The Lanthipeptide Synthetase-like Protein CA_C0082 Is an Effector of Agr Quorum Sensing in Clostridium acetobutylicum.

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described.

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

RRNPP-type quorum sensing affects solvent formation and sporulation in Clostridium acetobutylicum.

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum.