Cellulase Cel5H related reagents and their use in microorganisms
View Patent ↗The invention relates to applications of the cellulase Cel5H of Saccharophagus degradans and its homologues, functional fragments and/or variants and engineered forms thereof, in the context of recombinant, more particularly solventogenic microorganisms, more particularly C. acetobutylicum . The invention also characterizes a novel domain of the Cel5H cellulase with a putative cellulose-binding module function, and its uses in chimeric proteins for depolymerization of cellulose containing substrates.
1. A recombinant Clostridium bacterium transformed with and expressing the Cel5H polypeptide of Saccharophagus degradans strain 2-40 or a homologue thereof having at least 95% sequence identity with said Cel5H polypeptide of Saccharophagus degradans strain 2-40, wherein said recombinant bacterium is capable of secreting said Cel5H polypeptide and capable of degrading cellulose.
2. The recombinant bacterium according to claim 1 comprising a recombinant nucleic acid molecule encoding said Cel5H polypeptide of Saccharophagus degradans strain 2-40 or said homologue having at least 95% sequence identity with said Cel5H polypeptide of Saccharophagus degradans strain 2-40, operably linked to regulatory sequences which allow for expression in said microorganism.
3. The recombinant bacterium according to claim 1 , which is a solventogenic bacterium.
4. The recombinant bacterium according to claim 1 , which is Clostridium acetobutylicum.
5. The recombinant bacterium according to claim 1 , wherein said Cel5H polypeptide of Saccharophagus degradans strain 2-40 or said homologue having at least 95% sequence identity with said Cel5H polypeptide of Saccharophagus degradans , is fused to a glycoside hydrolase (GH) catalytic domain, a carbohydrate-binding module (CBM) domain, a cohesin-binding domain, or a hydrophilic (X module) domain of a cellulosomal scaffoldin protein.
6. The recombinant bacterium according to claim 1 , wherein said Cel5H polypeptide of Saccharophagus degradans strain 2-40 or said homologue thereof, and optionally the one or more enzymes capable of degrading a substance comprising cellulose, are comprised in a covalent cellulosome.
7. A method for the degradation of a substance comprising cellulose, wherein said method comprises contacting said substance with the recombinant Clostridium bacterium according to claim 1 , growing said recombinant bacterium on said substance comprising or enriched in crystalline or semi-crystalline cellulose and ensuring direct production of the degraded cellulose comprising substance.
8. A method for producing a solvent, fuel or chemical intermediate from a substance comprising cellulose, wherein said method comprises treating said substance with the recombinant microorganism of the genus Clostridium according to claim 1 , growing said recombinant microorganism on said substance comprising or enriched in crystalline or semi-crystalline cellulose, and ensuring direct production of said solvent, fuel or chemical intermediate from said substance comprising cellulose.
9. The method according to claim 7 , wherein said substance comprises or is enriched in crystalline cellulose.
10. The method according to claim 8 , wherein said substance comprises or is enriched in crystalline cellulose.
11. The recombinant bacterium according to claim 3 , wherein the solventogenic bacterium is an ethanologenic bacterium.
12. The recombinant bacterium according to claim 1 , wherein said Cel5H polypeptide of Saccharophagus degradans strain 2-40, or a homologue thereof, comprises one or more domains chosen from or corresponding to the GH5 domain, the CBM6 domain, or the DZ domain of Cel5H.
13. The method according to claim 7 , wherein the method is performed at less than 50° C.
14. The method according to claim 8 , wherein the method is performed at less than 50° C.