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Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with
Yoriko Watanabe1, Yousuke Ichinomiya, Daisuke Shimada
1Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
Journal of Bioscience and Bioengineering
|November 18, 2011
Summary
A new high-throughput screening method using HPLC enables rapid analysis of polyhydroxyalkanoate (PHA) composition. This system identified beneficial PHA synthase mutations, enhancing copolymer production from soybean oil.
Area of Science:
- Biotechnology
- Polymer Science
- Enzyme Engineering
Background:
- Polyhydroxyalkanoates (PHAs) are biodegradable polymers with tunable properties.
- Efficient methods for analyzing PHA composition and engineering PHA synthases are crucial for developing novel materials.
- Current methods for PHA compositional analysis can be time-consuming and labor-intensive.
Purpose of the Study:
- To develop a rapid and high-throughput method for the compositional analysis of polyhydroxyalkanoates (PHAs).
- To utilize this method for identifying improved PHA synthase mutants with altered substrate specificity.
- To engineer PHA copolymers with enhanced 3-hydroxyhexanoate (3HHx) fractions.
Main Methods:
- Development of a high-performance liquid chromatography (HPLC)-based assay with alkaline sample pretreatment in a 96-well plate format.
- Creation and screening of a mutant library of Aeromonas caviae PHA synthase (PhaC(Ac)) to identify variants with enhanced reactivity toward 3-hydroxyhexanoate (3HHx).
- Genetic engineering of Ralstonia eutropha PHB(-)4 with selected PHA synthase mutants to synthesize P(3-hydroxybutyrate-co-3HHx) using soybean oil as a carbon source.
Main Results:
- A rapid and reliable HPLC-based method for PHA compositional analysis was established.
- Mutants, including PhaC(Ac) D171G, exhibiting increased reactivity toward 3HHx were successfully isolated.
- Engineered strains produced P(3HB-co-3HHx) with significantly higher 3HHx fractions compared to wild-type enzymes, utilizing soybean oil.
Conclusions:
- The developed high-throughput screening system is a powerful tool for PHA research.
- This system facilitates the discovery of beneficial mutations for tailoring PHA copolymer composition.
- The identified PHA synthase variants enable enhanced production of specific PHA copolymers from sustainable feedstocks.
