Metabolic engineering of Ruminiclostridium papyrosolvens for 2,3-butanediol production from lignocellulose.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic bacterium capable of secreting cellulosomes for efficient lignocellulose degradation, making it a promising candidate for consolidated bioprocessing (CBP). However, its native cellulolytic activity is repressed by cellobiose, and its natural fermentation products are limited to low-value compounds. In this study, we firstly discovered that the cellulosomal cip-cel gene cluster is subject to cellobiose-mediated carbon catabolite repression (CCR). By replacing its native promoter with the endogenous cellobiose/cellodextrin-inducible promoter Pcel using ClosTron, CCR was eliminated and sustained cellulose hydrolysis was enabled. We subsequently introduced the heterologous genes encoding acetolactate decarboxylase (ALD) and 2,3-butanediol dehydrogenase (BDH) to construct a 2,3-butanediol (2,3-BDO) biosynthetic pathway in R. papyrosolvens, and stably integrated both genes into the genome via the serine integrase-mediated Att/Int system. Furthermore, disruption of the endogenous ethanol synthesis pathway redirected carbon flux and reducing equivalents toward 2,3-BDO production. The final engineered strain, cultivated on unpretreated corn stover as the sole carbon source, produced 140 μg/mL of 2,3-BDO in a batch CBP fermentation. These metabolic engineering strategies provided a valuable framework for the efficient bioconversion of renewable lignocellulosic biomass into high-value chemicals.