Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas

Area of Science:

• Microbial Engineering
• Synthetic Biology
• Metabolic Engineering

Background:

• Ligand-induced systems offer tunable gene expression control crucial for microbial engineering.
• A previously developed oleic acid (OA)-induced system utilized the FadR protein in Escherichia coli.
• FadR is a transcriptional regulator involved in fatty acid metabolism.

Purpose of the Study:

• To construct a synthetic FadR-based OA-induced system in Halomonas bluephagenesis.
• To optimize dynamic control range and reduce expression leakage.
• To demonstrate the system's application in producing polyhydroxyalkanoates (PHAs).

Main Methods:

• Hybridization of porin promoter core region with FadR-binding operator (fadO).
• Tuning FadR expression levels and fadO positioning for optimization.
• Testing ligand orthogonality and cross-species portability across bacterial genera.
• Application of the system to control minCD and 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathways.

Main Results:

• Achieved an optimized dynamic control range exceeding 150-fold with significantly reduced expression leakage.
• Demonstrated high linear correlation for ligand orthogonality and cross-species portability in Halomonas, Escherichia, and Pseudomonas species.
• Successfully produced over 10 g/L of poly-3-hydroxybutyrate (PHB) and 6 g/L of P(3HB-co-9.57 mol% 4HB) by controlling OA induction.

Conclusions:

• Developed a robust and tunable OA-induced system in Halomonas bluephagenesis.
• The system exhibits broad applicability and portability across different bacterial species.
• Provides a novel genetic tool for dynamic control in industrial biotechnology, enabling efficient PHA production.