Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis

Microbial Cell Factories

Research

Xiangjing Wang¹  Haiyan Wang²  Yanyan Zhang²  Hairong He³  Hui Liu³  Wensheng Xiang³ 

[1] School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, 150030, Harbin, China;State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, 100193, Beijing, China;State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, 100193, Beijing, China;School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, 150030, Harbin, China;

关键词: Milbemycin;    LAL;    MilR;    Overexpression;    Streptomyces bingchenggensis;   

DOI  :  10.1186/s12934-016-0552-1

 received in 2016-04-20, accepted in 2016-08-31,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundMilbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes.ResultsmilR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. β-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR’s function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.ConclusionsA LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics.

【 授权许可】

CC BY   
© The Author(s) 2016