Small and low but potent: The complex regulatory role of the small RNA SolB in solventogenesis in Clostridium acetobutylicum

Abstract

The recently revived Clostridium acetobutylicum-based acetone-butanolethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology. C. acetobutylicum has been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad, ctfA, ctfB, and adc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, "degenerate" cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion of solB resulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact on adc mRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reduced adhE1 and ctfA-ctfB mRNA levels, and abolished the ctfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of the ctfA transcript, and this was verified by expressing variants of the ctfA-ctfB genes to demonstrate the importance of SolB for acetone formation.