Methanotroph Methylotuvimicrobium alcaliphilum 20Z-3E as a fumarate producer: transcriptomic analysis and the role of malic enzyme.

The halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z is a promising biotechnological strain that has been repeatedly tested as a producer of high-added-value polycarbon compounds. The mutant M. alcaliphilum 20Z-3E lacking two fumarases and a malic enzyme is a potential fumarate producer. The analysis of strand-specific 3'-end sequencing of mRNA did not reveal any effects of the 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 associated with stress response. When the strain 20Z-3E grows at low salinity under methane, some part of fumarate is formed from aspartate, since the increase in salinity results in the biosynthesis of ectoine and the decrease in fumarate concentration. However, when the strain grows on methanol, the fumarate pool is lower and does not depend on the salinity of the medium. Our results have shown that deletion of the mae gene encoding malic enzyme makes a significant contribution to the fumarate accumulation. The strain 20Z-2F with the deletion of only two genes, fumI and fumII, demonstrated delayed growth under methane in comparison with 20Z and 20Z-3E strains. The branching of the tricarboxylic acid cycle due to the adenylosuccinate shunt, as well as the presence of malic enzyme, provides metabolic flexibility to M. alcaliphilum, which allows the methanotroph to adapt to a variety of external conditions and, on the other hand, us to modify its genome to obtain valuable products.