High throughput screening and metabolic engineering of Saccharopolyspora spinosa for spinosad production

Abstract

The macrolide antibiotics spinosad, synthesized by Saccharopolyspora spinosa, is a highly effective, environmentally-friendly insecticide. However, due to poor fermentation performance and difficulties in engineering S. spinosa strains, the production cost of spinosad is still high, which restricts its industrial application. The industrial strains used for antibiotic production primarily originate from mutagenic screening, whereas traditional screening methods are time-consuming and laborious. Here, an in vitro detection method for spinosad was established to accelerate the breeding of mutated strains of S. spinosa. Firstly, a broad substrate promiscuity glycosyltransferase OleD from Streptomyces antibioticus was selected and employed for the detection of pseudoaglycone (PSA), which serves as the precursor compound for spinosad, through the utilization of colorimetric reactions coupled with glycosylation. Subsequently, the in vitro PSA detection system was optimized and applied for S. spinosa high throughput screening. The final selected mutant strain DUA15 was obtained and showed a 0.80-fold and 0.66-fold increase in spinosad and PSA production compared to the original strain, respectively. Furthermore, genetic engineering technology was combined to obtain the engineered strain D15-102, which showed a 2.9-fold increase in spinosad production compared to the original strain.

Keywords: Saccharopolyspora spinosa; Genetic modification; High-throughput screening; Pseudoaglycone; Spinosad.