Engineering yeast for efficient cellulose degradation.
Saccharomyces cerevisiae produces several beta-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the beta-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefacies cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo-beta-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-beta-D-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities.
Van Rensburg
P
P
Institute for Wine Biotechnology, University of Stellenbosch, South Africa.
Van Zyl
W H
WH
Pretorius
I S
IS
D001426:Bacterial Proteins; D015152:Blotting, Northern; D015139:Blotting, Southern; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; C066565:ced1 protein, Butyrivibrio fibrisolvens; D001617:beta-Glucosidase; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase