What question did this study set out to answer?

To enhance the production of 4-coumarate in Pseudomonas taiwanensis using lignocellulosic sugars as substrates.

May 25, 2026Microbial Cell Factories1 citationsOpen Access

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

BWBenedikt Wynands SFSophia Feltes NSNadine Teófilo da Silva

Key Points

To enhance the production of 4-coumarate in Pseudomonas taiwanensis using lignocellulosic sugars as substrates.
Heterologous expression of tyrosine ammonia-lyase from Rivularia sp.
Deletion of phosphoenolpyruvate carboxylase gene ppc
Adaptive laboratory evolution and whole-genome sequencing for improved growth on xylose and arabinose.
Product yield on xylose increased approximately 3.5-fold with non-oxidative xylose isomerase pathway compared to oxidative pathway.
Final engineered strain yielded 38.2% on xylose and 39.7% on arabinose, outperforming glucose yield of 26.0%.
The best producer significantly exceeded reference strains in mixed substrate conditions mimicking lignocellulosic hydrolysate.

Abstract

Abstract 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-TYR2Δ ppc -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.

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