Establishment of CRISPR-Cas-based gene deletions in Bacillus methanolicus and metabolic engineering towards the production of N-methylglutamate and putrescine

The global shift towards a sustainable bioeconomy requires alternative feedstocks, with methanol emerging as a promising alternative carbon source for microbial biotechnology. The increasing popularity of renewable resources such as green methanol can be attributed to the fact that they do not compete with human nutrition, a factor which is of particular importance in the context of environmental sustainability goals. In recent years microorganisms have been engineered to utilize methanol for the production of valuable compounds. Notably, the thermophilic methylotroph _Bacillus methanolicus_ is naturally capable of metabolizing methanol as sole carbon source. The potential of _B. methanolicus_ is limited by the lack of robust genetic engineering tools. Developing advanced genome editing technologies, such as CRISPR-Cas systems, is key to improving this organism for industrial applications and increasing yields and productivity in line with sustainability goals. The first objectives in this study were i) to establish a CRISPR-Cas9-based gene deletion tool in _B. methanolicus_ with proof-of-principle deletion of the _spoIIE_ gene, ii) construction of a plasmid library for gene deletions, iii) targeting of central metabolism genes with CRISPR interference. The results can be summarized as follows: • The CRISPR-Cas9-based system was established in _B. methanolicus_. Demonstrated by successful deletion of the _spoIIE_ gene, resulting in a decreased spore formation. • A library of 19 plasmids for gene deletions in _B. methanolicus_ was constructed, including the sporulation genes _spoIIE_, _spo0A_, _spo0F_, _yaaT_ and _ydcC_ and the genes _argF_, _fbaC_, _fbaP_, _glpXC_, _glpXP_, _hps_, _mdh_, _mdh2_, _mdh3_, _mtlR_, _odhA_, _phi_, _tktC_ and _tkt_. • CRISPRi was used to successfully downregulate the central metabolic genes _pckA_ and _pdhA_ in _B. methanolicus_ by 3.5- and 84-fold, respectively. By the choice of sgRNA the strengths of gene repression can be adapted. Polyamines are important compounds used across various industries for their ability to enhance material properties, serving as intermediates in products like pharmaceuticals, and biodegradable polymers. A notable example is nylon 4,6, an industrial polyamide produced by condensing biogenic putrescine with adipic acid to create a highly stable material. Putrescine itself is a linear four-carbon diamine involved in various cellular processes and stress responses in organisms, and it can be synthesized via two primary biochemical pathways: the ornithine decarboxylase (ODC) pathway and the arginine decarboxylase (ADC) pathway. N-Methylation of amino acids is a crucial post-translational modification that adds a methyl group to the nitrogen atom of an amino acid, significantly altering its chemical properties and biological activity. N-Methylglutamate (NMG) is an N-functionalized L-glutamate derivative produced via the NMG pathway in certain methylotrophic bacteria. NMG is part of a broader class of N-alkylated amino acids, which are known for their enhanced proteolytic stability, increased membrane permeability, and prolonged half-lives. The production of NMG has gained significant interest, particularly in the pharmaceutical and chemical industries, due to its unique properties. Further objectives in this study were to iv) genetically engineer _B. methanolicus_ for the production of putrescine and N-methylglutamate. The results can be summarized as follows: • _B. methanolicus_ was tested as a host for the production of putrescine, with a IC50 value of 20 g/L L-ornithine and 1.7 g/L putrescine. Neither putrescine or L-ornithine could be used by _B. methanolicus_ as carbon or nitrogen source. • Through the heterologous expression of the ornithine decarboxylase gene speCI163T/E165T from _E. coli_ the strain was able to produce 22 mg/L of putrescine. • For establishing the production of N-methylglutamate an IC50 value of 5.7 g/L MMA, 28 g/L L-glutamate and 1.6 g/L NMG was measured for _B. methanolicus_. In addition, _B. methanolicus_ is not able to use MMA as a carbon or nitrogen source. • _B. methanolicus_ strain NMeGlu2 was capable of producing 170 mg/L NMG through the heterologous expression of the genes _gmaS_ and _mgsABC_, which encode for the enzymes of the NMG pathway.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Eikmeier, Julia

Repository record source_url

https://pub.uni-bielefeld.de/record/3004101

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3004101