title: Restriction modification system analysis and development of in vivo methylation for the transformation of Clostridium cellulovorans.
abstract: Clostridium cellulovorans, a cellulolytic bacterium producing butyric and acetic acids as main fermentation products, is a promising host for biofuel production from cellulose. However, the transformation method of C. cellulovorans was not available, hindering its genetic engineering. To overcome this problem, its restriction modification (RM) systems were analyzed and a novel in vivo methylation was established for its successful transformation in the present study. Specifically, two RM systems, Cce743I and Cce743II, were determined. R. Cce743I has the same specificity as LlaJI, recognizing 5'-GACGC-3' and 5'-GCGTC-3', while M. Cce743I methylates the external cytosine in the strand (5'-GACG(m)C-3'). R. Cce743II, has the same specificity as LlaI, recognizing 5'-CCAGG-3' and 5'-CCTGG-3', while M. Cce743II methylates the external cytosine of both strands. An in vivo methylation system, expressing M. Cce743I and M. Cce743II from C. cellulovorans in Escherichia coli, was then established to protect plasmids used in electrotransformation. Transformants expressing an aldehyde/alcohol dehydrogenase (adhE2), which converted butyryl-CoA to n-butanol and acetyl-CoA to ethanol, were obtained. For the first time, an effective transformation method was developed for metabolic engineering of C. cellulovorans for biofuel production directly from cellulose.
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA.|Department of Molecular Genetics, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA.
authors.forename: Xiaorui
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Xu
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA. yang.15@osu.edu.|Department of Molecular Genetics, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA. yang.15@osu.edu.
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D046974:Clostridium cellulovorans / Q000201:enzymology* / Q000235:genetics*; D019175:DNA Methylation; D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D018014:Gene Transfer Techniques; D060847:Metabolic Engineering; D014169:Transformation, Bacterial
chemical_list: D000440:Butanols; D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial; D000431:Ethanol; D000426:Alcohol Dehydrogenase
keywords: Butanol; Clostridium cellulovorans; Ethanol; Methylation; Restriction modification systems; Transformation