Overexpression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae

Glycerol dehydratase that catalyses the conversion of glycerol to 3-hydroxypropionaldehyde was previously supposed to be a limiting step in the 1,3-propanediol production from glycerol. In this study, glycerol dehydratase was over-expressed separately or coordinately with 1,3-propanediol oxidoreductase in Klebsiella pneumoniaeDSM2026 to investigate its effects on the glycerol fermentation. The overexpression of glycerol dehydratase surprisingly led to a serious impairment of growth of K. pneumoniae in both continuous and batch cultures. Instability of the plasmids bearing the genes encoding glycerol dehydratase and/or 1,3-propanediol oxidoreductase was observed in recombinant cells, especially in anaerobic cultures using glycerol as sole carbon source. It is postulated that an imbalanced conversion of glycerol to the intermediate 3-hydroxypropionaldehyde and its further conversion to 1,3-propanediol and the associated accumulation and toxicity of 3-hydroxypropionaldehyde are responsible for the phenomena observed. Furthermore, the putative regulatory gene dhaR of the dha regulon was also overexpressed in K. pneumoniae. The increased expression of 1,3-propanediol oxidoreductase confirmed the role of DhaR as a positive regulator of the dhaT gene.