Synthesizing glycine betaine via choline oxidation pathway as an osmoprotectant strategy in Haloferacales

The accumulation of organic compatible solutes, such as glycine betaine, is one of the osmoprotective strategies used by halophilic archaea to adapt to high salinity. The uptake of glycine betaine from the external environment using various transporters has been widely studied in different halophilic archaea. However, the de novo biosynthesis of glycine betaine and its distribution in halophilic archaea remain unclear. In this study, an extremely halophilic archaea strain, named Halorubrum sp. 2020YC2 and previously isolated from a salt-lake sample, was identified with complete choline oxidation pathway genes. Halorubrum sp. 2020YC2 could synthesize and accumulate 1.56-4.25 μmol per mg of protein of glycine betaine in a defined medium, with its content increasing along with increasing salinity. The intracellular content of glycine betaine remained relatively stable at different salinities when another exogenous solute such as trehalose was provided. The metabolic profile and transcriptional results strongly suggested that the intracellular glycine betaine was derived from serine, which came from the glycolytic intermediate 3-phosphoglycerate when glucose was used as the sole carbon source. Out of 205 available genomes of halophilic archaea, genes encoding the choline oxidation pathway were identified in 30 genomes, and more than half of the strains belonging to order Haloferacales contained the choline oxidation pathway. Phylogenetic analysis further indicated that this pathway evolved from halophilic Proteobacteria, and its absence in some genera indicated a possible gene loss event during evolution. The analysis of reported culture data of halophilic archaea strains eventually demonstrated that the presence of the choline oxidation pathway had no significant effects on the adaptation of Haloferacales to high salinity habitats. Therefore, the de novo biosynthesis of glycine betaine via the choline oxidation pathway could be an auxiliary osmoprotective strategy in halophilic archaea.