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This page was translated automatically using artificial intelligence (DeepL). The German version is binding. More information about automatic translation
Hydrogen is of immense importance as a raw material, as, in addition to its role as an energy storage medium, it can also be used as a feedstock in various industrial sectors, such as the chemical industry and steel production [1]. However, a large proportion of the hydrogen currently produced still comes from the decomposition of fossil fuels, which means that it cannot yet be described as sustainable hydrogen production. This is because the processes currently in use release carbon dioxide. There are various approaches to making hydrogen production carbon-neutral. One possibility, for example, would be the electrochemical splitting of water molecules into hydrogen and oxygen (electrolysis). This process produces what is known as ‘green hydrogen’, provided that the electricity used comes from renewable sources such as wind power or solar energy.
Another approach is the production of biogenic, so-called ‘brilliant’ hydrogen (biohydrogen). In this process, hydrogen is produced through the metabolism of microorganisms using light energy. One example of a microorganism capable of producing hydrogen is the purple bacterium *Cereibacter sphaeroides*. When nitrogen is in short supply, it produces hydrogen as a by-product during nitrogen fixation (Fig. 1).
There is a sub-strain of the bacterium that produces hydrogen even in the presence of ammonium ions. Previous studies suggest that a point mutation in the rpoN gene is responsible for this [2]. The gene encodes the transcription factor Sigma54, which is indirectly involved in the regulation of the enzyme nitrogenase by initiating, in the event of nitrogen deficiency, the transcription of genes required for the synthesis of nitrogenase [2,3,4]. The aim of this project is to demonstrate the causal link between the mutation and the altered hydrogen production. Introducing the mutation into the rpoN gene of a bacterial strain of C. sphaeroides and subsequently measuring the hydrogen production rate in the presence of ammonium ions should provide insight into this causal relationship. Base editors, which can edit DNA with high precision, are well-suited for the targeted introduction of a mutation.
DNA base editors are a further development of the CRISPR/Cas9 system, which enables DNA to be cut with precision using a single-guide RNA (sgRNA) (Fig. 2) [5].
To this end, the various components of the base-editing system – comprising sgRNA and the Cas9 base editor – are first synthesised and then tested in vitro on the isolated DNA of the rpoN gene from C. sphaeroides. The system, having been evaluated, is then introduced into living C. sphaeroides cultures using an expression plasmid, where the mutation is to be induced. A comparison is then carried out between strains of C. sphaeroides with and without the mutation, regarding the amount of hydrogen produced in the presence of ammonium ions.
About the person
Anna Nauruschat has been studying at Mittweida University of Applied Sciences since 2020, initially on the Bachelor’s degree programme in Biotechnology and subsequently on the Master’s degree programme in Genomic Biotechnology. As part of Prof. Röbbe Wünschiers’ research group, her research focuses on the development of a DNA base editor for C. sphaeroides.
Sources
[1] Allelein, Hans-Josef et al. (2022): Energy Technology: Systems for Conventional and Renewable Energy Conversion. Essential Knowledge for Study and Work, Richard Zahoransky (ed.), Wiesbaden [Heidelberg]: Springer Vieweg.
[2] Wappler, Nadine and Wünschiers, Röbbe (2024): Analysis of a σ54 transcription factor L420P mutation in the context of increased organic nitrogen tolerance in photofermentative hydrogen production in Cereibacter sphaeroides strain 2.4.1 substrain H2, in: *Synthetic Biology and Engineering*, 2, 1, 10001.
[3] Kustu, S. et al. (1989): Expression of sigma 54 (ntrA)-dependent genes is probably governed by a common mechanism, in: Microbiological Reviews, 53, 3, 367–376.
[4] Merrick, M. J. (1993): In a class of its own — the RNA polymerase sigma factor σ;54 (σN), in: Molecular Microbiology, 10, 5, 903–909.
[5] Qi, Lei S. et al. (2013): Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, in: Cell, 152, 2, 1173–1183.
Text and illustrations: Anna Nauruschat
. Photographs: Johanna Wicht and Jacob Golde