Assessing the potential of C1 enzymes in Limosilactobacillus reuteri towards catalyzing C2 substrates

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Kemian tekniikan korkeakoulu | Master's thesis
Biosystems and Biomaterials Engineering
Degree programme
Master’s Programme in Life Science Technologies
65 + 1
The population of the world is at a constant growth demanding more energy and fuel than ever before. To stop the dreaded effects global warming poses to the planet, novel innovations for ecological solutions are more important than ever. Methanogenic archaea have the ability to convert CO2 to methane, which can be used for fuel. This could be a promising solution for fuel production and a very promising candidate for green innovations. However, ethane, which is similar to methane, would serve as a better option for fuel. Ethane has higher energy density and it is both easily liquefied and stored compared to methane. To have archaea produce ethane instead of methane, a full understanding of methanogenesis is required. One proposed hypothesis is that the enzymes are promiscuous, meaning that they can work with more than one substrate. Enzyme substrate promiscuity for C2 com- pounds was proposed in the one carbon pool by folate in Limosilactobacillus reuteri. Therefore the aim of this thesis is to improve on the metabolite study performed earlier by an enzymatic study of promiscuity in L. reuteri. If enzyme substrate promiscuity was proven in the folate cycle, it could possibly shed some light on the enzyme substrate promiscuity of methanogenesis pathway in archaea. The experimental part consisted of producing enzymes and testing their activity with UV spectrometry. Three enzymes from the folate cycle were chosen from which only two corresponding genes were found in the genome of L. reuteri (folD and fhs). The two genes were heterologously expressed and purified in Escherichia coli. To test promiscuity an enzyme assay was designed and the activity was measured with C1 and C2 substrates. However, the enzymes showed no activity in the assays for neither substrate. Therefore, no conclusions about enzyme substrate promiscuity could be drawn from this enzymatic study. However, the successful production of the L. reuteri enzymes was presented and therefore, this thesis lays out a good basis for the continuation of enzyme substrate promiscuity studies in L. reuteri.
Scheller, Silvan
Thesis advisor
Laird, Maxime
enzyme substrate promiscuity, two-carbon metabolism, CO2–fixation, Limosilactobacillus reuteri
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