Promoter engineering for high ectoine production in a lower saline medium by Halomonas hydrothermalis Y2

For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2.

In H. hydrothermalis Y2, the σ⁷⁰- and σ³⁸-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P₂₆₅ that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/△ectD/△doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/△ectD/△doeA synthesized 11.5 g ectoine l^-1 in the presence of 60 g NaCl^-1 l, with a high 0.32 g ectoine l^-1 h^-1 productivity, a specific productivity of 512.2 mg ectoine per g cell dry weight (CDW)^-1, and an excretion ratio of 67 % ectoine.

As no impaired growth was observed in strain p/Y2/△ectD/△doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.