Metabolic engineering of rational screened Saccharopolyspora spinosa for the enhancement of spinosyns A and D production

Abstract

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L(-1) and 129 mg L(-1), which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L(-1) and 27 mg L(-1)), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L(-1) that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

Keywords: metK1-sp; metabolic engineering; rmbA; rmbB.

Fig. 1.

Schematic diagram of approaches used for enhancement of the spinosyn production in S. spinosa MUV

Fig. 2.

Morphology of (A) S. spinosa ATCC83543.1, (B) S. spinosa MUV RMB152, (C) S. spinosa MUV RMBIBR, (D) S. spinosa MUV, (E) S. spinosa MUV SAM152, and (F) S. spinosa MUV SIBR on 5 and 8 days.

Fig. 3.

Time course profiles of biomass obtained by introduction of pIBR25, pSIBR, pRMBIBR, pSAM152, and pRMB152 in S. spinosa MUV along with S.spinosa MUV and S. spinosa ATCC83543.1.

Fig. 4.

SEM microscopy of (A) wild type strain, (B) S. spinosa MUV, (C) S. spinosaMUV SIBR, (D) S. spinosa MUV RMBIBR, (E) S. spinosa MUV SAM152, and (F) S. spinosa MUV RMB152. Bars = 1 μm. The arrows indicate the sites of enhanced spore separation.