Metabolic engineering and scale-up of Klebsiella michiganensis for high-level 2,3-butanediol production and agricultural application

Abstract

2,3-Butanediol (2,3-BDO) is a versatile platform chemical with applications ranging from industrial solvents and fuel additives to agricultural biostimulants and biopesticides. However, its industrial deployment requires robust microbial production at large scale with high titer, yield, and productivity, as well as clear demonstration of functional value. In this study, we engineered Klebsiella michiganensis (strain KH001) by eliminating major by-product pathways (ldhA, ackA-pta, poxB) and enhancing the budABC operon via promoter replacement, thereby efficiently redirecting carbon flux toward 2,3-BDO synthesis. The engineered strain achieved 115.1 +/- 2.5 g/L of 2,3-BDO with a productivity of 3.84 +/- 0.18 g/L/h in a 5 L bioreactor, and maintained strong performance during scale-up, reaching 104.2 g/L and 2.31 g/L/h productivity in a 500 L pilot bioreactor. Beyond production, the cell-free fermentation broth containing predominantly meso2,3-BDO exhibited remarkable agricultural efficacy. Soil application of the broth suppressed rootknot nematode (Meloidogyne incognita) infection in tomato plants, achieving control comparable to or exceeding a commercial nematicide. In addition, treated plants showed improved growth under nematode stress and enhanced drought tolerance, maintaining shoot biomass and leaf turgor under water deficit conditions. These findings establish the dual role of fermentationderived 2,3-BDO as both a biopesticide and a biostimulant, addressing both biotic and abiotic stress.