Metabolic engineering of Escherichia coli and process optimization for high-level production of cinnamaldehyde

Abstract

Cinnamaldehyde derived from cinnamon bark has received big attention as a potent nematicide due to its high nematicidal activity. Previously in Master’s thesis, I succeeded in developing Escherichia coli (YHP05 strain) able to synthesize cinnamaldehyde. However, production titer (75 mg/L) was not enough high for commercialization, and it is desired to further engineer host for enhanced production of cinnamaldehyde. First, I engineered two amino acids-auxotrophic YHP05 strain to prototrophic strain for the economic process of cinnamaldehyde production. Next, for the efficient conversion of trans-cinnamic acid to cinnamaldehyde, I constructed a single-step conversion system by introducing co-expression system of carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase) genes. In addition, to prevent the spontaneous conversion of cinnamaldehyde to cinnamyl alcohol, total 10 endogenous reductases and dehydrogenases genes were deleted. Fourth, after comparison of promoters and integration positions, cinnamaldehyde production system was integrated into optimal positions of E. coli chromosomal DNA for development of antibiotic-free, inducer-free system. Fifth, reinforcement of cofactor generation was performed for enhanced production of cinnamaldehyde. Next, to reduce the inhibitory effect of cinnamaldehyde and improve the production titer, in situ product removal (ISPR) strategy was applied in cultivations. However, cell growth retardation was occurred by acetate accumulation. To solve this problem, acetate production pathway was deleted by newly constructed CRISPR/Cas9-based scarless genome editing system. Additionally, to decrease cell toxicity to cinnamaldehyde, cell recycle system with a polymer resin and promoter replacement of efflux pumps (TolC and AcrAB) were tested. Through these metabolic engineering and process optimization strategies, I successfully demonstrated the gram-scale production of cinnamaldehyde. I also confirmed that cinnamaldehyde produced by our engineered E. coli had a nematicidal activity similar to the activity of authentic cinnamaldehyde by nematicidal assays against Bursaphelenchus xylophilus.