Metabolic and fermentation engineering of Klebsiella Pneumoniae for high-level production of 3-hydroxypropionic acid

3-hydroxypropionic acid (3-HP) is an essential C3 platform chemical with considerable potential for synthesizing various chemicals and biodegradable materials. However, efficient production of 3-HP is still an urgent problem. In this study, a comprehensive approach involving the screening and mutation of KGSADH, along with the optimization of ribosome binding sites (RBS), promoters, and the downregulation of competing metabolic pathways, led to a marked improvement in 3-HP production, achieving a concentration of 6.67 g/L in shake flask cultures. Furthermore, innovatively changing inducible promoters to achieve constitutive protein expression. Then replacing yeast extract with corn steep liquor resulted in a notable increase in 3-HP titer, rising from 86.80 to 102.56 g/L. The reduction of residual glycerol concentrations to below 25 g/L further enhanced production, culminating in a final titer of 110.70 g/L, which is the highest titer for 3-HP production in Klebsiella pneumoniae to date. These findings underscore the potential of engineered microbial systems in optimizing the sustainable production of valuable platform chemicals from renewable feedstocks.