Metabolic Engineering Of Saccharopolysporaspinosa For The Production Of Spinosad And Study On Spinosad Biosynthesis

Spinosyns, which consist of21-carbon tetracyclic lactone containing two deoxysugars: tri-O-methylated rhamnose and forosamine, are polyketide-derived macrolides produced by Saccharopolyspora spinosa. Spinosyns A and D, which are defined as spinosad, are the two major components in the S. spinosa fermentation. Spinosad has shown broad-spectrum insecticidal activity and has an excellent environmental and mammalian toxicological. However, the low yield of spinosad has impeded its application. So we some researches have been done to resolve this problem. Some main results of this study are as following:1. A rational strain improvement strategy was developed to tune the unbalanced biosynthetic pathway of spinosad. First, we overexpressed spnK to increase the amount of the flux from rhamnosylated aglycone to pseudoaglycones (PSA). Then six close genes (spnP, spnO, spnN, spnQ, spnR, and spnS) involved in forosamine biosynthesis and spnK were co-expressed in S. spinosa LU102to convert the accumulated PSA to spinosad. The yield of spinosad in S. spinosa LU102was2.6-fold higher than that in the wild-type S. spinosa (82mg/L), reached214mg/L. Finally, the yield of spinosad in the tuned S. spinosa LU104was further increased to405mg/L, which was a5.0-fold enhancement compared with the wild-type S. spinosa, by combining spnP, spnO, spnN, spnQ, spnR, spnS, spnK, gtt, gdh and kre genes.2. Eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene(s). The number of amplification cycles of these candidate genes was calculated by Bestkeeper, Normfinder and geNorm. The results indicated that the most suitable reference genes for normalization during the whole fermentation of S. spinosa were16S rRNA and rbL13.3. In the presence of exogenous fatty acid, gene expression assays indicated that de novo fatty acid was significantly decreased and β-oxidation and spinosad biosynthesis were up-regulated. The decreased de novo fatty acid synthesis and the increased β-oxidation resulted in the increase of acetyl-CoA and malonyl-CoA. So it is the up-regulated spinosad pathway coupling with the enhancement concentration of acetyl-CoA and malonyl-CoA that contributed to the increase of spinosad. Taken together, a metabolic link between FAS, β-oxidation and spinosad biosynthesis at the presence of exogenous fatty acid was established. The results presented in this study enable researchers to better understand why fatty acid addition can increase polyketides production.