Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays.

Area of Science:

• Biotechnology and Synthetic Biology
• Polymer Science and Engineering
• Microbial Physiology

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by bacteria.
• Granule-associated proteins (GAPs), particularly phasins, are crucial for PHA granule formation and metabolism.
• Phasins are being explored for functionalizing PHA surfaces in various biotechnological applications.

Purpose of the Study:

• To investigate the binding affinity and influence of two specific phasins, PhaP1 and PhaI, on PHA accumulation and granule morphology.
• To assess these effects in engineered Pseudomonas putida KT2440 strains producing distinct PHA compositions (short-chain-length [scl]-PHA, medium-chain-length [mcl]-PHA, and hybrid scl/mcl-PHA).
• To explore the potential of phasins as bio-affinity tags for selective PHA functionalization.

Main Methods:

• Expression of phasin-msfGFP fusion proteins in engineered Pseudomonas putida strains.
• In vivo fluorescence binding assays to monitor phasin surface binding.
• Microscopy and Gas Chromatography-Mass Spectrometry (GC-MS) for PHA accumulation analysis.
• Transmission Electron Microscopy (TEM) for granule morphology assessment.
• Flow cytometry for differential surface affinity analysis.

Main Results:

• Phasin expression did not significantly alter overall PHA yield (%PHA/CDW) but influenced granule characteristics.
• PhaI expression in mcl-PHA-producing strain PP00_01 increased granule number and homogeneity.
• Flow cytometry revealed differential phasin surface affinities based on PHA type, with PhaP1 preferring scl-PHA and PhaI preferring mcl-PHA.
• Physicochemical properties of PHA and phasin type collectively impact binding strength.

Conclusions:

• Phasins play a dual role in modulating PHA production and enabling selective functionalization of PHA granules.
• The distinct binding preferences of PhaP1 and PhaI for different PHA types highlight their potential as specific bio-affinity tags.
• This study supports the use of phasins for targeted surface modification of PHA granules in biotechnological applications like enzyme immobilization and drug delivery.