How is ectoine obtained?

Theectoine, chemically known as 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, is an amino acid derived from aspartate. It is synthesised by halophilic bacteria that live in salt-rich environments, through the biosynthesis of aspartate family amino acids with the enzymatic reactions of three main enzymes: L-2,4-diaminobutyrate transaminase, L-2,4-diaminobutyrate acetyltransferase and ectoine synthase. A highly salty environment often triggers the expression of these ectoine-synthesising enzymes.

Industrial production method of ectoine.

The bacteria most commonly used for synthesising ectoine in industrial production belong to the Halomonas family, such as Halomonas salina, Halomonas boliviensis and Halomonas elongata. The various applications of ectoine across different industries have led to the development of its large-scale production to meet the growing market demand.

Intracellular production of ectoine.

Industries generally utilise "bacterial milking" through microbial fermentation. A high-salt content medium, supplemented with carbon and nitrogen, is used to stimulate the biosynthesis of ectoine. The bacterial cells are then shocked by a low-salt content medium to release the synthesised ectoine into the medium.

However, this technique generally yields low returns and requires several downstream process steps for the purification of ectoine in order to achieve acceptable high yields. Moreover, a downside of regular use of a high salt concentration is the corrosiveness of high salinity for materials and equipment, which subsequently reduces the growth of microbial cells and the overall production yields of ectoine.

Extracellular production of ectoine.

Certain strains, such as Halomonas salina, can synthesise extracellular ectoine with a low salt concentration. The growing cells are cultivated in a fermentation medium containing glutamate as the main source of carbon and nitrogen. When they have reached the late exponential growth phase, phosphate-limited growing cells are used for the production of ectoine.

Furthermore, heterologous producers of ectoine, that is, species different from those producing the natural molecule, have been developed by incorporating the ectoine synthesis gene into non-halophilic microorganisms, such as Escherichia coli. Ectoine can be synthesised and secreted extracellularly by these heterologous producers. This "recombination" is relevant, as they also do not require high salinity.

The recovery of extracellular ectoine from the fermentation medium is relatively straightforward compared to the recovery of intracellular ectoine from microbial cells, as it is directly excreted without the need for a cellular shock. The production cost of extracellular ectoine at an industrial scale can be reduced thanks to the minimal use of salt and the elimination of additional steps to extract intracellular ectoine from microbial cells.

However, to date, we only know of a limited number of bacterial strains that secrete ectoine in an extracellular manner.