RESUMO
Both Saccharomyces and non-Saccharomyces yeast strains are of great importance for the fermentation industry, especially with the flourishing of craft breweries, which are driving current innovations. Non-conventional yeasts can produce novel beverages with attractive characteristics such as flavour, texture, and reduced alcohol content; however, they have been poorly explored. A new method for screening the fitness of conventional and non-conventional yeast libraries utilising robotic platforms and solidified media representing industrial conditions is proposed. As proof of concept, a library formed of 6 conventional and 17 non-conventional yeast strains was distributed in 96, 384 and 1536 arrays onto a YPD agar medium. Following this, the library was replicated in different conditions mimicking beer and cider fermentation conditions. The colony size was monitored over time, and fitness values measured in maximum pixels/h and maximum biomass were calculated. Significant differences in growth were observed in between the different strains and conditions. As examples, Candida milleri Y-7245 displayed good performance in wort conditions, and Kazachstania yakushimaensis Y-48837 stood out for its performance in apple juice. The method is proposed to be used as a pre-screening step when studying vast yeast libraries. This would enable interested parties to discover potential hits for further study at a low initial cost. Furthermore, this method can be used in other applications where the desired screening media can be solidified.
RESUMO
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.
