Deletion of α-amylase genes via CRISPR/Cas9 decreases the side effects of hydrolysis towards nonreducing maltoheptaose preparation

Maltodextrin is a significant ingredient extensively used in food industries, but its application is limited due to uneven polymerization and high reducibility. Hence, preparation of nonreducing maltodextrin with narrow distribution of DP is essential. A dual-enzyme cascade enzymatic reaction for preparing nonreducing maltoheptaose (N-G7) is feasible via recombinant cyclomaltodextrinase (EC 3.2.1.54, CDase) and maltooligosyltrehalose synthase (EC 5.4.99.15, MTSase) heterologously overexpressed in Escherichia coli BL21(DE3). However, during this process, N-G7 further was hydrolyzed into small molecule maltooligosaccharides by the amylases homologously expressed in the host. In this study, two α-amylase genes (ycjM and malS) were deleted to avoid host hydrolysis in N-G7 via CRISPR/Cas9 system. Gene deletion had a significant decrease on the yield of maltooligosaccharides of 5 and 6 glucose units. The defective strains ΔycjM and ΔycjM-ΔmalS increased the yield of N-G7 by 21.94% and 25.47%, correspondingly. Marginal impact was made in cell growth and protein secretion of the host. YcjM and MalS were overexpressed in ΔycjM-ΔmalS and the hydrolytic activity of YcjM was twice that of MalS. They constituted a complex multiple hydrolysis system for N-G7 with the Mal series enzymes involved in a maltose system in the host.