Wood Hydrolysis by Cellulomonas Fimi Endoglucanase and Exogiucanase Coexpressed as Secreted Enzymes in Saccharomyces Cerevisiae

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Secretion of the active enzyme by S. cerevisiae was greatly increased when the leader signal of the K 1 killer toxin of melibiase was inserted immediately upstream of, and in frame with, the bacterial ␤-glucanase sequence. An S. cerevisiae strain expressing both the C. fimi cenA-encoded endo-␤-1,4-glucanase (Eng) and the cex-encoded exo-␤-glucanase (Exg) was able to saccharify filter paper and pretreated aspen wood chips in reaction mixtures that were supplemented with ␤-glucosidase (130,754). In a similar study, the endoglucanase gene (515) and the ␤-glucosidase genes (551,552) from Cellulomonas biazotea were also cloned and successfully expressed in S. cerevisiae.…”

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

“…The availability of host systems for the export of recombinant cellulases into the culture medium facilitates not only the production of the enzymes, but also the studies of their reconstitution and interaction. Although Eng and Exg have been commonly expressed as extracellular enzymes (Fu et al, 2006;Lam et al, 1997, Wang et al, 2010b, 2011Wong and Lam, 2000;Wong et al, 1988), the attainment of Cel as secreted or excreted products is not so common. The low frequency is attributable to the fact that cellulolytic microbes are commonly found to produce cell-bound Cel (Bhat and Bhat, 1997;Wakarchuk et al, 1984), thus hindering the progress of developing recombinant strains expressing extracellular Cel.…”

“…Our laboratory has been involved in research and application of microbial cellulases for a long time (Fu et al, 2005(Fu et al, , 2006Gilkes et al, 1984;Lam et al, 1997Lam et al, , 1998Lau and Wong, 2001;Skipper et al, 1985;Wang et al, 2010bWang et al, , 2011Warren et al, 1986;Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1986Wong et al, , 1988. Over these years, we have cloned and expressed the cex and cenA genes from Cellulomonas fimi, which encode an Exg and an Eng, respectively, in a wide range of host systems (Fu et al, 2006;Lam et al, 1997Lam et al, , 1998Wang et al, 2010bWang et al, , 2011Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1988).…”

“…Over these years, we have cloned and expressed the cex and cenA genes from Cellulomonas fimi, which encode an Exg and an Eng, respectively, in a wide range of host systems (Fu et al, 2006;Lam et al, 1997Lam et al, , 1998Wang et al, 2010bWang et al, , 2011Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1988). To search for Cel that might work co-operatively with Exg and Eng, we have put our focus on Cellulomonas biazotea due to the reasons that i) it secretes high levels of Cel activity to the culture medium (Lau and Wong, 2001;Saratale et al, 2010;, and ii) it is closely related to C. fimi (Wood and McCrae Sheila, 1979).…”

“…To achieve the goal, a number of approaches involving the use of cellulase saccharification in conjunction with fermentation (Öhgren et al, 2007), fermenting organisms expressing transgenic cellulase genes (Wong et al, 1988), and modified cellulolytic microbial strains with enhanced fermentative ability have been contrived (Blanchard et al, 2010). However, regardless of the approach employed, the largest stumbling block is the availability of a practical enzymatic process for cellulose hydrolysis (Merino and Cherry, 2007), which requires a cooperative action among three different types of cellulase: endoglucanase (Eng; EC 3.2.1.4), exoglucanase (Exg; EC 3.2.1.91), and cellobiase (Cel; EC 3.2.1.21).…”