The small noncoding RNA sr8384 determines solvent synthesis and cell growth in industrial solventogenic clostridia

Abstract

20Small noncoding RNAs (sncRNAs) are crucial regulatory molecules in organisms and 21 are well known not only for their roles in the control of diverse essential biological 22 processes but also for their value in genetic modification. However, to date, in 23 gram-positive anaerobic solventogenic clostridia (which are a group of important 24 industrial bacteria with exceptional substrate and product diversity), sncRNAs remain 25 minimally explored, leading to a lack of detailed understanding regarding these 26 important molecules and their use as targets for genetic improvement. Here, we 27 performed large-scale phenotypic screens of a transposon-mediated mutant library of 28 Clostridium acetobutylicum, a typical solventogenic clostridial species, and 29 discovered a novel sncRNA (sr8384) that functions as a determinant positive regulator 30 of growth and solvent synthesis. Comparative transcriptomic data combined with 31 genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator 32and controls multiple targets that are associated with crucial biological processes, 33 through direct or indirect interactions. Notably, modulation of the expression level of 34 either sr8384 or its core target genes significantly increased the growth rate, solvent 35 titer and productivity of the cells, indicating the importance of sr8384-mediated 36 regulatory network in C. acetobutylicum. Furthermore, a homolog of sr8384 was 37 discovered and proven to be functional in another important Clostridium species, C. 38 beijerinckii, suggesting the potential broad role of this sncRNA in clostridia. Our 39 work showcases a previously unknown potent and complex role of sncRNAs in 40 clostridia, providing new opportunities for understanding and engineering these 41 3 anaerobes, including pathogenic Clostridium species.42 4 IMPORTANCE 43The discovery of sncRNAs as new resources for functional studies and strain 44 modifications are promising strategies in microorganisms. However, these crucial 45 regulatory molecules have hardly been explored in industrially important 46 solventogenic clostridia. Here, we identified sr8384 as a novel determinant sncRNA 47 controlling cellular performance of solventogenic Clostridium acetobutylicum and 48 performed detailed functional analysis, which is the most in-depth study of sncRNAs 49 in clostridia to date. We reveal the pleiotropic function of sr8384 and its multiple 50 direct and indirect crucial targets, which represents a valuable source for 51 understanding and optimizing this anaerobe. Of note, manipulation of these targets 52 leads to improved cell growth and solvent synthesis. Our findings provide a new 53 perspective for future studies on regulatory sncRNAs in clostridia. 54 5 93 importance of this sncRNA as well as the related gene network in genetic 94 improvement. Furthermore, we also identified a functional sr8384 homolog in C. 95 beijerinckii, another important Clostridium species that is widely used in the 96 fermentation of lignocellulose hydrolysates, indica...