Background: The complete hydrolysis of cellulosic residues requires three types of cellulases: endoglucanases [1], exoglucanases [2], and cellobiases (Cels) [3]. Before 2012, only two cellobiase (cel) genes from Cellulomonas biazotea (C. biazotea), cba and cba2, were sequenced and studied. The characterization and understanding of the functional roles of C. biazotea Cels may enable their utilization in various industrial applications, such as cellulosic waste management, and the production of biofuels and biomaterials. Consequently, my research studies have focused on the cloning and characterization of novel cel genes from C. biazotea, and the expression of recombinant Cels in Escherichia coli (E. coli).

Methodology/Principal Findings: To search for novel cels in C. biazotea,...[ Read more ]

Background: The complete hydrolysis of cellulosic residues requires three types of cellulases: endoglucanases [1], exoglucanases [2], and cellobiases (Cels) [3]. Before 2012, only two cellobiase (cel) genes from Cellulomonas biazotea (C. biazotea), cba and cba2, were sequenced and studied. The characterization and understanding of the functional roles of C. biazotea Cels may enable their utilization in various industrial applications, such as cellulosic waste management, and the production of biofuels and biomaterials. Consequently, my research studies have focused on the cloning and characterization of novel cel genes from C. biazotea, and the expression of recombinant Cels in Escherichia coli (E. coli).

Methodology/Principal Findings: To search for novel cels in C. biazotea, a shotgun approach and PCR-based probing were employed. From the experiments, four cel candidates, designated cba3, cba4, cba5 and Cbgh3d, were obtained. The cba3 [3], cba4 and cba5 genes were fully characterized, and their encoded enzymes were demonstrated to be bona fide secretory Cels, despite their absence of classical secretion signals. Biochemical studies on Cba, Cba3, Cba4 and Cba5 support that Cba, Cba3 and Cba4 are multifunctional enzymes, which exhibit not only Cel, but also hemicellulase activities. The results suggest that the four Cels are unlikely to be functionally redundant in C. biazotea. The strategy of extracellular production (excretion) of cellulases has many advantages over its intracellular counterpart in biotechnological applications [4]. In my dissertation, I have also introduced and discussed an innovative approach of employing histidine-tag sequences to greatly enhance the secretion of recombinant Cba, Cba3 and Cba4 in E. coli.

Conclusions/Significance: A total of six cel genes have been cloned by our research group from C. biazotea, of which cba3, cba4 and cba5 have been characterized in detail in my studies. Moreover, an innovative approach is introduced for the efficient excretion of non-classical secretory proteins in E. coli.