Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

Abstract

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.

Cell surface engineering of industrial microorganisms for biorefining applications

“…Using this system, several proteins, including xylose isomerase (Ota et al, 2013), laccase (Nakanishi et al, 2012), and expansin-like protein , have been displayed on the yeast cell surface. Although numerous technologies for surface display systems have been reported of late (Fushimi et al, 2013;Qiu et al, 2014;Wilde et al, 2012;Yi et al, 2013;Zhang et al, 2013), the development of a wider variety of display systems would broaden the potential applications of surface engineered microorganisms toward industrial applications.…”

Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium

“…The resistance to Cd 2+ by displayed SMT yeast results from the enhanced adsorption of toxic heavy metal ions on their surface (Fig. 2), which is similar to the resistance to Cd 2+ by YMT and hexa-His-displaying yeast cells [17,18] Binding heavy metal ions at the cell surface is probably a mechanism of resistance like intracellular sequestration and efflux pumping [9,24,28]. The cell division of nonengineered yeasts was inhibited by 80 µM Cd 2+ [16] whereas the engineered ones lived in the 0.5 mM Cd 2+ -containing medium (Fig.…”

High β-Glucosidase Secretion in Saccharomyces cerevisiae Improves the Efficiency of Cellulase Hydrolysis and Ethanol Production in Simultaneous Saccharification and Fermentation

“…Other studies have also shown that the expression of β-glucosidase genes with signal sequence in S. cerevisiae can also confer the recombinant strain to secrete β-glucosidase and utilize cellobiose [13,27]. Wilde et al compared the activity of 35 β-glucosidases from 12 filamentous fungi such as Aspergillus niger, and Aspergillus oryzae expressed in S. cerevisiae, and found the β-glucosidase from A. niger had the highest cellobiase activity [34]. The BGL1 gene from S. fibuligera is also frequently expressed in S. cerevisiae owing to its high activity [4,11,28,36].…”