Abstract
BackgroundThe interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.ResultsIn this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E.limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth.ConclusionsThe addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E.limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
Highlights
The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions
In this study, we implemented the fluorescent activating and absorption shifting tag as an oxygen-independent fluorescent reporter protein in E. limosum. We showed that this reporter protein is functional under various growth conditions and can be adapted to the natural spectral properties of the organism using different fluorogens
We showed that the fluorescence of feg-expressing cells can be determined using a microplate reader, fluorescence microscopy, or flow cytometry
Summary
The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Acetogens are promising biocatalysts for the sustainable production of biocommodities since their ability to use carbon dioxide (CO2)- and carbon monoxide (CO)-containing industrial waste gases as feedstock contributes to the reduction of greenhouse gas emissions. Fluorescent reporter systems which are well-established and often used tools in molecular biology to study gene expression [23, 24], promoter activities [25, 26], or the dynamics in microbial populations and co-cultures [27,28,29] are still restricted for acetogens, basically due to the lack of proteins that show bright fluorescence under anaerobic conditions. FAST is a small-sized protein with a mass of 14 kDa that only shows fluorescence when it forms a non-covalent reversible complex with a fluorogenic ligand, a so-called fluorogen [30, 33]. Such fusion proteins enable investigations of protein localization and intracellular dynamics [31, 34,35,36]
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