Sustainable bio-based production of a performance-advantaged chemical such as cis,cis-muconate through microbial bioconversion has the potential to replace petroleum-derived platform chemicals. Researchers developed microbial cell factories to produce cis,cis-muconate from the biomass-derived feedstock. We identified the novel robust bacterium Erwinia aphidicola LJJL01 as a potential host strain to produce advanced bio-functionalized chemicals such as cis,cis-muconic acid from biomass-derived substrates. Given the ability of the strain to grow and utilize catechol, we demonstrated that by knocking out the muconate cycloisomerase gene (catB), the strain can produce cis,cis-muconic acid from catechol. However, the yield of cis,cis-muconic acid was low from catechol, and we explored the metabolic engineering approaches to increase the production. Firstly, we deleted another gene, muconalactone isomerase (catC), together with catB gene. Secondly, we overexpressed catechol-1,2-dioxygenase (catA) gene to catalyze the conversion of catechol into cis,cis-muconate. Thirdly, we introduced the aroY gene encoding protocatechuate decarboxylase that converts protocatechuate (PCA) to catechol, and concurrently enables the production of cis,cis-muconate. The strain enables the production of cis,cis-muconate from biomass-derived aromatics. Currently, we are engineering the shikimate pathway to convert sugars into PCA. Given that E. aphidicola LJJL01 natively enables the catabolism of C6 and C5 sugars, the pathway enable the production of cis,cis-muconate from biomass-derived glucose, xylose, and arabinose. Collectively, we demonstrated the re-wiring of E. aphidicola LJJL01 catabolic pathway(s) to biofunnel biomass-derived substrates to high-value cis,cis-muconate. Our study suggests that E. aphidicola LJJL01 is a promising candidate to develop as a microbial cell factory to produce performance-advantaged chemicals.

Lakshika Dissanayake

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