Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

摘要

The α-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 carrying ØC31-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.