Cloning, Function And Expression Of Related Gene Involyed In Pristinamycin Biosynthesis By Streptomyces Pristinaespiralis

The antibiotic pristinamycin, produced by Streptomyces pristinaespiralis, is a mixture of two types of macrocylic lactone peptolides:pristinamycinⅠ(PI) and pristinamycin II (PII) and is particularly active against gram-positive bacteria including drug-resistant pathogens. There are three types gene:resistant gene, structural gene and regulatory gene, involved in the biosynthesis of antibiotic. Streptomyces undergo complex morphological differentiation and secondary metabolism. The intricate metabolism system is belived to be linked by a network composed of biosynthesis enzyme and common regulatory elements, including some specific signaling molecules and some different types of regulatory proteins. So far, only seventeen gens involved in resistant gene, the structural and regulatory gene of PⅠand PⅡ, were identified in S. pristinaespiralis. So the discovery of novel genes governing secondary metabolism is of particularly practical inerest, because they can be expoited to improve the production of valuable antibiotic.A series of research aiming to expoit novel genes and to improve the pristinam-ycin-producing strain were carried out and the results are as follows.First, the function of spy1 gene was investigated by strategy of homologous recombination and conjugation. The spyl gene locates on a about 8 kb DNA fragment screened from cosmid library of pristinamycin-producing 5. pristinaespiralis F618 using partial afsk-like gene involved in pristinamycin biosynthsis as a probe. The spyl gene disruptants were successfully obtained, and pristinamycin production of disruptants and S. pristinaespiralis F618 was examined by HPLC after fermentation experiment, the results indicated there may be a positive correlation between the product of spyl gene and PIA biosynthesis, and a negative correlation to PⅡA biosynthesis.Clustering location is a general rule for the genes encoding the biosynthesis of antibiotics, pigments and other secondary metabolites in Streptomyces. Based on it, a 8kb DNA fragment was analysised again, and the other four ORFs located on it were obtained and designated sprl, spr3, spr4, spr5, respectively. Four genes disruptants were obtained by the same methods as above, respectively. Finally, the results indicated that there may be a positive correlation between the product of every gene and pristinamycin, PⅡA. PⅠA biosynthesis.To improve the production of pristinamycin, a resistant gene ptr was placed under the constitutive and strongly expressing ermEp* promoter, then the DNA fragment containing the ermEp*-ptr cassette was cloned from pGH112 into a integrated vector pSET152. The resulting recombinant plasmid was introduced into S. pristinaespiralis F618 by conjugation and intergrated into the S. pristinaespiralis F618 chromosome by site-specific intergration into the attB site. One high-yield strain was obtained by primarily screening and rescreening with pristinamycin resistance, and its production of pristinamycin, PⅡA and PIa was 335.49 mg/L, 286.12 mg/L,49.37 mg/L, respectively, with an increase of 2.29 fold,2.56 fold,1.41 fold compared to that of S. pristinaespiralis F618. Moreover, its hereditary property is stable after five subcultures.