Elimination of D-Lactate Synthesis Increases Poly(3-Hydroxybutyrate) and Ethanol Synthesis from Glycerol and Affects Cofactor Distribution in Recombinant Escherichia coli

Abstract

The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP ratio was higher than the NADH/NAD ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP. In 60-h fed-batch cultures, K24KL reached 41.9 g / liter biomass and accumulated PHB up to 63% (wt/wt), with a PHB yield on glycerol of 0.41 g /g, the highest reported using this substrate.