Jha Amit Kumar, Pokhrel Anaya Raj, Chaudhary Amit Kumar, Park Seong-Whan, Cho Wan Je, Sohng Jae Kyung
Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Asan 333-708, Korea.
Mol Cells. 2014 Oct 31;37(10):727-33. doi: 10.14348/molcells.2014.0168. Epub 2014 Sep 26.
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.
多杀菌素A和D是控制昆虫的有效成分，对非靶标生物具有极高的安全性。它由一个21碳的四环内酯与福乐胺和三-O-甲基化鼠李糖组成，这些糖源自S-腺苷甲硫氨酸。尽管先前的研究已经揭示了metK1（S-腺苷甲硫氨酸合成酶）、rmbA（葡萄糖-1-磷酸胸苷酰转移酶）和rmbB（TDP-D-葡萄糖-4,6-脱水酶）参与多杀菌素的生物合成，但迄今为止，这些基因在经过合理筛选的刺糖多孢菌（S. spinosa MUV）中的表达情况尚未阐明。在本研究中，开发了S. spinosa MUV用于代谢工程。S. spinosa MUV中多杀菌素A和D的产量分别为244 mg L(-1)和129 mg L(-1)，分别比野生型（50 mg L(-1)和27 mg L(-1)）高4.88倍和4.77倍。为了实现更好的产量；分别使用整合载体pSET152和表达载体pIBR25，在强ermE*启动子的控制下，将来自天蓝色链霉菌的正调控因子metK1-sp、rmbA和rmbB基因在S. spinosa MUV中进行表达和共表达。由此，经过基因工程改造的S. spinosa MUV菌株产生的多杀菌素A和D高达372/217 mg L(-1)，分别比野生型高7.44/8.03倍。这一结果证明了利用代谢工程对合理开发的高产天然变体进行生产的可行性。
