Development of industrial cellobiose fermenting Saccharomyces cerevisiae strains for the lignocellulosic bioethanol.

In recent years increasing attention has been devoted to the production of bioethanol from lignocellulose biomass, as cheap and abundant feedstock. To date a cost-effective method for the industrial production of bioethanol from vegetal biomass has not been developed. One of the most attractive strategies is the construction of a CBP (Consolidated BioProcessing) microbe able both to hydrolyze the complex polymers of lignocellulosic biomass and to convert the resulting sugars into ethanol. The aim of this work was to develop an industrial S. cerevisiae yeast able to convert cellobiose into bioethanol. To achieve this goal, two distinct strategies were followed, namely: 1- The selection of a robust newly isolate yeast strain, having both excellent fermenting abilities and inhibitor tolerance. 2- The engineering of the selected yeast for the secretion of the β-glucosidase BglI of Saccharomycopsis fibuligera. Grape marcs supplemented with an inhibitors cocktail was found to be a great source of yeasts . ITS sequencing indicated that the obtained 336 isolates belong to the following four major species: Candida glabrata, C. zemplinina, Issatchenkia orientalis and S. cerevisiae. All the yeasts identified as I. orientalis were isolated at 40°C, while the other strains have been obtained at 30 °C. Twenty-one yeasts, identified as S. cerevisiae, showed promising fermentative vigour in MNS medium with glucose and xylose at 25 and 40 °C. Their tolerance to inhibitors commonly present in lignocellulosic hydrolysates was also tested. S. cerevisiae T2, T9, T11 and T12, selected as the most proficient fermenting yeasts, showed good tolerance to the inhibitors cocktail B. As a result, S. cerevisiae T2 was selected in order to start a molecular biology programme for the development of an efficient cellulolytic yeast. Four bechmark S. cerevisiae strains were used: 27P, F12 and Fp96 and YI30. The wild type S. cerevisiae strains were transformed with the XhoI digested pBKD1_BGL1. Recombinant cells were plated onto YPD with sorbitol plates, supplemented with geneticin, in order to select the positive transformed yeasts. Once grown, the yeast colonies of greater size were selected for further tests of β-glucosidase activity.Recombinant strains were transferred onto fresh YPD plates formulated with 4-methyl-umbelliferyl-β-D-glucopyranoside (4-MUG) as substrate. T2[pBKD1_BGL1] and 27P[pBKD1_BGL1] exhibited the largest hydrolysis halos and both yeasts were found to be mitotically stable. The enzymatic activity of engineered yeasts was then detected in liquid assays, using p-nitrophenyl-β-D-glucopyranoside (pNPG). Their parental yeasts, S. cerevisiae T2 and 27P, did not produce any detectable activity while the engineered strains exhibited interesting hydrolytic abilities. In this work a new method for the selection of yeasts suitable for the production of second generation bioethanol has been developed. This procedure allowed to obtain promising yeasts having both high fermentative abilities, at 25 and 40 °C, and interesting inhibitor tolerance. Moreover, the S. cerevisiae T2, selected as the most promising candidate, has been genetically modified for the produce of S. fibuligera β-glucosidase. The resulting recombinant strain produced interesting and improvable cellobiose-hydrolyzing activities.