Engineering Clostridium ljungdahlii for direct acetoin production from one-carbon gases

Autotrophic gas-fermenting Clostridium species are recognized as key chassis for constructing C1 (CO/CO₂) cell factories. However, their limited natural product spectrum restricts broader industrial applications, necessitating an expansion of their product repertoire. Acetoin is widely used in the food, pharmaceutical, and chemical synthesis industries, but its conventional sugar‑based production route is cost-prohibitive. Thus, low-cost alternative carbon sources are needed, and C1 gases offer a promising solution. In this study, using the gas‑fermenting bacterium Clostridium ljungdahlii as the chassis, we engineered a synthetic pathway that integrates acetate reassimilation with pyruvate decarboxylase (PDC)‑catalyzed carboligation, while simultaneously disrupting the native acetoin consumption pathway. This enabled the strain to produce acetoin directly from syngas (CO₂ - CO - H₂). We further deleted key genes responsible for ethanol and 2,3-butanediol synthesis and introduced an NADH oxidase to modulate the intracellular reducing equivalent balance. These modifications effectively suppressed byproduct accumulation and improved acetoin production. The optimal strain, Clj‑acet07, produced 2.52 g·L^-1 of acetoin within 36 h under syngas-fermenting conditions, achieving a production rate of 0.07 g·L^-1·h^-1. Collectively, this study demonstrates a strategy for converting C1 gases into acetoin using gas-fermenting clostridia, offering a new biosynthetic pathway for this valuable chemical.