2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae.

Abstract

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process. Key Points • A biological route of 2,3-dihydroxyisovalerate production was setup. • Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae. • Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell. • 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation.