Cloning, Heterologous Expression And Their Structural And Functional Study Of Bacterial Active Product Gene Clusters

Bacteria (especially actinomycetes and myxobacteria) can produce an immense variety of natural products with good biological activities. Many of these compounds belong to the polyketides and nonribosomal peptide families of natural products that are synthesized by multi-enzyme complex known as polyketide synthases and nonribosomal peptide synthetases, respectively. Although assembled from simple building blocks, there is an extraordinary structural diversity among PKS-, NRPS-and mixed PKS/NRPS derived compounds. The complex structural and stereochemistry of polyketides and nonribosomal peptides limits efficient synthetic production and leaves microbial fermentation as the predominant economical route to these bacterial active products. However, this approach is often limited by objective conditions such as growth rate, culture conditions, compound yield and genetic manipulation tools associated with the original microbial producer. Therefore, using advanced molecular biology techniques, the secondary metabolic pathways from the original producer transferred to the appropriate host strain to produce expected secondary metabolites heterologously become research focus of the biotechnology and pharmaceutical research gradually.Rhizoxin compound from Pseudomonas fluorescens Pf-5is a kind of polyketide macrolide antibiotics and it can exert its destructive effect by binding to β-tubulin and thus inhibiting microtubule assembly until cell death. Due to rhizoxin exhibits excellent antifungal and antitumor activities, it has propelled an immense research endeavour; moreover, it has once undergone phase Ⅱ clinical trials as a potential antitumor drug candidate. In this study, the full-length rhizoxin biosynthetic gene cluster was directly cloned from digested genomic DNA of Pseudomonas fluorescens Pf-5into two minimal linear vectors via Red/ET recombineering separately, followed by stitching both together into single BAC vector by T4DNA ligase. Combined with stepwise insertion of a conjugation origin, transpositional cassette, even promoter exchange, and the resultant constructs were integrated into genomic DNA of Pseudomonas putida and Burkholderia glumae, respectively. The heterologous expression of the engineered biosynthetic gene cluster was confirmed via HPLC-MS, but the production level has been a little lower (～1.4-fold and1.8-fold low) than that in the native rhizoxin analogs producer P. fluorescens Pf-5, respectively. Then a series of rhizoxin derivatives were further identified and effect of fermentation temperature, fermentation time and the host cell secretory function on rhizoxin derived compounds yield was investigated as well.The tubulysins from myxobacteria Cystobacter sp. SBCb004are a family of natural compounds with promising cytotoxic activity (including tubulysins A-Z and so on) and potently disrupt microtubule assembly leading to apoptosis. Tubulysins show better antitumor activity than anticancer drugs vinblastine, taxol and epothilone and show antiangiogenic effects as well. Together, these properties make them promising candidates for development as novel antineoplastic agents. So it will become another research focus after taxol and epothilone. This study focuses on post-modification and heterologous expression of tubulysin biosynthetic gene cluster. In tubulysin core biosynthesis gene tubB-tubF, the start codon of tubC is TTG. Because TTG is not commonly used in most heterologous host, it is a good idea to determine specific differences between tubulysin constructs pTub-ATG and pTub-TTG. Two complete tubulysin expression constructs harbouring start codon ATG and TTG in tubC were separately introduced to the Pseudomonas putida KT2440, The heterologous expression of the engineered biosynthetic gene cluster was confirmed via HPLC-MS, but the expression product is not tubulysin family compounds but their precursor-Pretubulysin. Then, we insert oxidase gene633P1(p450oxidase) from myxobacteria Cystobacter sp. SBCb004into pTub-ATG expression construct by Red/ET recombination and the resultant construct was integrated into genomic DNA of P. putida KT2440to be able to express tubulysin family compouds. Although the construct failed to express tubulysin compounds (still express pretubulysin), this study has laid a solid foundation for functional genomics research.In this study, the error-prone PCR, staggered extension shuffling together with Red/ET recombination technology were used to successfully construct gene library of CrylAc insecticidal toxin from Bacillus thuringensis4.0718. The variant T524N showed1.46-fold increased insecticidal activities against Spodoptera exigua and could also retain similar insecticidal activity against Helicoverpa armigera compared with the wild type. The improved insecticidal activity of variant T524N was explained using a molecular modeling. The combination between Red/ET recombination and DNA shuffling maybe pioneer a feasible way to shuffle a large-size gene. Firstly, small fragments (less than3kb) of this gene are modified by DNA shuffling. Then, those small shuffled fragments can be seamlessly connected by using Red/ET recombination to form an intact shuffled large-size gene.