Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid,CAS: 96702-03-3), a natural protective molecule, exhibits an exceptional water-retention capacity-80 times higher than that of glycerol. It effectively balances osmotic pressure across cell membranes, stabilizes the structures of proteins and macromolecules, and protects biological systems under extreme environmental conditions. Due to these remarkable properties, ectoine has been widely applied in industries such as cosmetics and agriculture.
In 2023, the global market size of ectoine reached 70million,anditis expectedtocontinuegrowingatacompoundannualgrowthrate(CAGR)of5.4100 million by 2032.
Recently, a research team led by Professor Jiang Min from Nanjing Tech University published a study in the prestigious journal ACS Synthetic Biology. The paper is titled "High Ectoine Production from Lignocellulosic Hydrolysate by Escherichia coli through Metabolic and Fermentation Engineering".
Researchers constructed an ectoine synthesis module in E. coli and relieved the rate-limiting steps, enabling the synthesis of 115.15 g/L ectoine using glucose in a 5 L bioreactor. When wheat straw hydrolysate was used as the carbon source, the ectoine yield reached 134.08 g/L (with a productivity of 0.33 g/g). This represents the highest level of ectoine synthesis by microorganisms using low-cost biomass to date, providing technical support for the industrial production of ectoine. Doctoral candidate Feng Yifan is the first author of the paper, while Professor Xin Fengxue and Associate Professor Jiang Wankui are the corresponding authors.
First, the researchers integrated the ectABC gene cluster derived from the halophilic bacterium Halomonas venusta ECT into the genome of E. coli MG1655, achieving the heterologous synthesis of ectoine in E. coli.
Figure | Metabolic Engineering Construction of Ectoine Production in E. coli
To address the issue of phosphoenolpyruvate (PEP) metabolic flux diversion in the host, the researchers used CRISPR-Cas9 technology to knock out the crr gene-a key gene in the phosphotransferase system (PTS). This redirected the metabolic flux toward oxaloacetate (OAA), increasing the ectoine yield from 0.56 g/L to 1.27 g/L.
Through rate-limiting enzyme analysis combining fermentation and overexpression experiments, it was found that aspartate kinase LysC was the metabolic bottleneck. Therefore, after overexpressing its feedback-resistant mutant EcLysC*, the shake flask yield of ectoine was further increased to 2.51 g/L.
Figure | Elimination of Rate-Limiting Steps in the Ectoine Synthesis Pathway
Systematic optimization of medium components-including the types and concentrations of carbon sources and nitrogen sources, as well as the concentrations of sodium chloride and magnesium sulfate-further significantly improved the synthesis efficiency. In a 5 L bioreactor, the researchers found that the inhibitory effect of glucose on ectoine synthesis could be alleviated by adopting a dynamically controlled feeding strategy (maintaining a residual glucose concentration of 1.0 g/L). Meanwhile, after optimizing the addition time of the inducer to balance cell growth and ectoine synthesis, the ectoine yield reached 115.15 g/L.
Figure | Optimization of Fermentation Process
To achieve low-cost production, glucose was replaced with wheat straw hydrolysate. Through precise control of carbon source supplementation strategies and nutritional regulation, the final yield reached 134.08 g/L (yield of 0.33 g/g sugar, production efficiency of 3.7 g/L/h), an increase of 17% compared to the pure glucose system, and it reached the highest level reported so far for the synthesis of ectoine using lignocellulose hydrolysate.
Figure 1 | Using lignocellulosic hydrolysate as a carbon source for ectoine production
This research demonstrates a strategy where Escherichia coli, through metabolic and fermentation engineering modifications, efficiently utilizes lignocellulosic hydrolysate to produce ectoine. It provides a new pathway for low-cost and environmentally friendly biomanufacturing. By employing multi-dimensional engineering strategies, it combines inexpensive raw materials with efficient biosynthesis. This not only advances the industrial potential of ectoine production but also offers a reference for the biomanufacturing of other high-value-added compounds.
Disclaimer: This article aims to convey the latest information in synthetic biology and does not represent the platform's stance. It does not constitute any investment advice or suggestions, and official/company announcements shall prevail. This article is also not a treatment plan recommendation. For treatment plan guidance, please consult a regular hospital.
