Cellulases and coding sequences

Details Cellulases and coding sequences Cellulases and coding sequences

2000-10-12

2001-07-31

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S252300, C435S252330, C435S252500, C536S023200

Reexamination Certificate

active

06268198

ABSTRACT:

BACKGROUND OF THE INVENTION
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), &bgr;-glucosidase (also called cellobiase, E.C. 3.2.1.21). Endoglucanases hydrolyze &bgr;-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). &bgr;-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 2×10
6
to 6.5×10
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 &bgr;-glucosidase which act synergistically on the substrate.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a cellulase coding sequence for a cellulase selected from the group consisting of CelA, CelB and CelC. Besides the specifically exemplified coding sequences isolated from Orpinomyces PC2, the present invention also encompasses all synonymous coding sequences for each of the exemplified amino acid sequences disclosed herein and coding sequences for cellulase enzymes having at least about 90% amino acid sequence identity with an exemplified sequence.
Also provided by the present invention are recombinant host cells genetically engineered to contain and express the foregoing cellulase coding sequences. Such recombinant host cells can be fungal or bacterial. Preferred fungal host cells include, but are not limited to,
Saccharomyces cereviside, Aspergillus niger
, Aspergillus, Penicillium,
Pichia pastoris
and
Trichoderma reesei
. Bacterial host cells for cellulase expression can include
Bacillus subtilis, Bacillus stearothermophilus, Escherichia coli
and
Staphylococcus aureus
and Streptomyces, among others.
It is a further object of this invention to provide purified cellulase enzymes (CelA, CelB and CelC) as defined herein. As specifically exemplified, CelA, CelB and CelC have amino acid sequences as provided in SEQ ID NO:2, SEQ ID NO:12 and SEQ ID NO:4. Cellulases of equivalent biological activity and enzymatic specificity having at least about 75% amino acid sequence identity with the exemplified CelA and CelC sequences and at least about 85% amino acid sequence identity with the exemplified CelB are within the scope of the present invention.


REFERENCES:
patent: 5591619 (1997-01-01), Li et al.
Barichievich and Calza (1990) “Supernatant Protein and Cellulase Activities of the Anaerobic Ruminal FungusNeocallismastix frontalisEB188”Applied and Environmental Microbiology56:43-48.
Béguin, P. (1990) “Molecular Biology of Cellulose Degradation”Annu. Rev. Microbiol.44:219-248.
Borneman et al. (1989) “Fermentation Products and Plant Cell Wall-Degrading Enzymes Produced by Monocentric and Polycentric Anaerobic Ruminal Fungi”Applied and Environmental Microbiology55:1066-1073.
Borneman and Akin (1994) “The Nature of Anaerobic Fungi and Their Polysaccharide Degrading Enzymes”Mycoscience35:199-211.
Chen et al. (1995) “A Cyclophilin from the Polycentric Anaerobic Rumen Fungus Orpinomyces sp. Strain PC-2 is Highly Homologous to Vertebrate Cyclophilin B”Proc. Natl. Acad. Sci. USA92:2587-2591.
Chen et al. (1995) “Biomass Degrading Enzymes from Anaerobic Rumen Fungi” In: Southern Association of Agricultural Scientists Bulletin: Biochemistry and Biotechnology, Ballal, S.K. (Ed.). 8:1-6.
Chen et al. (1994) “Isolation and Properties of an Extracellular &bgr;-Glucosidase from the Polycentric Rumen Fungus Orpinomyces sp. Strain PC-2”Applied and Environmental Microbiology60:64-70.
Choi and Ljungdahl (1996) “Dissociation of the Cellulosome ofClostridium thermocellumin the Presence of Ethylenediaminetetraacetic Acid Occurs with the Formation of Truncated Polypeptides”Biochemistry35:4897-4905.
Choi and Ljungdahl (1996) “Structural Role of Calcium for the Organization of the Cellulosome ofClostridium thermocellum” Biochemistry35:4906-4910.
Chow et al. (1994) “The cel3 Ge

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