Boosting Escherichia coli's heterologous production rate of ectoines by exploiting the non-halophilic gene cluster from Acidiphilium cryptum.

The compatible solutes ectoine and hydroxyectoine are synthesized by many microorganisms as potent osmostress and desiccation protectants. Besides their successful implementation into various skincare products, they are of increasing biotechnological interest due to new applications in the healthcare sector. To meet this growing demand, efficient heterologous overproduction solutions for ectoines need to be found. This study is the first report on the utilization of the non-halophilic biosynthesis enzymes from Acidiphilium cryptum DSM 2389T for efficient heterologous production of ectoines in Escherichia coli. When grown at low salt conditions (≤ 0.5% NaCl) and utilizing the cheap carbon source glycerol, the production was characterized by the highest specific production of ectoine [2.9 g/g dry cell weight (dcw)] and hydroxyectoine (2.2 g/g dcw) reported so far and occurred at rapid specific production rates of up to 345 mg/(g dcw × h). This efficiency in production was related to an unprecedented carbon source conversion rate of approx. 60% of the theoretical maximum. These findings confirm the unique potential of the here implemented non-halophilic enzymes for ectoine production processes in E. coli and demonstrate the first efficient heterologous solution for hydroxyectoine production, as well as an extraordinary efficient low-salt ectoine production.

The hydroxyectoine gene cluster of the non-halophilic acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic α-Proteobacterium which thrives in acidic, metal-rich environments (e.g. acid mine drainage). Recently, an ectABCDask gene cluster for biosynthesis of the compatible solutes ectoine and hydroxyectoine was detected in the genome sequence of A. cryptum JF-5. We were able to demonstrate that the type strain A. cryptum DSM 2389(T) is capable of synthesizing the compatible solute hydroxyectoine in response to moderate osmotic stress caused by sodium chloride and aluminium sulphate, respectively. Furthermore, we used the A. cryptum JF-5 sequence to amplify the ectABCDask gene cluster from strain DSM 2389(T) and achieved heterologous expression of the gene cluster in Escherichia coli. Hence, we could for the first time prove metabolic functionality of the genes responsible for hydroxyectoine biosynthesis in the acidophile A. cryptum. In addition, we present information on specific enzyme activity of A. cryptum DSM 2389(T) ectoine synthase (EctC) in vitro. In contrast to EctCs from halophilic microorganisms, the A. cryptum enzyme exhibits a higher isoelectric point, thus a lower acidity, and has maximum specific activity in the absence of sodium chloride.