Function of the Glycoside Hydrolase Family 8 Endoglucanases in Cytophaga Hutchinsonii Cellulose Utilization
Start Date
15-4-2021 2:00 PM
End Date
15-4-2021 3:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Yongtao Zhu
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, digests crystalline cellulose efficiently, but the mechanism by which it does this is unclear and is different from other well studied cellulolytic bacteria. The two well understood mechanisms involve secreting soluble extracellular cellulases or producing cell-associated cellulosomes. In both cases, endoglucanases (EGs) cleave the disordered amorphous regions and cellobiohydrolases (CBHs) attack the free ends generated by the EGs processively, releasing cellobiose. β-glucosidases (BGs) then digest cellobiose to glucose, finalizing the process of cellulose breakdown. C. hutchinsonii appears to employ a unique cellulose utilization mechanism. It does not produce cellulosomes, but its cellulolytic enzymes are primarily cell associated. Genomic analysis revealed predicted EGs belonging to the glycoside hydrolase families 5, 8, and 9 (GH5, GH8, and GH9). No CBHs, essential in digestion of crystalline regions of cellulose used by other bacteria, were predicted in the genome. C. hutchinsonii may utilize novel enzymes to cleave crystalline cellulose and transport fragments of cellulo-oligosaccharides across its outer membrane, then digesting these internally in the periplasm.
We employed a sacB-mediated deletion strategy to create mutants lacking cel8A, one of the GH8 encoding genes. Cells of the cel8A mutants were able to grow on cellulose as well as the wild type, indicating this gene is not essential for C. hutchinsonii cellulose utilization. We plan to delete all the other GH8 encoding genes including cel8B, cel8C, cel8D, and cel8E individually and in combinations. Growth of the mutants on various carbohydrate substrates, especially cellulose, will aid in the determination of GH8 gene function as it relates to cellulose utilization by C. hutchinsonii.
Key words: Cytophaga hutchinsonii, cellulose, cellulase
Function of the Glycoside Hydrolase Family 8 Endoglucanases in Cytophaga Hutchinsonii Cellulose Utilization
Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, digests crystalline cellulose efficiently, but the mechanism by which it does this is unclear and is different from other well studied cellulolytic bacteria. The two well understood mechanisms involve secreting soluble extracellular cellulases or producing cell-associated cellulosomes. In both cases, endoglucanases (EGs) cleave the disordered amorphous regions and cellobiohydrolases (CBHs) attack the free ends generated by the EGs processively, releasing cellobiose. β-glucosidases (BGs) then digest cellobiose to glucose, finalizing the process of cellulose breakdown. C. hutchinsonii appears to employ a unique cellulose utilization mechanism. It does not produce cellulosomes, but its cellulolytic enzymes are primarily cell associated. Genomic analysis revealed predicted EGs belonging to the glycoside hydrolase families 5, 8, and 9 (GH5, GH8, and GH9). No CBHs, essential in digestion of crystalline regions of cellulose used by other bacteria, were predicted in the genome. C. hutchinsonii may utilize novel enzymes to cleave crystalline cellulose and transport fragments of cellulo-oligosaccharides across its outer membrane, then digesting these internally in the periplasm.
We employed a sacB-mediated deletion strategy to create mutants lacking cel8A, one of the GH8 encoding genes. Cells of the cel8A mutants were able to grow on cellulose as well as the wild type, indicating this gene is not essential for C. hutchinsonii cellulose utilization. We plan to delete all the other GH8 encoding genes including cel8B, cel8C, cel8D, and cel8E individually and in combinations. Growth of the mutants on various carbohydrate substrates, especially cellulose, will aid in the determination of GH8 gene function as it relates to cellulose utilization by C. hutchinsonii.
Key words: Cytophaga hutchinsonii, cellulose, cellulase
