title: 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.
authors.affiliation: Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Asan 333-708, Korea.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Jha
authors.affiliation: Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Asan 333-708, Korea.
authors.forename: Anaya Raj
authors.initials: AR
authors.lastname: Pokhrel
authors.affiliation: Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Asan 333-708, Korea.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Chaudhary
authors.forename: Seong-Whan
authors.initials: SW
authors.lastname: Park
authors.forename: Wan Je
authors.initials: WJ
authors.lastname: Cho
authors.affiliation: Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Asan 333-708, Korea.
authors.forename: Jae Kyung
authors.initials: JK
authors.lastname: Sohng
mesh_terms: D000818:Animals; D004338:Drug Combinations; D006801:Humans; D007300:Insect Control / Q000379:methods; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism*; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D019076:Transgenes
chemical_list: D004338:Drug Combinations; D007306:Insecticides; D018942:Macrolides; C114883:spinosyn A; C114884:spinosyn D; D008716:Methionine Adenosyltransferase; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; C415329:spinosad
keywords: metK1-sp; metabolic engineering; rmbA; rmbB