Создание рекомбинантных штаммов дрожжей для получения биоэтанола из целлюлозосодержащего сырья

S. M. Taipakova; I. T. Smekenov; A. K. Bissenbaev

Authors

S. M. Taipakova Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Kazakhstan, Almaty
I. T. Smekenov Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Kazakhstan, Almaty
A. K. Bissenbaev Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Kazakhstan, Almaty

Keywords:

Saccharomyces cerevisiae, cellobiohydrolase, β-glycosidase, endo-1, 4-β-glucanase, signal peptide of yeast α-factor, genomic integration.

Abstract

Biodegradation of the cellulose with the formation of soluble sugars is catalyzed by specific multi-enzyme cellulase system composed of endo-β-1,4-glucanase, exo-1,4-β-glucanase (cellobiohydrolase), exo-1,4-β-glucosidase. Properties of individual enzymes and their interactions in these cellulase complexes determine the efficiency of the hydrolytic degradation of cellulose substrates to soluble oligomeric and monomeric sugars. Glucose and other sugars produced by enzymatic hydrolysis of lignocellulosic biomass can be further converted by certain microorganisms into bioethanol and/or biobutanol.

By means of gene engineering approaches the pHO-GAPDH-eng1-GAPDH-α-cel7A-myc-6xHis-GAPDH-α-bglI-flag-KanMX4-HO integral cassette, including endo-β-1, 4-glucanase, exo-1, 4-β-glucanase (cellobiohydrolase), exo-1, 4-β-glucosidase genes was constructed. Each gene included in integral vector are comprises the signal peptide of yeast α-factor and expressed under the control of a promoter of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The constructed vector provides targeted integration of cellulose genes at S. cerevisiae HO locus. Integral vector contain KanMX selectable marker, and integrants can be selected by resistance to G418.

New stable yeast strains carrying cellulose genes in yeast genome were developed. Chromosomal integration of cellulase genes into HO locus of yeast genome was confirmed by PCR. The strain shows continuous expression of cellulase genes and secretion of the protein products into surrounding medium. It has been shown that recombinant S. cerevisiae expressing cellulase genes became able to grow in synthetic medium containing cellobiose or carboxymethyl-cellulose as the single carbon source. Moreover, the recombinant strain produced 15,6 g/L ethanol from 20% cellobiose and 20% CMC. These results suggest that recombinant S. cerevisiae strains may be applicable to the simultaneous saccharification and fermentation cellulose biomass.