A new split GFP-based linker and biosensor system for in vivo protein immobilization

In vivo immobilization of proteins on biogenic carriers, such as polyhydroxybutyrate (PHB) granules, is a commonly used technique to enhance their stability and reusability in industrial applications. Although many strategies for protein immobilization have been developed, e.g. the use of linker systems instead of direct fusion of the protein of interest (POI) to a matrix, in vivo immobilization still represents a major challenge. Moreover, online-analysis of the immobilization process is yet not feasible. Here, we present a new approach for PHB-based in vivo immobilization of POIs in Escherichia coli where split fluorescent proteins such as split GFP are used as a linker and biosensor. The split GFP system consists of the superfolder GFP that is split into two non-fluorescent fragments GFP1-10 and GFP11 that have the ability to specifically self-assemble resulting in a green fluorescent signal. In our approach, GFP11 is fused as an anchor protein to the PHB synthase PhaC that is covalently bound to the surface of the PHB granule, while the POI is linked to GFP1-10, resulting in a green fluorescence signal after successful immobilization. To test the system, the red fluorescent protein mCherry, the squalene synthase (SQS) from Methylococcus capsulatus and the ester hydrolase LipD from Alcanivorax borkumensis were used as target proteins. The immobilization process was verified using fluorescence spectrometry, microscopy, and fluorescence lifetime imaging. Online-monitoring of the corresponding immobilization processes in E. coli was performed by determining fluorescence signals during the cultivation and production process. The activity of immobilized enzymes could successfully be demonstrated via HPLC analyses (analysis of squalene formation by the SQS) as well as by the conversion of 4-nitrophenyl butyrate (analysis of LipD activity).

In summary, we successfully showed that the split GFP-mediated system for in vivo immobilization of POIs on PHB granules represents a promising alternative to existing concepts for protein immobilization. Furthermore we could demonstrate that successful immobilization can be online monitored and quantified via the biosensor function of split GFP even when using candidate enzymes that have not yet been used as target proteins for split GFP-mediated immobilization. The new system therefore enables a detectable decoration of the PHB surface and thus represent a promising approach for future biotechnological applications.