Obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z-3E as a fumarate producer

Methanotrophic bacteria are a unique group of microorganisms that use CH4 as the sole source of carbon and energy. Methanotrophs are important participants in the global carbon cycle, reducing the levels of methane and other greenhouse gases in the atmosphere. Methane is the main component of natural gas and is found in abundance in oil and coal fields and is formed during the biological decomposition of organic matter under anaerobic conditions. Methane is a suitable substrate for biotechnological processes as it does not compete with food demand. The World Bank estimates the loss of natural gas alone at 92 Mt per year when flared or vented to the atmosphere, which provides a huge opportunity to transform methane into more valuable products. All over the world, aerobic methanotrophs attract the attention of biotechnologists by the possibility of obtaining products with high added value due to methane bioconversion. Methanotrophic cultures have been tested for the production of single cell protein, poly-beta-hydroxybutyrate, components for nanotechnology applications, lipids and carotenoids, and as a carbohydrate feedstock for fermentation. A kind of renaissance in the study of the biotechnological potential of methanotrophs as producers of various valuable metabolites began with the sequencing of the genome sequences of these bacteria and the development of genetic engineering approaches for targeted modification of metabolism, which was also facilitated by the emergence (isolation, description) of strains exhibiting high resistance to influences of external factors - variations in salinity, pH, concentrations of substrates (methanol, O2). This project is aimed at studying the biotechnological potential of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z, which has a high growth rate and stability in a wide range of growth conditions - in the presence of 0.5 - 6% NaCl and in the pH range of 7.5-10.5, thereby being a good candidate for a scalable process. By introducing marker-free mutations into the chromosome of M. alcaliphilum 20Z, we obtained a strain with deletions of three genes simultaneously: two nonhomologous genes encoding the enzyme of the tricarboxylic acid cycle, fumarase classes I and II, and the gene for decarboxylating malate dehydrogenase (malic enzyme). Analysis of strand-specific 3-end sequencing of mRNA did not reveal an effect of the introduced mutations on the central metabolism of the methanotroph, however, it showed a dramatic change in the expression of putative iron transport genes, as well as some genes encoding stress protein.