(129e) Design and Construction of Artificial Pathways for 4,6-Dihydroxycoumarin Production in Escherichia coli | AIChE

(129e) Design and Construction of Artificial Pathways for 4,6-Dihydroxycoumarin Production in Escherichia coli

Authors 

Gan, Q. - Presenter, University of Georgia
Jiang, T., University of Georgia
Li, C., University of Georgia
Gong, X., University of Georgia
Zhang, J., University of Georgia
Yan, Y., University of Georgia
Desai, B., University of Georgia
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.