Improvement Of Streptomyces Pristinaespiralis By Genome Shuffling, Optimization Of Pristinamycin Fermentation And Analyses On Mechanisms For The Yield Enhancement

Pristinamycin which produced by streptomyces pristinaespiralis is a streptogramin of natural origin comprising two components, prsitinamycin I and prsitinamycin II. Pristinamycin not only has strong antibacterial activity against gram-positive bacteria, including methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, but also exhibits a prolonged post-antibiotics effect. So it is considered as the specially selected medicament against the stubborn gram-positive infection. Pristinamycin has been commercially produced in overseas and have not been accomplished in China currently. Therefore, it is very valuable to investigate and develop pristinamycin further as a new generation of antibiotic.The aim of this work is to increase the productivity of pristinamycin fermentation. After thorough and particularity study in strain improvement by genome shuffling, medium composition and culture condition optimization, pristinamycin productivity is increased greatly. Moreover, molecular mechanisms of yield enhancement of pristinamycin and proteomics in Streptomyces pristinaespiralis were anlaysed in the paper.The main research contents and results are as follows:1. Streptomyces pristinaespiralis CGMCC 0957, used as an original strain, was stepwise treated with ultraviolet light (UV). Four mutant strains with higher yield and tolerance of pristinamycin were selectively starting strain of genome shuffling. Then the formation and regeneration of protoplasts were studied. When streptomyces pristinaespiralis were cultured with 0.6 % glycine in advance and treated with 2% lysozyme for 1.5h at 32℃, the protoplast formation rate was 95.8%. The protoplast regeneration was 18.1%.2. Four mutant strain, M-23,M-79,M-113,M-156, were subjected for recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-μg/mL pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/L, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable.3. The pristinamycins fermentation in shake flasks by Streptomyces pristinaespiralis G4-17 was studied. The production medium was optimized by orthogonal design experimental methods. Also, the effect of culture conditions in shake flasks on the pristinamycins production was evaluated. The optimized medium and culture conditions were as follows: soluble starch 3.0%, sucrose 1.0%, glucose 0.5%, maltose 0.8%, soybean flour 1.2%, peptone 0.4%, fish flour 1.2%, yeast extract 0.6%, (NH₄)₂SO₄ 0.2%, MgSO₄·7H₂O 0.35%, KH₂PO₄ 0.02%, NaNO₃ 0.075%, CaCO₃ 0.4%; inoculum age of 36 h; inoculum level of 6%; initial pH value of 6.5; temperature of 25℃; shaking speed of 220 rpm. Under the above optimized conditions, S. pristinaespiralis G4-17 could produce 1.21g/L pristinamycins after 60 h cultivation in the shake flask, which was 40.0% higher than that before optimization.4. Amplified fragment length polymorphism (AFLP) with the double-enzyme ApaI/TaqI was used to analyze genomic variability between high pristinamycin-producing recombinants of S. pristinaespiralis obtained by genome shuffling and their ancestral strain CGMCC 0957. The AFLP fingerprints showed together that there were some polymorphism between these high yield recombinants and their ancestor, and that there was similar polymorphism among these recombinants. Nevertheless, the unique polymorphic bands, which be absent in the ancestor, could be distinguished from all the recombinants. In addication, AFLP analysis showed that variation of organism at the genomic level happens more extensively and easily by genome shuffling than by induced mutagenesis. 5. In order to analyze gene expression changes related to the enhanced antibiotic yield, genes involved in the biosynthesis of PI or PII, and resistance to pristinamycin were investigated by reverse transcription PCR (RT-PCR) between the high-yield recombinants and their ancestral strain CGMCC 0957. The results showed the persistent expression of snbA and snaB involved in the biosynthesis of PI and PII component respectively in the recombinant had a close correlation to the increase of the antibiotic production. It might imply that snbA was a key gene for the biosynthesis of PI component, while snaB was another key gene for the PII biosynthesis. Other two genes snaA and snaC displayed no obvious changes at this time course of fermentation between the recombinant and the ancestor. The ptr gene involved in pristinamycin resistance started ahead high-level expression before the onset of pristinamycin production for the recombinant, also leading to the increase of the antibiotics yield. The spbR gene regulating the antibiotic production did not showed obvious expression change between the recombinant and the ancestor.6. To extract and identify the total protein in the cell of Streptomyces pristinaespiralis, the application and effects of some conventional proteomics methods are compared. The results showed that liquid nitrogen grounding method and high lysis buffer were much suitable for Streptomyces pristinaespirali. And the content of total protein in the cell of G4-17 was more than that of CGMCC 0957 in the whole fermentation process. Compared with fermentation anaphase, the content of total protein in the same strain was higher in earlier fermentation.