Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain

Area of Science:

• Biotechnology and Polymer Science
• Microbial Biochemistry

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable polymers offering sustainable alternatives to petroleum-based plastics.
• PHA synthase (PhaC) enzyme activity and substrate specificity are critical for PHA production and properties.
• Understanding substrate side chain modifications is key to unlocking new PHA functionalities.

Purpose of the Study:

• To synthesize and evaluate 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues with modified side chains.
• To investigate the substrate specificity and activity of different PHA synthases (Class I and Class III) with these analogues.
• To explore the potential of modified PHAs for new material applications and biogenesis studies.

Main Methods:

• Synthesis of various 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues.
• Enzymatic assays using Class I PHA synthases (PhaCCs, A479S-PhaCCs, PhaCCc) and Class III PHA synthase (PhaECAv).
• Measurement of enzyme activity and catalytic efficiency (kcat/Km) with modified substrates.

Main Results:

• PHA synthases exhibited preferences for specific alkyl side chain lengths.
• Enzymes tolerated moderate side chain modifications, including unsaturated bonds and azide groups.
• Specific activity of PhaCCs with a propynyl analogue was only 5-fold lower than with HBCoA.
• PhaECAv showed a catalytic efficiency of 2.86 × 10^5 M⁻¹s⁻¹ for an azide analogue.

Conclusions:

• Tailoring PHA side chains is feasible and can be achieved using modified HACoA analogues.
• Modified PHAs hold promise for developing novel materials with unique properties.
• These modifications facilitate the study of PHA biogenesis, crucial for optimizing economic production through metabolic engineering.