Mechanism Of Acarbose Over-production And Metabolic Engineering Of High-yield Strains

Acarbose,an ?-glucosidase inhibitor,is produced in large scale by strains derived from Actinoplanes sp.SE50 and used widely for the treatment of type-2 diabetes.As one of the high-yield derivatives,Actinoplanes sp.SE50/110 shows 2-fold improved acarbose yield and 4 to 8-fold improved transcription of the acb biosynthetic gene cluster compared with the wild-type strain SE50.In order to elucidate the overproduction mechanism,the genome of the SE50 was fully sequenced and compared with that of SE50/110.Strain SE50/110 has 15 mutation sites affecting 15 CDSs,including 11 SNVs and 4 InDels.Apart from synonymous substitutions and variations occurring in intergenic regions,8 sites related to 8 CDSs were chosen for inactivation in the wild-type.Individual deletion of gene ACWT₄325?putative alcohol dehydrogenase?and gene ACWT₇629?putative elongation factor G protein?in SE50 resulted in improved acarbose yields from 1.87 to 2.34 g/L?63% of SE50/110?and 2.54 g/L?68% of SE50/110?,respectively.In addition,the biomass of ?ACWT₄325 was improved by 18% and the expression of acb cluster of ?ACWT₇629 was substantially increased compared with the wild-type SE50.Subsequently,combined deletions of ACWT₄325 and ACWT₇629 was performed in SE50,which resulted in an 2.83 g/L yield of acarbose?76% of SE50/110?.What's more,a comprehensive analysis and utilization of high-yield mechanism was carried out,and the yield of acarbose was further increased from 3.73 g/L to 4.21 g/L.In addition,the effect of different maltose concentrations on acarbose production was determined.The results showed that the optimal maltose concentration for cell growth and acarbose production is 50 g/L.Meanwhile,the transcription of acb biosynthetic genes,esp.acbW encoding transporter,was dramatically enhanced by increased maltose concentrations.To further investigate the involvement of acbW in acarbose production,it was inactivated in the wild-type,and the accumulation of acarbose was abolished in the mutant.Moreover,the overexpression of acbW and acbWXY improved the acarbose yield by 6.4% and 8%,respectively.In conclusion,we clarified the mechanism for acarbose overproduction via comparative genomics,and derivative of the high-yield strain SE50/110 with higher yield was constructed via genetic engineering.In addition,optimization of fermentation process and functional analysis of acarbose biosynthetic genes were also carried out,suggesting that the transporter AcbWXY is necessary for acarbose production,and the its overexpression is an efficient strategy for acarbose yield improvement.