Improvement of oxytetraclyline biosynthesis in S.rimosus M4018 with zwf2 disruption

Abstract:

Genes of zwf1 and zwf2 encode two isozymes of glucose-6-phosphate dehydrogenase (G6PDH) of Streptomyces, respectively. G6PDH is the first enzyme in the oxidative pentose phosphate pathway (PPP) and the key enzyme for NADPH generation.Based on thermal sensitive plasmid pKC1139, a recombinant plasmid pKC1139-zwf2′was constructed and verified with restriction enzyme digestion. The plasmid pKC1139-zwf2′was electropolated into competent Streptmyces rimosus M4018 cells after it was demethylated by E.coli GM2929. Transformants grown on TSA plate containing 500ug/mL apramycin were selected, and identified using dot hybridization analysis and PCR amplification with apramycin resistant gene as primers.A positive clone was then selected and designated M4018-△zwf2. With parent strain S. rimosus M4018 as control, mutant M4018-△zwf2 was cultured in shaking flask. Specific acitivity of G6PDH of M4018-△zwf2 was only half of that of parent strain whereas yield of oxytetracycline (OTC) of mutant was 27% higher. to the mutant had a similar biomass profileto that of the control Oxytetracycline biosynthesis started when the growth entered stationary phase on the 4th day. However, specific oxytetracycline production of mutant was 31% higher thanthat of the parent strain, indicating that zwf2 disruption could enhance oxytetracycline biosynthesis in S. rimosus M4018-△zwf2.