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Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using
Manami Ishii-Hyakutake1, Tetsuo Sakurai1, Takeharu Tsuge1
1Department of Materials Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
Microorganisms
|September 28, 2021
Summary
A new high-throughput screening method using HPLC accurately measures polyhydroxyalkanoate (PHA) degree of polymerization (DP). This method successfully identified beneficial PHA synthase mutants producing high molecular weight PHA.
Area of Science:
- Biotechnology
- Polymer Chemistry
- Microbial Engineering
Background:
- Polyhydroxyalkanoates (PHAs) are biodegradable polymers with diverse applications.
- Controlling the molecular weight, specifically the degree of polymerization (DP), is crucial for tailoring PHA properties.
- Efficient screening methods are needed to identify PHA-producing microorganisms with desirable polymer characteristics.
Purpose of the Study:
- To develop a high-throughput screening method for identifying polyhydroxyalkanoate (PHA) synthase mutants that produce polymers with a high degree of polymerization (DP).
- To validate the accuracy of the developed screening method using established analytical techniques.
- To apply the screening method to discover novel PHA synthase variants with enhanced high molecular weight PHA production capabilities.
Main Methods:
- A novel high-throughput screening method was established utilizing high-performance liquid chromatography (HPLC).
- Recombinant *Escherichia coli* was employed for polyhydroxyalkanoate (PHA) production, with benzyl alcohol used as a chain-terminating agent.
- Alkaline treatment of cultured cells released PHA monomers and terminal benzyl alcohol, which were quantified by HPLC to determine PHA DP based on peak ratios.
Main Results:
- The developed HPLC-based method accurately determined the degree of polymerization (DP) of polyhydroxyalkanoates (PHAs).
- The DP measurements were validated against other instrumental analyses using purified PHA samples.
- Screening of *Bacillus cereus* YB-4 PHA synthase (PhaRCYB4) mutants identified several enzymes capable of producing PHA with a higher DP than the wild-type.
- Two identified PHA synthase mutants exhibited PHA yields comparable to the wild-type enzyme.
Conclusions:
- The developed high-throughput screening method based on HPLC is effective for determining polyhydroxyalkanoate (PHA) degree of polymerization (DP).
- This method is suitable for selecting beneficial PHA synthase mutants that yield high molecular weight PHAs.
- The identified mutants offer potential for producing advanced PHA materials with improved properties.
