Permeabilized whole cells containing co-expressed cyclomaltodextrinase and maltooligosyltrehalose synthase facilitate the synthesis of nonreducing maltoheptaose (N-G7) from β-cyclodextrin.

Background: Maltodextrin is an important bulk ingredient in food and other industries; however, drawbacks such as uneven polymerization and high reducibility limit its utilization. Nonreducing maltoheptaose (N-G7) is a good substitute for maltodextrin owing to its single degree of polymerization and its nonreducing properties. In this study, in vitro cell factory biotransformation of β-cyclodextrin (β-CD) to N-G7 is demonstrated using coexpressed cyclomaltodextrinase (CDase, EC 3.2.1.54) and maltooligosyltrehalose synthase (MTSase, EC 5.4.99.15). However, the cell membrane prevents β-CD from entering the cell owing to its large diameter.

Results: The amylase-deficient permeabilized host ΔycjM-ΔmalS-ΔlpxM is utilized for the coexpression of recombinant CDase and MTSase. Deletion of lpxM effectively allows the entry of β-cyclodextrin into the cell, despite its large diameter, without requiring any relevant cell membrane permeability-promoting reagent. This results in a 28.44% increase in the efficiency of β-CD entry into the cell, thus enabling intracellular N-G7 synthesis without the extracellular secretion of recombinant CDase and MTSase. After reacting for 5.5 h, the highest purity of N-G7 (65.50%) is obtained. However, hydrolysis decreases the purity of N-G7 to 49.30%, thus resulting in a conversion rate of 40.16% for N-G7 when the reaction lasts 6 h. Precise control of reaction time is crucial for obtaining high-purity N-G7.

Conclusion: Whole-cell catalysis avoids cell fragmentation and facilitates the creation of an eco-friendly, energy-efficient biotransformation system; thus, it is a promising approach for N-G7 synthesis. © 2023 Society of Chemical Industry.