Microbial Secretion Platform for 3-Hydroxybutyrate Oligomer and Its End-Capped Forms Using Chain Transfer...

Area of Science:

• Biotechnology and Metabolic Engineering
• Polymer Science
• Microbial Production Systems

Background:

• 3-hydroxybutyrate (3HB) polymer [P(3HB)] is a biodegradable polyester synthesized intracellularly in recombinant Escherichia coli.
• 3-hydroxybutyrate oligomers (3HBOs) are naturally occurring, physiologically active substances with potential applications.

Purpose of the Study:

• To engineer a microbial system for the secretion of 3HB homo-oligomers (3HBOs) using native polyhydroxyalkanoate (PHA) synthases.
• To investigate the formation of different end-structures of 3HBOs during microbial secretion.
• To establish a platform for producing 3HBOs with desirable end structures by controlling chain transfer agents.

Main Methods:

• Utilized native PHA synthases from Aeromonas caviae and Bacillus cereus YB4 in recombinant E. coli for 3HBO secretion.
• Employed an alcohol dehydrogenase gene (adhE)-deficient mutant strain to obtain carboxyl-terminated 3HBOs.
• Conducted in vitro polymerization assays with various chain transfer (CT) agents, including ethanol and free 3HB.

Main Results:

• Achieved high secretory production of 3HBOs, particularly with PHA synthases from A. caviae and B. cereus YB4.
• Identified an ethyl ester end-group in 3HBOs produced by most tested PHA synthases.
• Demonstrated that endogenous ethanol in E. coli acts as a CT agent, generating ethyl ester forms, while free 3HB yields carboxyl forms.
• Showcased the ability to control 3HBO end-structures by supplementing with specific CT agents.

Conclusions:

• Established a versatile microbial platform for the high-yield secretion of 3HBOs with tunable end structures.
• The platform allows for the production of both ethyl ester and carboxyl forms of 3HBOs by strategic use of CT agents.
• The secreted 3HBOs and their end-capped derivatives hold promise as physiologically active substances and building blocks for advanced polymeric materials.