Development of genetic transformation and heterologous expression system in carboxydotrophic thermophilic acetogen Moorella thermoacetica
Open accessAbstract
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.
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PyrF has been shown to be functional in thermophilic acetogen M. thermoacetica.
“PyrF has also been shown to be functional in thermophilic acetogen M. thermoacetica (Kita et al., 2013).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Kita et al., 2013
Heterologous expression of ldh from Thermoanaerobacter pseudethanolicus in M. thermoacetica results in production of 6.8 mM lactate on fructose.
“The gene ldh (encodes lactate dehydrogenase) from Thermoanaerobacter pseudethanolicus was inserted into the genome, resulting in the production of 6.8 mM lactate. Experiments were, however, performed on fructose rather than gas (Kita et al., 2013).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Kita et al., 2013
M. thermoacetica with genome insertion of ldh from Thermoanaerobacter pseudethanolicus into the pyrF locus produced up to 613 mg/L lactate from glucose in bottles under heterotrophic conditions; autotrophic production was not reported.
“| M. thermoacetica | Genome insertion of ldh from Thermoanaerobacter pseudethanolicus into pyrF locus | Heterotrophic production of up to 613 mg/L lactate from glucose in bottles; Autotrophic production not reported | Kita et al., 2013 |”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Kita et al., 2013
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References 31 · Cited by 75
- Authors
- Kita, A., Iwasaki, Y., Sakai, S., Okuto, S., Takaoka, K., Suzuki, T., Yano, S., Sawayama, S., Tajima, T., Kato, J., Nishio, N., Murakami, K., & Nakashimada, Y.
- Affiliations
- National Institute of Advanced Industrial Science and Technology (JP)
- Hiroshima University (JP)
- Mitsui Engineering and Shipbuilding (Japan) (JP)
- Gifu University (JP)
- Kyoto University (JP)
- Corresponding author
- Nakashimada, Yutaka
- ORCID
- Venue
- Journal of Bioscience and Bioengineering
- Publisher
- Elsevier BV
- Published
- 2013-04
- Volume
- 115
- Issue
- 4
- Pages
- 347-352
- ISSN
- 1347-4421 (ISSN-L)
- 1389-1723
- OpenAlex
- W1980696141
- Cited by
- 75
- References
- 31
- Citations by year
- 2026: 3 · 2024: 7 · 2023: 4 · 2022: 7 · 2021: 6 · 2020: 3 · 2019: 7 · 2018: 3 · 2017: 11 · 2016: 7 · 2015: 4 · 2014: 7 · 2013: 6
- FWCI
- 2.18
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Journal of Bioscience and Bioengineering (acceptedVersion)
- PubMed (publishedVersion)
- Funders
- Japan Science and Technology Agency
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Physical Sciences › Engineering › Building and Construction › Anaerobic Digestion and Biogas Production
- Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
- Keywords
- Moorella thermoacetica, genetic transformation, heterologous expression, uracil auxotrophy, restriction-modification systems, homologous recombination, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase promoter, lactic acid production, fructose, hydrogen production, CO2 utilization
- MeSH terms
- Carbon Dioxide, Carboxy-Lyases, Fermentation, Hydrogen, L-Lactate Dehydrogenase, Lactates, Plasmids, Transformation, Bacterial, Gene Expression, Moorella
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