Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde

Abstract

Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

Keywords: Cinnamaldehyde; Escherichia coli; Fed-batch cultivation; Metabolic engineering; in situ product recovery (ISPR).