Presentation Information
[P03-310]Engineering a Vibrio Chassis for Robust 5-Hydroxyectoine Production at Elevated Salinity
○yeeun Mun1, DONG KYU SEO1, Yong Hee Han1 (1. Department of Biological Sciences and Biotechnology, Chonnam National University (Korea))
Keywords:
Adaptive Laboratory Evolution,5-Hydroxyectoine,Vibrio,Metabolic pathway Engineering,Halotolerance
corresponding author:Yong Hee Han
A central challenge in modern biomanufacturing is developing robust microbial cell factories that can stably produce high-value biomaterials under harsh conditions. Specifically, 5-hydroxyectoine is highly valued in the cosmetic and pharmaceutical industries. It acts as an excellent protectant, shielding cells from thermal and oxidative stress. In this study, we utilized a fast-growing marine Vibrio as a chassis.
However, Vibrio has two main limitations for industrial use: low natural ectoine-synthesizing activity and the absence of the ectoine hydroxylase enzyme (EctD). To overcome these limitations, we first conducted adaptive laboratory evolution (ALE) to improve the host's physiological fitness and robustness. By iteratively culturing the strain with gradually increasing salinity, we isolated evolved mutants that maintained high viability and rapid growth even under severe osmotic stress. Remarkably, the evolved strain recovered its specific growth rate to approximately 80% of that of the wild-type under these high-salinity conditions.
Building on this optimized host background, we strategically engineered the 5-hydroxyectoine biosynthetic pathway to maximize productivity. While Vibrio sp. possesses an endogenous ectABC operon for ectoine synthesis, it naturally lacks the ectoine hydroxylase (EctD) required for the final conversion to 5-hydroxyectoine. To bridge this metabolic gap, we introduced a codon-optimized ectD gene derived from Halomonas sp. and placed it under a strong constitutive promoter system. This design decoupled the hydroxylation step from the native salt-dependent regulatory network, ensuring consistent enzymatic activity regardless of external osmotic fluctuations. The evolved strain maintained a robust precursor flux through its inherent pathways, which, in synergy with the introduced EctD module, resulted in a markedly enhanced conversion of ectoine into 5-hydroxyectoine.
In conclusion, this study fully addressed the low productivity and genetic limitations of the parental Vibrio. This was achieved by integrating physiological improvements from ALE with the rational design of a Halomonas-derived heterologous pathway. Our results provide a clear example that optimizing the physiological background of a microbial chassis is a critical determinant of metabolic engineering performance. Therefore, this work is expected to serve as a foundational reference for future bioprocesses leveraging marine microorganisms to produce diverse functional biomaterials.
However, Vibrio has two main limitations for industrial use: low natural ectoine-synthesizing activity and the absence of the ectoine hydroxylase enzyme (EctD). To overcome these limitations, we first conducted adaptive laboratory evolution (ALE) to improve the host's physiological fitness and robustness. By iteratively culturing the strain with gradually increasing salinity, we isolated evolved mutants that maintained high viability and rapid growth even under severe osmotic stress. Remarkably, the evolved strain recovered its specific growth rate to approximately 80% of that of the wild-type under these high-salinity conditions.
Building on this optimized host background, we strategically engineered the 5-hydroxyectoine biosynthetic pathway to maximize productivity. While Vibrio sp. possesses an endogenous ectABC operon for ectoine synthesis, it naturally lacks the ectoine hydroxylase (EctD) required for the final conversion to 5-hydroxyectoine. To bridge this metabolic gap, we introduced a codon-optimized ectD gene derived from Halomonas sp. and placed it under a strong constitutive promoter system. This design decoupled the hydroxylation step from the native salt-dependent regulatory network, ensuring consistent enzymatic activity regardless of external osmotic fluctuations. The evolved strain maintained a robust precursor flux through its inherent pathways, which, in synergy with the introduced EctD module, resulted in a markedly enhanced conversion of ectoine into 5-hydroxyectoine.
In conclusion, this study fully addressed the low productivity and genetic limitations of the parental Vibrio. This was achieved by integrating physiological improvements from ALE with the rational design of a Halomonas-derived heterologous pathway. Our results provide a clear example that optimizing the physiological background of a microbial chassis is a critical determinant of metabolic engineering performance. Therefore, this work is expected to serve as a foundational reference for future bioprocesses leveraging marine microorganisms to produce diverse functional biomaterials.
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