Metabolic engineering of Klebsiella oxytoca for highly selective meso-2,3-butanediol production with minimized byproducts.

Abstract

Producing enantiopure meso-2,3-butanediol (meso-2,3-BDO) with minimal organic acid byproducts is critical for its commercial applications in specialized polymers and cosmetics. In this study, we developed a highly selective and robust Klebsiella oxytoca platform using stepwise metabolic engineering and bioprocess optimization. Starting with a lactate- and formate-deficient parent strain (Δldh/ΔpflB), we sequentially deleted poxB and frdBC to suppress acetate and succinate byproducts. To resolve recalcitrant succinate accumulation caused by NADH surplus during micro-aerobic fed-batch fermentation, we implemented dynamic redox control strategies (nutrient supplementation and agitation shifts) and permanently sealed the reductive TCA branch by deleting mdh. Furthermore, to achieve ultra-high stereoisomeric purity, we disrupted the isomer interconversion network by knocking out dar and gldA. The resulting engineered strain (GSC-P8) was cultivated using the optimized redox-control bioprocess. This integrated scale-up fermentation completely abolished undesired stereoisomers, strictly maintaining meso-2,3-BDO selectivity at approximately 97.9%. We achieved a prominent meso-2,3-BDO titer of 125.2 g/L and a volumetric productivity of 2.84 g/L/h, while restricting each acidic byproduct to below 2.0 g/L. This study demonstrates a commercially viable strategy for pure meso-2,3-BDO production through rigorous intracellular redox homeostasis.