Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains.
Beta-glucosidase genes of fungal origins were isolated and heterologously expressed in Saccharomyces cerevisiae to enable growth on the disaccharide, cellobiose. To promote secretion of the beta-glucosidases, the genes were fused to the secretion signal of the Trichoderma reesei xyn2 gene and constitutively expressed from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The two most promising enzymes, BGL1 from Saccharomycopsis fibuligera and BglA from Aspergillus kawachii, were anchored to the yeast cell surface by fusing the mature proteins to the alpha-agglutinin (AGalpha1) or cell wall protein 2 (Cwp2) peptides. The maximum specific growth rates (mu(max)) of the recombinant S. cerevisiae strains were determined in batch cultivation. S. cerevisiae secreting the recombinant S. fibuligera BGL1 enzyme sustained growth aerobically and anaerobically, in minimal medium containing 5g L(-1) cellobiose at 0.23 h(-1) (compared to 0.29 h(-1) on glucose) and 0.18 h(-1) (compared to 0.25 h(-1) on glucose), respectively. Substrate consumption and product formation were determined to evaluate product yields in glucose and cellobiose.
van Rooyen
Ronél
R
Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, Stellenbosch, South Africa.
Hahn-Hägerdal
Bärbel
B
La Grange
Daniël C
DC
van Zyl
Willem H
WH
D000373:Agglutinins / Q000737:chemistry / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D019149:Bioreactors; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D002475:Cellobiose / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D004271:DNA, Fungal; D005285:Fermentation; D005800:Genes, Fungal; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D012442:Saccharomycopsis / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature; D014242:Trichoderma / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism
D000373:Agglutinins; D003470:Culture Media; D004271:DNA, Fungal; D011994:Recombinant Proteins; D002475:Cellobiose; D001617:beta-Glucosidase; D005947:Glucose