Engineering Eubacterium limosum towards commodity production
Publication Date
March 15, 2024
Creator
Abstract
Climate change and sustainability have come to the fore in recent years, with a greater interest in transition away from fossil usage where possible. One of the most difficult uses of fossil fuels to replace is the production of precursors for the synthesis of chemicals essential for our modern civilization. C1 utilising organisms may be able to aid in the replacement of fossil fuels for this purpose, with the added benefit of potentially producing carbon neutral or carbon negative products.
Eubacterium limosum is an acetogenic bacterium which is not used currently in any industrial setting. It possesses interesting properties which may make it suitable for future use, from its utilisation of multiple energy sources, such as sugars, synthesis gas, carbon monoxide/CO2 mixes and methanol, to its native production of chemicals such as butanol and vitamin B12. This work sought to develop this organism to be more amenable to industrial fermentation with the goal of addressing some of the organism’s limitations and introducing a non-native chemical to its product spectrum. These limitations included the organism requiring yeast extract supplementation of growth media, significant adhesive properties, and a lack of a robust genetic toolset. All three of these issues were addressed as part of this work.
An improved strain was created by directed evolution using continuous fermentation. This resulted in a strain which no longer required complex medium supplementation (with yeast extract), and no longer had significant extracellular adhesive properties. In the investigation of the cause of the altered phenotype observed changes in the epigenome, hitherto not considered, suggested themselves as a likely explanation. Improved CRISPR-Cas9 tools were developed, and a toxin-antitoxin-based counter selection system employed. The use of and development of genetic modification tools produced many mutants with the following mutants created as part of this work, Δact, Δcop, ΔhisI, Δmti, ΔmtiI, Δpbt1, Δpbt2, ΔactΔpbt1, ΔactΔpbt2 and ΔactΔpbt1Δpbt2. The generated mutants bettered the understanding of the internal biochemistry of the organism as related to butyrate production and methanol usage, with the essentiality of the gene pair metVmetF confirmed by an inability to create a knockout strain. The creation of auxotrophic mutants allowed for use of allele-coupled exchange, a genetic modification technique, culminating in the genomic integration of genes from Clostridium acetobutylicum which allowed for the production acetone and isopropanol.
This work serves as a proof of concept for E. limosum, with the primary conclusion of this work being that E. limosum is an organism which is suitable for further development towards industrial use.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
77035
UoN Repository URI
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Alexander Agius - 14316128 - thesis.pdf
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Full-text
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Examined
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13.56 MB
Format
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