Expanding the substrate spectrum in engineered Pseudomonas taiwanensis for efficient production of 4-coumarate from...

Aromatics are important building blocks for polymers, pharmaceuticals, and advanced materials, but their current production relies on petrochemical processes. Biotechnological de novo production from renewable bio-based feedstocks with microbial cell factories provides a sustainable alternative. In this study, we enhanced 4-coumarate production in Pseudomonas taiwanensis from glucose and glycerol compared to previously published producers. This was achieved through heterologous expression of tyrosine ammonia-lyase (TAL) from Rivularia sp. PCC7116, which debottlenecked the specific deamination of tyrosine. Moreover, deletion of the phosphoenolpyruvate carboxylase-encoding gene ppc further increased the production. Subsequently, the substrate spectrum for efficient aromatics production was expanded to include the abundant pentoses, xylose and arabinose. Heterologous non-oxidative assimilation pathways were integrated into P. taiwanensis GRC3 chassis strains and growth on xylose and arabinose was improved through adaptive laboratory evolution, whole-genome sequencing, and reverse engineering. Optimized catabolic modules were then transferred to producer strains to enhance or enable 4-coumarate production from xylose and arabinose. Notably, the product yield on xylose increased approximately 3.5-fold with the non-oxidative xylose isomerase pathway compared to the oxidative native Weimberg pathway, without compromising yields on glucose. For the final strain, P. taiwanensis GRC3Δ6-TYR2Δppc-REXA-attTn7::P_14f-RpcTAL, product yields were significantly higher on xylose (38.2% (Cmol/Cmol)) and arabinose (39.7% (Cmol/Cmol)) than on glucose (26.0% (Cmol/Cmol)). 4-Coumarate production was characterized on mixtures of glucose, xylose, and arabinose to mimic lignocellulosic hydrolysate feedstocks, with the best reverse-engineered xylose- and arabinose-metabolizing 4-coumarate producer significantly outperforming the reference strain.