Metabolic Flux Analysis And Genetic Engineering Of Pentose Phosphate Pathway Of Streptomyces Roseosporus

Daptomycin is a novel lipopeptide antibiotic which was produced by Streptomyces roseosporus and can resist to most gram-positive bacteria. Cubicin, the commercial name of daptomycin, had a sales of 538 million dollars in year 2009. The application of metabolic flux analysis of Streptomyces roseosporus to genetic engineering had not been reported. In this work, the metabolic network of Streptomyces roseosporus was constructed. A strategy was developed based on metabolic flux analysis and a genetic engineering strain was constructed with molecular biology technology. Then the effect on daptomycin production was also investigated. And the genetic engineering strain of Chinese intellectual property rights was obtained.An initial reaction list was built by blasting the open reading frame collected in the metabolic network of Streptomyces coelicolor A3(2) against the genome sequence of Streptomyces roseosporus NRRL 11379. With adding daptomycin synthesis reaction, biomass synthesis reaction, transport reaction and gap filling, the metabolic network of Streptomyces roseosporus was constructed. And the main nodes and metabolic pathways were recognized by topological analysis.Based on the metabolic network, metabolic flux analysis was conducted. The effect of dilution rate on the carbon flux through pentose phosphate pathway, Embden-Meyerhof-Parnas pathway and tricarboxylic acid cycle was investigated. The flux distribution around the nodes pyruvate andα-ketoglutarate was also studied. Through the simulation of improving the specific daptomycin production rate, it was found that the flux through pentose phosphate pathway pathway was enhanced most significantly in central metabolism. The assumption was made that the enzyme in pentose phosphate pathway should be overexpressed to generate more NADPH.By sequence blast, the 6-phosphgluconate dehydrogenase encoding gene zwf3 was found in the genome sequence of Streptomyces roseosporus. The primers were designed for PCR of zwf3 and the gene was cloned. The plasmid pTA2-zwf3 was constructed by TA cloning and the express vector pIB139-zwf3 was also constructed. The zwf3-overexpressed strain was obtained by conjugal transfer and antibiotics screening. In the medium containing glucose and inorganic nitrogen, the daptomycin yield increased from 365.5 mg/L to 411.6mg/L, which was increased by 12.6%. This demonstrated that the assumption was right and the metabolic flux analysis could successfully guide the genetic engineering to enhance the product yield.In 7.5 L fermentor the cell growth and daptomycin production were monitored under different agitation speeds. And a two-stage dissolved oxygen controlled strategy was developed. The daptomycin yield increased from 655.8 mg/L (best result at 300 rpm) to 758.2 mg/L, which was increased by 15.6%. The kinetic model was developed and could describe the fermentation process adequately.