Rapid Quantification of Polyhydroxyalkanoates Accumulated in Living Cells Based on Green Fluorescence Protein-Labeled Phasins: The qPHA Method.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that have the potential to replace nonbiodegradable petroplastics. A real-time in situ PHA quantification method has long been awaited to replace the traditional method, which is time- and labor-consuming. Quantification of PHA in living cells was finally developed from fluorescence intensities generated from the green fluorescence protein (GFP) fused with the <i>Halomonas bluephagenesis</i> phasin proteins. Phasins PhaP1 and PhaP2 were used to fuse with GFP, which reflected PHA accumulation with an <i>R</i>-square of over 0.9. Also, a standard correlation was established to calculate PHA contents based on the fluorescence and cell density recorded via a microplate reader with an <i>R</i>-square of over 0.95 when grown on various substrates. The PhaP2-GFP containing <i>H. bluephagenesis</i> was applied successfully to quantify PHA synthesis in a 7.5 L fermenter with high precision. Moreover, the method was found to be feasible in non-natural PHA producers such as <i>Escherichia coli</i>, demonstrating its broad applicability.