Enhancement Of Oxytetracycline Production In Industrial Streptomyces Rimosus By G6PDH Gene Disruption Or Increased Copy Of Self-resistance Gene

The aromatic polyketide antibiotic oxytetracycline (OTC) produced by Streptomyces rimosus is one of important secondary polyketide metabolites.Currently, regulatory gene manipulation has been developed one of effective ways to improve antibiotic biosynthesis. Taken industrial Streptomyces rimosus(SRI) strain as an example, in this thesis the investigation of gene disruption of a key enzyme in primary metabolic pathways and enhancement of the cell's self-resistant genes were carried out to improve the OTC productivity.Based on the facts that perturbation in primary metabolic pathways can affect both the secondary metabolism and physiological characterization, and that zwf2 gene knock-out in a type strain M4018 enhanced OTC production in our previous work, zwfl encoding Glucose-6-phosphate dehydrogenase(G6PDH) was disrupted to explore its effects on oxytetracycline biosynhtesis in an industrail OTC producer SRI. The results were obtained as follows:(1) A mutant with zwf1 disruption, designated S-z8, was genetically constructed and successfully screened, The maximum oxytetracycline level in S-z8 was 36.2%higher than that in parent strain SRI.(2) Experimental results showed that the biomass of SRI was 90%more than S-z8 on the sixth day when cultured in the shaking flask, however, there were no significant differences in substrate consumption rate between mutant and parent strain.In addition, to improve OTC production, extra copy of two self-resistant genes otrA and otrB was introduced into SRI OTC, respectively, the results were as follows:(1) otrA or otrB gene was introduced into SRI genome by site-specific integration at attB site and a mutant with high-yield OTC performance was screened, and designated S-B80;(2) OTC Yield on substrate (YP/S,mg/g) of S-B80 was 81.83%higher than that of SRI whereas Yx/s(mg/g) of the mutant was 38.58%lower than SRI;(3) Compared with SRI, the intracellular OTC contents in SRI-B80 was 13.8% lower, however, total OTC produced by SRI-B80 was 14.2%higher, indicating that otrB could exflux intracelluler OTC into ouside the cell so that it could elevated OTC production by SRI.