High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium

As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications.

The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L^-1 NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L^-1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L^-1), and also exceeds the production of Y2/ΔectD (2.21 g L^-1). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na⁺/H⁺ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L^-1 or 500 mg (g DCW) ^-1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L^-1 NaCl stress, a maximum 10.5 g L^-1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)^-1 and a yield of 0.21 g g^-1 monosodium glutamate.

The remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na⁺/H⁺ antiporters in their halophilic producers and thus lowering the medium salinity.