We herein introduce a strategy that leverages and integrates the attributes of whole-cell catalysis with enhanced stability of extracellular immobilized enzymes for rapid, robust, recyclable enzyme cascade reactions in a scalable fashion. We demonstrated the utility of the integrative strategy for catalytic synthesis of trehalose from soluble starch with two-step sequential bioconversion enzymatic reactions, implemented by coupling the enzymatic immobilization of β-amylase (BA), based upon E. coli biofilm curli display technique, with intracellular expression of trehalose synthase (TreS) within the same cells. This integrative strategy, compared with a strategy based on cells coupled with isolated BA, enabled a 103.5 ± 18.7% increase in the maximum trehalose formation rate by efficiently reducing the average distance of BA to intracellluar TreS enzyme. In addition, the maximum yield of starch into trehalose reached as high as 59.0 ± 1.3% at a relatively high starch concentration (10% w/v) with 15 g/L of engineered cells. We further showed that the productivity of trehalose and the percentages of cell viability remained 89.1 ± 4.4% and 85.2 ± 3.6%, respectively, even after 8 continuous rounds of biocatalysis. In addition, this strategy exhibited superb operational stability even under harsh conditions, for example, solutions rich in high amount of organic solvents. The strategy demonstrated here opens up research opportunities of combining extracellular catalysis with intracellular reactions for rapid and robust production of various value-based products.

To develop cost-effective, biobased, industrial trehalose production from maltose, an integrated bioprocess for trehalose production and separation from maltose with recombinant trehalose synthase in permeabilized cells is proposed in this study. We have successfully established an efficient production system for recombinant trehalose synthase (TreS) (9234 U/mL) in a 50 L fermentor and efficient processes for separation and purification to achieve comprehensive utilization of the raw material and products. The trehalose conversion rate of 75% at 30 °C can be reached with 6% glucose generated as byproduct using maltose syrup (30 wt %) as substrate in the bioreactor system via whole-cell biocatalysis. After two-stage simulated moving bed chromatography (SMB), the trehalose could be successfully separated, crystallized, and identified with a 97.6% purity and 95.9% recovery yield, respectively. The yield of trehalose produced was 0.675 g per gram of maltose consumed in the whole process within 80.5 h.