The field of the present invention is the area of cellulolytic enzymes, nucleotide sequences encoding them and recombinant host cells and methods for producing them.
Cellulose, the most abundant structure of plant cell walls, exists mostly as insoluble microfibril which are formed by hydrogen bonds between individual cellulose chains. Conversion of cellulose to glucose provides readily available carbon sources for fuel and chemical production. Such conversion requires several types of enzymes including endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (also called exoglucanase, E.C. 3.2.1.91), .beta.-glucosidase (also called cellobiase, E.C. 3.2.1.21). Endoglucanases hydrolyze .beta.-glycoside bonds internally and randomly along the cellulose chains whereas cellobiohydrolases remove cellobiose molecules from the reducing and non-reducing ends of the chains (Barr et al., 1996). .beta.-Glucosidases hydrolyze the cellobiose to two molecules of glucose, and therefore eliminate the inhibition of cellobiose on cellobiohydrolases and endoglucanases.
Microorganisms have evolved diverse strategies for efficient break down of plant cell wall constitutes, particularly cellulose. Aerobic organisms tend to secrete individual enzymes whereas some anaerobic bacteria produce high molecular weight enzyme complexes on the cell surface. Examples of such enzyme producers are the fungus Trichoderma reesei and bacteria Cellulomonas fimi and Thermomonospora fusca. Cellulases of these organisms consist of cellulose binding domains (CBD) and catalytic domains joined by linker sequences. Anaerobic bacteria whose cellulolytic systems received extensive investigations include Clostridium thermocellum (Felix and Ljungdahl. 1993. Ann. Rev. Microbiol. 47:791-819; Aubert et al. 1993. In: M. Sebald (ed.) Genetics and Molecular Biology of Anaerobic Bacteria. p. 412-422. Springer-Verlag, NY) and C cellulovorans (Doi et al. 1994. Crit. Rev. Microbiol. 20:87-93). The high molecular weight cellulase complex, more often called the cellulosome, of C. thermocellum contains about 26 polypeptides with a mass in a range of 2.times.10.sup.6 to 6.5.times.10.sup.6 Da (Lamed et al., 1983). These polypeptides include at least one scaffolding protein termed cellulosome integrating protein A (CipA) and a number of catalytically active proteins. The protein and protein interactions forming the cellulosome are effected by conserved duplicated regions (CDR) of the catalytically active proteins and nine internal repeated elements (IRE) of CipA.
Highly efficient cellulases of anaerobic fungi have been demonstrated (Wood et al. 1986. FEMS Microbiol Lett. 34:37-40; Lowe et al. 1987. Appl. Environ. Microbiol. 53:1216-1223; Bomeman et al. 1989. Appl. Environ. Microbiol. 55:1066-1073). A high molecular weight cellulase/hemicellulase complex has been isolated from Neocallimastix frontalis (Wilson and Wood. 1992. Enzyme Microb. Technol. 14:258-264). No individual native cellulases have been purified from anaerobic fungi. On the basis of morphology of sporangia, mycelia and zoospores, anaerobic fungi have been classified into two groups, monocentric and polycentric (Bomeman et al., 1989, supra; Bomeman and Akin. 1994. Mycoscience 35:199-211). Monocentric fungi have only one sporangium developed from one zoosporium, whereas polycentric isolates have multiple sporangia originating from one zoosporium. Most investigations on anaerobic fungi have focused on monocentric isolates, particularly isolates of the genera Neocallimastix and Piromyces. Gene cloning and sequencing of polysaccharidases from the monocentric anaerobic fungi have shown that multiple cellulases and hemicellulases of these fungi may form high molecular weight complexes (HMWC) similar to the cellulosomes of Clostridia (Gilbert et al. 1992. Mol. Microbiol. 6:2065-2072; Zhou et al. 1994. Biochem. J. 297:359-364, Fanutti et al. 1995. J. Biol. Chem. 270:29314-29322). Evidence provided by these studies is three fold: 1) Most of the hydrolases lack cellulose binding domains; 2) They have repeated peptide (RP) domains at the carboxyl termini or between two catalytic domains although they lack sequence homology with the CDRs of cellulosomal catalytic proteins. These regions are not required for catalysis; and 3) The RP domain of a Piromyces xylanase binds to other proteins in the Neocallimastix and Piromyces HMWCs. More recently, however, a cellulase (CelA) of Neocallimastix, which lacks the RP domain but contains a typical fungal CBD and a cellobiohydrase catalytic domain, has been reported (Denman et al., 1996).
By contrast, the polysaccharide hydrolyzing enzymes of aerobic fungi are generally secreted as individual enzymes, including endoglucanases, cellobiohydrolases and .beta.-glucosidase which act synergistically on the substrate.