Metabolic engineering of Escherichia coli for efficient osmotic stress-free production of compatible solute hydroxyectoine.

Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.

Highly Efficient Production of N-Acetyl-glucosamine in Escherichia coli by Appropriate Catabolic Division of Labor in the Utilization of Mixed Glycerol/Glucose Carbon Sources.

Currently, microbial production is becoming a competitive method for N-acetyl-glucosamine production. As the biosynthesis of N-acetyl-glucosamine originating from fructose-6-P directly competes with central carbon metabolism for precursor supply, the consumption of glucose for cell growth and cellular metabolism severely limits the yield of N-acetyl-glucosamine. In this study, appropriate catabolic division of labor in the utilization of mixed carbon sources was achieved by deleting the pfkA gene and enhancing the utilization of glycerol by introducing the glpK mutant. Glycerol thus mainly contributed to cell growth and cellular metabolism, and more glucose was saved for efficient N-acetyl-glucosamine synthesis. By optimizing the ratio of glycerol to glucose, the balancing of cell growth/cellular metabolism and N-acetyl-glucosamine synthesis was achieved. The resulting strain GLALD-7 produced 179.7 g/L N-acetyl-glucosamine using mixed glycerol/glucose (1:8, m/m) carbon sources in a 5 L bioreactor, with a yield of 0.458 g/g total carbon sources (0.529 g/g glucose) and a productivity of 2.57 g/L/h. Coherent high titer/yield/productivity was obtained, with the highest values ever reported, suggesting that an appropriate catabolic division of labor using mixed glycerol/glucose carbon sources is a useful strategy for facilitating the microbial production of chemicals originating from glucose or metabolites upstream of glycolysis.