Screening And Cloning Of High Production Related Gene Of Pristinamycin

Pristinamycin, a member of the streptogramin family of antibiotics, is produced by Streptomyces pristinaespiralis. For the present, little research on genetic engineering of pristinamycin producing strain has been conducted overseas and there is no research as yet at home, which blocks the development of improvement of pristinamycin production through genetic engineering. In this article, we did some exploratory research on genetic engineering of pristinamycin producing strain.Firstly, amplified fragment length polymorpHism (AFLP) technique with two sets of single restriction enzyme ApaI and SacII digests was performed in this experiment, and four polymorpHic DNA fragments only could be distinguished from the genomes of the high-yield recombinants, which was absent in the initial stain ATCC25486. After cloning, sequencing and similarity analysis of these DNA fragments, two homologues of high yield-related genes were found for the amplification treatment of ApaI selective primers. One was the homologue of afsR regulatory gene and another was transposase gene. Two homologues of high yield-related genes were also amplified with SacII selective primers. One was the homologue of afsK regulatory gene and another was exodeoxyribonuclease encoded gene exoSC.Secondly, selecting the cosmid supercos-1 as the vector, a genomic cosmid library of pristinamycin-producing strain of F618 was successfully constructed. In this part of experiment, the method of extracting genomic DNA was improved to obtain the high molecular genomic DNA. Also the condition of partially digesting of genomic DNA was optimized to obtain 35-45kb digested fragments. Through connecting partial fragments with vector, packaging the recombinant cosmid to be bacteriopHages, and transducting the bacteriopHages into E. coli, a high quality cosmid genomic library was successfully constructed.Finally, with the DIG labelled gene fragment of afsk as a probe, a novel gene was screened from the cosmid library through colony in situ hybridization. Then a restriction fragment which contains the target gene was determined by restriction enzymes analysis and southern blotting. Through sequencing, a gene of afsK with full-length sequence was successfully subcloned and was named as spy1, which contains an open reading frame (ORF) with a length of 2127bp. Preliminary analysis of the novel gene by bioinformatics shows that the ORF encodes a protein of serine/threonine protein kinase which contains 708 amino acids.