(129e) Design and Construction of Artificial Pathways for 4,6-Dihydroxycoumarin Production in Escherichia coli
AIChE Annual Meeting
2024
2024 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Advances in Biocatalysts and Biocatalytic Processes
Monday, October 28, 2024 - 2:04pm to 2:22pm
Coumarins and their derivatives possess remarkable biochemical properties and pharmacological significance. However, the exploration of the biosynthetic pathways of coumarins is still limited, constraining their microbial biosynthesis potential. Here, we designed and implemented several innovative artificial pathways aimed at synthesizing the valuable compound 4,6-dihydroxycoumarin (4,6-DHC) within Escherichia coli (E. coli). Firstly, the synthesis of novel hydroxycoumarins was pursued by leveraging enzyme promiscuity through the analysis of retrosynthetic pathways and catalytic mechanisms. Multiple pathways were further developed and confirmed to produce the direct precursor 2,5-dihydroxybenzoic acid (2,5-DHBA) by extending the shikimate pathway, screening candidate enzymes, and characterizing the relevant enzymes. Additionally, rare codon optimization and protein engineering approaches were employed to enhance the efficiency of the rate-limiting step. Subsequently, complete artificial pathways were assembled in E. coli and de novo biosynthesis of 4,6-DHC was achieved using the cheap carbon source glycerol. Finally, a significant increase in 4,6-DHC production was accomplished by controlling the expression of critical pathway genes using a tetracycline-inducible controller. This work not only established a microbial platform to produce 4,6-dihydroxycoumarin, but also demonstrated a generalizable pathway design approach for the de novo biosynthesis of valuable compounds.