Programming Integrative Extracellular and Intracellular Biocatalysis for Rapid, Robust, and Recyclable Synthesis of Trehalose

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.