) Optimization of L-arginine biosynthetic pathway from L-glutamate. The arginine operon was optimized for enhancing L-arginine biosynthesis from L-glutamate. First, the argBEc gene(encoding feedback inhibition-resistant N-acetylglutamate kinase(NAGK) from Escherichia coli) was integrated into the genome of C. crenatum SYPA5-5(CA0) at the argR(encoding ArgR, the repressor of arginine operon) locus, for argR deletion and expression of NAGK from E. coli. Then, the genes involved in L-arginine biosynthesis were overexpressed by replacing the native promoters Parg C and PargG of argCJBDFR and argGH operons with the promoter Peftu of eftu, encoding elongation factor Tu, and the strain CA1 was constructed. Fed-batch fermentation of the CA1 strain resulted in the production of 45.3 g/L L-arginine with a yield of 0.271 g/g. In comparison, fed-batch fermentation of the CA0 strain resulted in the production of 37.5 g/L L-arginine with a yield of 0.228 g/g.(2) Enhancing the precursor L-glutamate supply. The precursor L-glutamate supply was enhanced by regulating the enzyme activities of glutamate dehydrogenase(GDH), ?-ketoglutarate dehydrogenase complex(ODHC) and pyruvate carboxylase(PYC) in CA1 strain. First, the GDH was overexpressed by implementation of an additional copy of gdh gene in genome, for enhancing L-glutamate biosynthesis from ?-ketoglutarate. Next, in order to reduce the carbon flux consumption in TCA cycle, the ODHC activity was attenuated by optimization of odhA(encoding the E1 o subunit of the ODHC) ribosome binding site(RBS). Then, in order to alleviate the negative impacts of un-smooth TCA cycle on cell growth and L-arginine biosynthesis, the anaplerotic enzyme PYC was overexpressed by substitution of the pyc native start codon GTG by ATG, and the strain CA2 was constructed. Fed-batch fermentation of the CA2 strain resulted in the production of 51.6 g/L L-arginine with a yield of 0.318 g/g.(3) Increasing the NADPH supply and reducing the byproducts formation. The NADPH supply was increased and the byproducts formation was reduced by regulating the enzyme activities of phosphoglucoisomerase(PGI), aspartokinase(AK) and glutamate kinase(GK). First, the expression level of the pgi gene encoding the first glycolysis-specifc enzyme PGI was downregulated by RBS optimization for increasing the metabolic flux of pentose phosphate pathway, and enhancing the NADPH regeneration. Next, the AK activity was downregulated by RBS optimization for reducing the formation of L-lysine and L-isoleucine. Then, the proB gene encoding GK was deleted for blocking the L-proline biosynthesis, and the strain CA3 was constructed. Fed-batch fermentation of the CA3 strain resulted in the production of 58.1 g/L L-arginine with a yield of 0.371 g/g, and the levels of L-lysine and L-isoleucine were decreased signifcantly and the L-proline was undetectable.(4) The identification and function studies of NAD(P)H-dependent H2O2-forming flavin reductases. The frd1 and frd2 genes encoding putative NADPH-dependent FMN reductases were amplified via PCR from genomic DNA of C. crenatum SYPA5-5, according to the cg3223 and cg1150 genes of C. glutamicum ATCC13032. After DNA sequencing and analysis, the frd1 and frd2 genes were expressed in E. coli BL21, and the target proteins Frd181 and Frd188 were purified. The purified Frd181 and Frd188 were used for investigation of the catalytic properties and H2O2 formation, and the results showed that the Frd181 and Frd188 were NAD(P)H-dependent H2O2-forming flavin reductases. The frd1 and frd2 genes were overexpressed and deleted in CA3 strain, the results showed that deletion of frd1 and frd2 genes could reduce the H2O2 formation and useless NAD(P)H oxidation. Fed-batch fermentation of the frd1 and frd2 genes deletion strain CA4 resulted in the production of 64.2 g/L L-arginine, and the biomass was signifcantly increased compared to CA3 strain.(5) Increasing the ATP supply for enhancing L-arginine biosynthesis. First, the noxA gene encoding NADH oxidase was deleted in CA4 strain for reducing the non-energy generating NADH oxidation, and supply more NADH for oxidative phosphorylation. Next, the amn gene encoding AMP nucleosidase was deleted for enhancing ATP regeneration, and obtained CA5 strain. Then, the 3-phosphoglycerate kinase(PGK, encoded by pgk) and pyruvate kinase(PYK, encoded by pyk) were co-overexpressed in CA5 strain for enhancing the substrate-level phosphorylation, and obtained CA5(pyk-pgk) strain. Fed-batch fermentation of the CA5(pyk-pgk) strain resulted in the production of 71.8 g/L L-arginine.(6) Improvement of the ammonia assimilation for enhancing L-arginine biosynthesis. First, the effects of nitrogen atom donor(L-glutamate, L-glutamine and L-aspartate) addition on L-arginine production of CA5 strain was studied, and the addition of L-glutamine and L-aspartate was beneficial for L-arginine production. Next, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in CA5 strain for increasing the L-glutamine and L-aspartate synthesis, and the L-arginine production was effectively increased. In addition, the L-glutamate supply re-emerged as a limiting factor for L-arginine biosynthesis due to the increased L-glutamine and L-arginine synthesis. Then, the GDH gene gdh was co-overexpressed for further enhancement of L-arginine production, and obtained CA6 strain. Fed-batch fermentation of the CA6 strain resulted in the production of 76.8 g/L L-arginine, which was signifcantly higher than CA5 strain(69.5 g/L).