Heterologous Expression Of Daptomycin Biosynthetic Gene Cluster And Preliminary Study On The Regulatory Mechanism Of Daptomycin Biosynthetic

Daptomycin, a novel cyclic lipopeptide antibiotic from Streptomyces roseosporus that provides rapid bactericidal activity against gram-positive pathogens in vitro, with novel structure and the special mechanism. Due to its unique cyclic lipopeptide structure and mechanism of action, it will not generate cross-resistance from other approved antibiotics, instead, has a broad market prospect. As a secondary metabolite, Daptomycin is synthesized by a nonribosomal peptide synthetase (NRPS) via a thiotemplate mechanism. A128kb region of S.roseosporus GC-rich DNA contains the daptomycin biosynthetic gene cluster.Currently, the methods of daptomycin preparation mainly include natural fermentation, precursor induction, chemical synthesis, semi-synthetic and so on. As to its unique structure, containing special amino acid composition, lead to microbial fermentation be commonly used.But the expression level of daptomycin is too low to meet the industry needs with this method. So it’s necessary to improve the daptomycin expression level of Streptomyces roseosporus.In addition, the rapid development of synthetic biology provides a novel direction for the screening,reforming and preparation of antibiotics. The concept of synthetic biology is systematic design and engineering, aim at obtaining remodeling or natural biological systems by reasonable combination of a variety of natural or artificial biological components. The biosynthetic pathway of daptomycin and related functional gene has been successfully resolved, the application of synthetic biology techniques can manipulate its biosynthesis gene cluster artificially, such as screening of novel antibiotics by gene replacement, elevation of expression amount by changing the host and so on. But synthetic biology research is also part of the initial stage, there is no large DNA fragments, especially the large fragment of DNA derived from Streptomyces genetic manipulation successfully reported.In order to handle the key technology of synthetic biology system, especially the large fragment of gene manipulation techniques, lay the foundation for subsequent daptomycin synthetic biology research,in this research, We preliminarily improved the production capacity of the original producing bacteria and establish a system of fermentation, separation and purification, separation and detection technology by daptomycin original producing bacteria (ATCC31568) UV mutagenesis combined with antibiotic resistance screening process. Moreover, we Construct the daptomycin production of strain----Streptomyces roseosporus genome BAC library and screen the daptomycin biosynthetic gene cluster at the same time, through conjugative transfer method, daptomycin can be expressed in Streptomyces lividans in order to grasp the large gene fragment manipulation technology.Streptomyces roseosporus genomic library was constructed successfully, which then was inserted by fragment larger than140kb. To meet the needs of next experiment, the library consists of2496-well plates with a total of2304clones. The BAC plasmid containing the complete daptomycin biosynthetic gene cluster was successfully screened by PCR, named pESPBAC-DAP then. The recombination plasmid pESPBAC-DAP was transformed into ET12567(pUZ8002),a donor host of conjugal transfer, daptomycin biosynthetic gene cluster was transfered into Streptomyces lividans TK24. Because pESPBAC carries the phage genome attP sites, the integrase can mediate site-specific recombination between attP and attB (bacterial attachment site in Streptomyces), integrating daptomycin biosynthetic gene cluster into the genome of the Streptomyces lividans TK24. Fermentation of Streptomyces lividans TK24indicats that the expression level of daptomycin is30.2mg/L by HPLC, following detected its activity.We have made a further improvement of the strains, increasing strain resistance marker, quickly and effectively improving the production capacity of the strain, By UV mutagenesis combineed with resistance screening of rifampin (rifampicin) and gentamicin (gentamicin); A large number of Resistant Mutant strains were selected, the highest yield of the mutant Ge-27reached79.44mg/L, improving42.2%compared to the original strain. The results proved that using the UV mutagenesis combined with antibiotic resistance screening can efficiently enhance the production levels of daptomycin. We initially screen out ORF-12, ORF-53, ORF-21, ORF-31and ORF-22five ORFs which would play a regulatory role in its biosynthesis By real-time quantitative PCR analysis of daptomycin ORFs, wherein three ORFs,ORF12, ORF21and ORF-53, may play a rate-limiting role in the initial stage of daptomycin synthesis process; ORF-31may participate in the positive regulation of the daptomycin post-synthesis, and ORF-22could be a negtive regulator in the synthesis process. Our screening would lay down a fundation for the following analysis of the single ORF function, or the improvement of the daptomycin production level by knockout or overexpression of some ORFs.