Swapping of Phasin Modules To Optimize the In Vivo Immobilization of Proteins to Medium-Chain-Length Polyhydroxyalkanoate Granules in Pseudomonas putida

PhaF is a bimodular protein of Pseudomonas putida KT2442 exhibiting multiple functions within the polyhydroxyalkanoate (PHA) apparatus. It behaves as phasin or PHA granule binding protein (by BioF domain) and also as nucleoid-associated protein involved in granule localization and segregation during cell division (by C-terminal domain). This work addresses the function of the PhaI phasin in the PHA granule formation machinery. Epifluorescence microscopy and flow cytometry studies of P. putida phasin mutant cells producing recombinant phasin domains fused to GFP protein demonstrated a balanced granule distribution after cell division only when low dosage of PhaF, and BioF domain or PhaI, are expressed together, revealing the exchangeability of phasins modules. These findings show the precise combination of phasin module production that leads to the optimal PHA production and granule localization and distribution, concomitantly to in vivo immobilization of recombinant proteins to PHA (22 mg of protein/g PHA)