Evaluating LDH and BUDB gene knockouts in Klebsiella pneumoniae for improved 1,3-propanediol production

Metabolic engineering has become a powerful tool to enhance 1,3-propanediol (1,3-PDO) production. In this study, we knocked out genes involved in by-product formation in Klebsiella pneumoniae TWO to improve 1,3-PDO yield. We constructed ΔLDH and ΔBUDB mutants by knocking out ldh and budB, respectively. Flask experiments using 20 g/l glycerol under microaerobic conditions showed that the ΔBUDB mutant produced 8.71 g/l of 1,3-PDO with a yield of 0.51 mol/mol glycerol, outperforming the ΔLDH mutant, which produced 6.93 g/l 1,3-PDO with a yield of 0.41 mol/mol glycerol. Notably, the mutants exhibited different by-product profiles, particularly in formic acid and ethanol levels. An optimized medium supplemented with 0.0025 g/l cyanocobalamin further improved 1,3-PDO yield in the ΔbudB mutant to 0.60 mol/mol glycerol. Further supplementation with a vitamin solution increased 1,3-PDO level to 10.08 g/l, yielding 0.62 mol/mol glycerol and reducing residual glycerol to 0.08 g/l. This approach resulted in a 29% increase in 1,3-PDO production compared to the wild-type strain. Our findings highlight the single knockout of the budB gene, combined with cyanocobalamin and a vitamin solution, significantly enhances the yield and level of 1,3-PDO with a low by-product profile, leaving no residual glycerol, offering a promising strategy for industrial bioprocessing.