Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways

Moreira, João P.C.; Heap, John T.; Alves, Joana I.; Domingues, Lucília

Abstract

Background: Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO2, and H2, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands.
Results: This work reports the development and optimization of an electrotransformation protocol for A. wieringae strain JM, which can also be used in A. wieringae DSM 1911, and A. woodii DSM 1030. We also show the functionality of the thiamphenicol resistance marker, catP, and the functionality of the origins of replication pBP1, pCB102, pCD6, and pIM13 in all tested Acetobacterium strains, with transformation efficiencies of up to 2.0 × 103CFU/μgDNA. Key factors affecting electrotransformation efficiency include OD600 of cell harvesting, pH of resuspension buffer, the field strength of the electric pulse, and plasmid amount. Using this method, the acetone production operon from Clostridium acetobutylicum was efficiently introduced in all tested Acetobacterium spp., leading to non-native biochemical acetone production via plasmid-based expression. Conclusions: A. wieringae can be electrotransformed at high efficiency using different plasmids with different replication origins. The electrotransformation procedure and tools reported here unlock the genetic and metabolic manipulation of the biotechnologically relevant A.wieringa estrains. For the first time, non-native acetone production is shown in A. wieringae.

Citation

Moreira, J. P., Heap, J. T., Alves, J. I., & Domingues, L. (2023). Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways. Biotechnology for Biofuels and Bioproducts, 16(1), Article 24. https://doi.org/10.1186/s13068-023-02259-6

• Journal Article Type: Article
• Acceptance Date: Jan 5, 2023
• Online Publication Date: Feb 14, 2023
• Publication Date: Feb 14, 2023
• Deposit Date: Feb 20, 2023
• Publicly Available Date: Feb 20, 2023
• Journal: Biotechnology for Biofuels and Bioproducts
• Electronic ISSN: 2731-3654
• Publisher: Springer Science and Business Media LLC
• Peer Reviewed: Peer Reviewed
• Volume: 16
• Issue: 1
• Article Number: 24
• DOI: https://doi.org/10.1186/s13068-023-02259-6
• Keywords: Management, Monitoring, Policy and Law; Energy (miscellaneous); Applied Microbiology and Biotechnology; Renewable Energy, Sustainability and the Environment; Biotechnology
• Public URL: https://nottingham-repository.worktribe.com/output/17648411
• Publisher URL: https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02259-6
• Additional Information: Received: 5 May 2022; Accepted: 5 January 2023; First Online: 14 February 2023; Not applicable.; Not applicable.; The authors declare that they have no competing interests.