Simultaneous utilization of glucose and xylose by metabolically engineered Pseudomonas putida for the production of 3-hydroxypropionic acid

[Display omitted] •A P. putida strain was engineered to utilize xylose via the Weimberg pathway.•An adaptive laboratory-evolved strain showed similar growth on both glucose and xylose.•Identified key mutations responsible for higher growth on xylose through Weimberg pathway.•A reverse-engineered strain capable of co-utilization of glucose and xylose was constructed.•The engineered P. putida produced 29 g/L of 3HP from glucose and xylose in fed-batch fermentation. Pseudomonas putida,a robust candidate for lignocellulosicbiomass-based biorefineries, encounters challenges in metabolizing xylose. In this study, Weimberg pathway was introduced intoP. putidaEM42 under a xylose-inducible promoter, resulting in slow cell growth (0.05 h−1) on xylose.Through adaptive laboratory evolution, an evolved strain exhibited highly enhanced growth on xylose (0.36 h−1), comparable to that on glucose (0.39 h−1). Whole genome sequencing identified four mutations, with two key mutations located inPP3380andPP2219. Reverse-engineered strain 8EM42_Xyl, harboring these two mutations, showed enhanced growth on xylose but co-utilizing glucose and xylose at a rate of 0.3 g/L/h. Furthermore, 8EM42_Xyl was employed for 3-hydroxypropionic acid (3HP) production from glucose and xylose by expressing malonyl-CoA reductase and acetyl-CoA carboxylase, yielding 29 g/L in fed-batch fermentation. Moreover, the engineered strain exhibited promising performance in 3HP production from empty palm fruit bunch hydrolysate, demonstrating its potential as a promising cell factory forbiorefineries.