High Ectoine Production from Lignocellulosic Hydrolysate by Escherichia coli through Metabolic and Fermentation Engineering

Ectoine, a major compatible solute in halophilic micro-organisms, shows great potential in cosmetics and pharmaceuticals areas owing to its water-binding properties and capability to prevent oxidative damage. In this study, the ectABC gene cluster responsible for the ectoine synthesis originated from halophilic bacterium Halomonas venusta was first assembled into Escherichia coli. Subsequently, the crr gene in PTS was knocked out to further drive the metabolic flux from phosphoenolpyruvate to oxaloacetate, resulting in 1.27 g/L of ectoine. Then, the rate-limiting enzyme LysC in the ectoine synthesis pathway was identified and modified. The recombinant E. coli with the further overexpression of feedback-insensitive mutant EclysC* increased the ectoine titer to 2.51 g/L with a yield of 0.37 g/g in shake flasks. After the medium optimization including the carbon and nitrogen source, sodium chloride, and magnesium sulfate concentration, the ectoine titer was improved to 4.55 g/L. 115.15 g/L of ectoine with a yield of 0.23 g/g was obtained in the 5.0 L bioreactor through the optimization of substrate feeding and IPTG supplementation in the fed-batch fermentation. To achieve the cost-effective production of ectoine, lignocellulosic hydrolysate from wheat straw was adopted. 134.08 g/L of ectoine with a yield of 0.33 g/g sugar and a productivity of 3.7 g/L/h was finally produced, representing a relatively high level of ectoine production from renewable resources compared to other studies. This study provides valuable insights into a cost-effective and efficient method for industrial-scale ectoine production.