Improvement Of Pristinamycin Production By Transferring Ptr Gene In Streptomyces Pristinaespiralis And Family Shuffling Of Ptr Gene

Pristinamycin, produced by Streptomyces pristinaespiralis, is a mixture composed of two structurally dissimilar synergistic antibiotics, pristinamycins I (PI) and pristinamycins II (PII). It is effective against Gram-positive bacteria including drug-resistant pathogens. Now, the absence of high pristinamycin-producing S. pristinaespiralis with stable hereditary character has become a main cause hindrance to pristinamycin fertermation and clinical application. Therefore, it is necessary to obtain high-yield strains with good stable genetic stability. In this dissertation, intention of breeding high-yielding strains was carried out by amplifying the copies of ptr resistant gene based on the relationship between strain's production level and resistance to antibiotic. Furthermore, directed evolution of ptr resistant gene and its homologous were performed with DNA family shuffling method, which lay the foundation for increasing the yield of pristinamycin.1. An effective conjugal transfer system from E. coli ET12567 to Streptomyces pristinaespiralis was established using an integrated vector, pSET152. The procedure was optimaized including the spore germination, the screening concentration of antibiotics, the spore concentration, the optimum medium used in conjugation, and the concentration of Mg²⁺, etc., and the conjugation frequency was up to 1.36×10^-3 under the optimal condition. The PCR experiments verified that the plasmid vector pSET152 was integrated in S. pristinaespiralis and was stable after ten successive subcultivation, which provide a powerful technological base for genetic modification of S. Pristinaespiralis.2. In order to breed high pristinamycin-yield strains, the experiment for increasing the copies of ptr resistant gene was performed in S. pristinaespiralis ATCC25486. At first, ptr was amplified by an optimized PCR technique, and then transferred into S. pristinaespiralis, resulting in 146 transformants of S. pristinaespiralis. These transformants were primarily screened on the pristinamycin resistant plates, and then were screened for high-yield strains by fermentation test. Finaly, two high-yield strains SPR1 and SPR2 with 6-8 fold increase of Pristinamycin yield were obtained, and their yield was 0.11 and 0.15 g/l respectively, six to eight times more than that of ATCC25486. Moreover, it keeps good hereditary stability even after five subcultures.3. In order to further enhance the Pristinamycin yield, DNA family shuffling of ptr resistant gene was studied. The ptr gene and its homologous actV,mmr were cloned in E. coli by T vector. Because of low homology of three genes, their recombinant plasmids were used for gene reassembly. At first, the plasmids was digested by DNaseI enzyme and the digest fragments of 100—600 bp size was purified. The purified fragments were reassembled into a full-length gene in the absence of primers. After addition of primers, a single PCR product of 1.8 kb size is typically obtained. These products were cloned and a shuffled gene library with concentration of 1.83×10⁵ was obtained. It provided a new method for family shuffling of low homologous genes by proposing recombinant plasmids as templates in DNA family shuffling.