Continuous process technology for bottom-up synthesis of soluble cello-oligosaccharides by immobilized cells co-expressing three saccharide phosphorylases.

Background: Continuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative β-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP).

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production.

Background: Soluble cello-oligosaccharides (COS, β-1,4-D-gluco-oligosaccharides with degree of polymerization DP 2-6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose.

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol.

Background: Glucosylglycerol (2-O-α-D-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented.

Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes Whole-Cell Production of Cellobiose.

Catalyst development for biochemical cascade reactions often follows a "whole-cell-approach" in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two-enzyme co-expression in E.