Abstract
Converting lignocellulosic biomass into value-added products is one of the challenges for the development of a sustainable economy. Contrary to Saccharomyces cerevisiae, B. raffinosifermentans naturally assimilates xylose and arabinose, two pentoses that composed the xylan found in the cereals and hardwoods hemicellulose. Additionally, this yeast species has a strong tolerance to various environmental conditions such as high temperatures and high salt concentrations. To use B. raffinosifermentans as a cell factory to hydrolyse hemicellulosic polysaccharides and convert released sugars into high value products, we first developed efficient expression and secretion vectors. We characterized new promoters : one of them, the TDH3 promoter, confers high level constitutive gene expression ; the HSP26, PCK1 and XYL1 promoters were shown to be induced by high temperature, glycerol and xylose polymers respectively. Two endogenous protein secretion signals were then used to ensure the secretion of xylan-degrading enzymes. Using these tools, strains constitutively expressing either a β-endoxylanase from a new Blastobotrys species, a -arabinofuranosidase from Apiotrichum siamense or the endogenous -glucuronidase were finally designed. Capacities of these strains to grow on different xylan sources such as by-products of several industries will be presented in this poster. In order to be able to follow each of the three strains in a synthetic consortia during hemicellulose deconstruction, we developed three fluorescent reporter cassettes. For the purpose, the YFP, CFP and RedStar coding sequences were cloned downstream of the XYL1 promoter and co-expressed with each of the enzymes. In parallel, we plan to inactivate the PEP4 gene by a CRISPR-Cas9 approach to increase the levels of secreted proteins. These engineered yeasts optimized for efficient bioconversion of various plant by-products could be used to produce any substrates of high added value.