Utilization of excess NADH in 2,3-butanediol-deficient Klebsiella pneumoniae for 1,3-propanediol production.

Abstract

To utilize excess NADH for 1,3-propanediol production by 2,3-butanediol-deficient mutants, the effect of dhaT overexpression in two distinct 2,3-butanediol-deficient mutants was investigated. Two 2,3-butanediol-deficient mutants, KG1-3 (blocking of the 2,3-butanediol pathway only) and KG1-5 (blocking of both of 2,3-butanediol and lactate pathways) were constructed. Our results showed that although the intracellular redox balance (NADH/NAD(+)) was extremely high at the end of fermentation for both mutants, the status of intracellular redox in KG1-5 was maintained at a normal level following the first stage of fermentation. Analysis of cell growth and metabolite formation confirmed the inhibition of excess lactate in 2,3-butanediol pathway-deficient mutants. Furthermore, dhaT was overexpressed in two 2,3-butanediol-deficient mutants (KG1-3T and KG1-5T). In KG1-5T, the intracellular redox balance was restored to normal and 1,3-propanediol production increased. The yield of 1,3-propanediol from glycerol in KG1-5T was also restored to a normal level of 0·6. The excess NADH in both the 2,3-butanediol- and lactate-deficient mutants can be used by overexpresstion of dhaT. The metabolic flux tended to increase lactate production by the abolishment of the 2,3-butanediol pathway in Klebsiella pneumoniae, and the high accumulation of lactate prevented the cell from using excess NADH, thereby inhibiting cell growth and 1,3-propanediol production.