title: Metabolic engineering of Clostridium tyrobutyricum for n-butanol production.
abstract: Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Mingrui
authors.initials: M
authors.lastname: Yu
authors.forename: Yali
authors.initials: Y
authors.lastname: Zhang
authors.forename: I-Ching
authors.initials: IC
authors.lastname: Tang
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D000084:Acetate Kinase / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D046969:Clostridium acetobutylicum / Q000201:enzymology / Q000235:genetics; D048014:Clostridium tyrobutyricum / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D008353:Mannitol / Q000378:metabolism / Q000494:pharmacology; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D010709:Phosphate Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D013549:Sweetening Agents / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D013549:Sweetening Agents; D008353:Mannitol; D020001:1-Butanol; C446050:AdhE2 protein, Clostridium acetobutylicum; D010088:Oxidoreductases; D010709:Phosphate Acetyltransferase; D000084:Acetate Kinase