Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering

ABSTRACT In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non‐ATP‐forming chimeric glycolytic pathway were assembled in an artificial operon and co‐expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell‐free extract of the multiple‐gene‐expression E. coli were 5.0–1,370 times higher than those in an enzyme cocktail prepared from a mixture of single‐gene‐expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple‐gene‐expression cells led to the denaturation of indigenous proteins and one‐step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2O‐forming NADH oxidase or the malate/lactate dehydrogenase facilitated one‐pot conversion of glucose to pyruvate or lactate, respectively. Biotechnol. Bioeng. 2015;112: 189–196. © 2014 Wiley Periodicals, Inc. Nine genes encoding thermophilic glycolytic enzymes were assembled in an artificial operon and coexpressed in Escherichia coli. By a simple heat‐treatment of the crude extract of the recombinant cells, indigenous mesophilic proteins were denatured and an in vitro artificial metabolic pathway comprising of only a limited number of thermotolerant enzymes could be constructed.