title: Development of genetic transformation and heterologous expression system in carboxydotrophic thermophilic acetogen Moorella thermoacetica.
abstract: To develop a microbial production platform based on hydrogen and carbon dioxide, a genetic transformation system for the thermophilic acetogen Moorella thermoacetica ATCC39073 was developed. The uracil auxotrophic strain dpyrF was constructed by disrupting pyrF for orotate monophosphate decarboxylase. The transformation plasmids were methylated by restriction methylases of M. thermoacetica to avoid the decomposition of introduced plasmids by restriction-modification system. Reintroduction of native pyrF into the mutant by homologous recombination ensured recovery from uracil auxotrophy. To test heterologous gene expression in dpyrF, the lactate dehydrogenase (LDH) gene (T-ldh) from Thermoanaerobacter pseudethanolicus ATCC33223 was electroporated into dpyrF with a promoter of the glyceraldehyde-3-phosphate dehydrogenase (G3PD) gene of M. thermoacetica ATCC39073. The resulting transformant (C31) successfully transcribed T-ldh and exhibited higher LDH activity than ATCC39073 and dpyrF, yielding 6.8 mM of lactate from fructose, whereas ATCC39073 did not produce lactate.
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, Hiroshima, Japan.
authors.forename: Akihisa
authors.initials: A
authors.lastname: Kita
authors.forename: Yuki
authors.initials: Y
authors.lastname: Iwasaki
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Sakai
authors.forename: Shinya
authors.initials: S
authors.lastname: Okuto
authors.forename: Kazue
authors.initials: K
authors.lastname: Takaoka
authors.forename: Tohru
authors.initials: T
authors.lastname: Suzuki
authors.forename: Shinichi
authors.initials: S
authors.lastname: Yano
authors.forename: Shigeki
authors.initials: S
authors.lastname: Sawayama
authors.forename: Takahisa
authors.initials: T
authors.lastname: Tajima
authors.forename: Junichi
authors.initials: J
authors.lastname: Kato
authors.forename: Naomichi
authors.initials: N
authors.lastname: Nishio
authors.forename: Katsuji
authors.initials: K
authors.lastname: Murakami
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nakashimada
mesh_terms: D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D006859:Hydrogen / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D058845:Moorella / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D007773:Lactates; D002245:Carbon Dioxide; D006859:Hydrogen; D007770:L-Lactate Dehydrogenase; D002262:Carboxy-Lyases