Cloning And Construction Of Heterologoues Expression Vector Of Spinosad Biosynthesis Gene Cluster

Spinosad is a mixture of novel secondary metabolites produced by actinomyceteSaccharopolyspora spinosa. It is used in agriculture as a potent insect control agentand has an excellent environmental and mammalian toxicological profile. In this work,artificial neural network (ANN) modeling, was applied to optimize theconcentrations of medium components for spinosad production from fermentation ofSaccharopolyspora spinosa strain ATCC49460. Experiments were performed using arotatable central composite design (RCCD), and the data obtained were used toconstruct ANN model and RSM models. The regression coefficients (R2) for the bothmodels were0.9866and0.9458, respectively, indicating that the fitness of the ANNmodel was higher. Using a genetic algorithm (GA), the input space of the ANN modelwas optimized to obtain optimal values of medium component concentrations. Themaximal spinosad yield (401.26mg/L) was obtained using the followingGA-optimized concentrations: cottonseed protein28.22g/L, corn steep liquor21.61g/L,(NH4)2SO41.13g/L, glucose56.43g/L, soluble starch66.43g/L. The spinosadyield obtained using the ANN-GA model was13.2%higher than that using the RSMmodel. The hybrid ANN-GA approach provides a viable alternative to theconventional RSM approach for the modeling and optimization of fermentationprocesses.Most of the S. spinosa genes involved in spinosad biosynthesis are found in one74kb cluster. The cloning of the spinosad biosynthetic gene cluster provides thestarting materials for the molecular genetic manipulation of spinosad yields, and forthe production of novel derivatives containing alterations in the polyketide core. Toobtain the whole spinosad biosynthetic gene cluster of S. spinosa, a library of S.spinosa genomes and screening the library are needed. To obtain size collecting highmolecular weight DNA, genomes were partially digested with Sau3AI followed byselecting with pulsed field gel electrophoresis, and then DNA fragments in suitablesize were recovered. The partially digested genomic DNA was ligated with treatedBAC vector pCC1BAC/BamHI. The competent TransforMax EP3100bacteria was electroporated with ligation mixtures. Electroporations were plated on LB-agarcontaining chloramphenicol, IPTG, and X-gal. White clones were picked to constructthe library. The average insert size is100kb in the library which corresponds toapproximately8.93genome equivalents. Two couples of primers were designedaccording to gene sequences of spinosad biosynthetic gene cluster in GenBank underaccession number AY007564.1, and PCR was performed to screen the library forpositive clones containing entire spinosad biosynthetic gene cluster. As a result, fourclones with inserted fragments which were larger than spinosad biosynthetic genecluster sequences in GenBank were found. And the heterologous expression vector ofspinosad biosynthesis gene cluster was constructed by Red/ET recombineeringstrategy. This work provides the foundation for exploring of the heterologousexpression of spinosad biosynthetic gene cluster.