Engineering a High-Activity Photosensitive Synthase for Optogenetic Control of c-di-GMP and Biofilm Dynamics

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a key bacterial second messenger regulating diverse cellular processes.
• Controlling intracellular c-di-GMP levels is essential for understanding bacterial signaling and developing biotechnological tools.
• Optogenetic methods offer precise spatiotemporal control over biological processes, including c-di-GMP metabolism.

Purpose of the Study:

• To engineer a highly active and photosensitive c-di-GMP synthase for optogenetic applications.
• To develop a tool for precise, light-inducible regulation of bacterial c-di-GMP dynamics.
• To demonstrate the utility of the engineered enzyme in controlling bacterial biofilm formation.

Main Methods:

• Directed evolution involving error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis.
• Engineering of a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase (BphS).
• Characterization of the diguanylate cyclase (DGC) activity and light responsiveness of the engineered variant (BphS-13).

Main Results:

• An engineered BphS variant, BphS-13, with 13 mutations was generated through two rounds of directed evolution.
• BphS-13 exhibited approximately 13-fold higher DGC activity compared to the original BphS.
• BphS-13 demonstrated tightly regulated DGC activity in response to NIR light with minimal dark activity.

Conclusions:

• BphS-13 is a highly active and photosensitive c-di-GMP synthase suitable for optogenetic control.
• This engineered enzyme provides a powerful tool for manipulating bacterial signaling and biofilm dynamics.
• BphS-13 holds potential for future applications in mammalian systems for precise gene expression control.