|Skory, Christopher - Chris|
Submitted to: Molecular and General Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/1996
Publication Date: N/A
Citation: N/A Interpretive Summary: Agricultural biomass such as plant stalks, grasses, wood chips, and paper refuse represents an abundant renewable resource that can be used for the production of fuel ethanol. The first requirement before these products can be utilized is that they be broken down into simple sugars that yeasts can use for fermenting ethanol. One particular enzyme important in this breakdown scheme is called beta-glucosidase. Because of the unique and desirable characteristics not found with other beta-glucosidase enzymes, our laboratory has been involved in studying this enzyme. We have previously isolated the gene that is responsible for making beta-glucosidase. In this paper, we describe the modification and expression of this gene in the common brewing yeast. This brewing yeast is unsurpassed for its ability to produce ethanol in commercial facilities. However, this organism does not possess any beta-glucosidase activity. As a result, if it is to be used in a simultaneous biomass breakdown and fermentation process, beta-glucosidase levels must be supplemented at an additional cost. We were able to design several different genetically engineered strains that now have beta-glucosidase activity. With additional improvements on expression levels, these strains will be beneficial to the fuel ethanol industry.
Technical Abstract: The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2 um replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.