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

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