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

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.

Development of Cupriavidus necator H16 as a host for heterologous production of formate dehydrogenase I of Methylorubrum extorquens: Possibilities and limitations.

The discovery of formate dehydrogenase (Me-FDH1) from Methylorubrum extorquens has provided an avenue for sustainable CO2 fixation and utilization. However, the mass production of Me-FDH1 is challenging due to the presence of its unique tungsto-bis-metalopterin guanine dinucleotide (W-bis-MGD) cofactor, limiting its practical applications. In this study, C. necator H16 is proposed as a host for the large-scale production of Me-FDH1, utilizing fructose as a carbon source and its inherent machinery for cofactor synthesis. In a minimal salt medium, C. necator H16 could produce active Me-FDH1, which exhibited a specific activity of 80 to 100 U/mg for CO2 conversion to formate. In fed batch bioreactor experiments, approximately 50 g CDW/L (cell dry weight/L) and 10,000 U/L Me-FDH1 were achieved within 50 h. This study highlights C. necator H16 as the recombinant host for Me-FDH1, paving the way for the future development of efficient mass-production methods for this crucial enzyme.