摘要
Theα-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp.and used as a potent drug in the treatment of type-2 diabetes.In order to improve the yield of acarbose,an efficient genetic manipulation system for Actinoplanes sp.was established.The conjugation system between E.coli carryingØC31-derived integrative plasmids and the mycelia of Actinoplanes sp.SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture(mycelia and E.coli),quantity of recipient cells,donor-to-recipient ratio and the concentration of MgCl2,which resulted in a high conjugation efficiency of 29.4%.Using this integrative system,a cloned acarbose biosynthetic gene cluster was introduced into SE50/110,resulting in a 35%increase of acarbose titer from 2.35 to 3.18 g/L.Alternatively,a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm)was constructed for gene inactivation,which has a conjugation frequency as high as 0.52%.Meanwhile,almost all 5-flucytosine-resistant colonies were sensitive to apramycin,among which 75%harbored the successful deletion of targeted genes.Using this replicating vector,the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated,and component C was eliminated as detected by LC-MS.Based on an efficient genetic manipulation system,improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.
基金
We are grateful to Prof.Yuhui Sun from Wuhan university,China,and the late Prof.Keqian Yang from Institute of Microbiology,Chinese Academy of Sciences,for providing plasmids pWHU2653 and pDR-4-K^*,respectively.This work was supported by grants from the National Natural Science Foundation of China(No.31470157,21661140002)
the Ministry of Science and Technology of China(No.2012AA02A706).
