Development of a Synthetic, Light-controllable Biofilm for Environmental Biotechnology

Abstract

<p>Biofilms are self-immobilized systems that exhibit high tolerance to harsh conditions and long-term activity. These inherent biofilm features facilitate continuous processing in industrial operations. Hence, biofilms have great potential as industrial workhorses for many applications in sectors ranging from environmental to synthetic chemistry and biopharmaceutical. Because of the intrinsically heterogeneous and dynamic properties of biofilms, one fundamental challenge in biofilm biotechnology is to precisely control biofilm development and hence, performance of biofilm-mediated bioprocesses. Through optogenetically modulating the c-di-GMP biofilm signaling system, we have recently engineered biofilms whose growth and dispersal can be controlled using light and demonstrated their potential applications in environmental biotechnology.</p> <p>We constructed a synthetic near-infrared (NIR) light (660 nm) responsive c-di-GMP module through synthetic biology approaches. The NIR light responsive c-di-GMP module comprises an engineered protein BphS (a light-activated diguanylate cyclase) and BphO (a chromophore biliverdin (BV) synthase). The conformational change of BV-bound BphS arises under NIR light, which results in sequential changes including the activation of its DGC activity, increased c-di-GMP level, and enhanced biofilm formation. As the overexpression of bphO-bphS operon is toxic for cells, we placed this module under the control of the isopropyl β-D-thiogalactoside (IPTG)-inducible promoter Ptac. The expression and activation of BphO are respectively controlled by IPTG and NIR light. The expressed BphS has DGC activity only when IPTG and NIR light are present simultaneously, which accomplishes AND logic gate gene circuits. To characterize our synthetic module, we introduced the NIR light responsive c-di-GMP module into <em>Shewanella oneidensis</em> and demonstrated a biofilm-based AND logic gate in microbial fuel cells (MFCs). </p> <p>To dynamically control biofilm formation and dispersal, we constructed a dichromatically controllable c-di-GMP module comprising our previously constructed NIR light-responsive module and a blue (450 nm) light-activated PDE gene. The blue light-activated PDE, designated as EB1, is a derivative protein from <em>Magnetococcus marinus</em> (Mmc1_2641). EB1 contains two main domains, i.e., a typical PDE domain (EAL domain) which can degrade intracellular c-di-GMP to reduce biofilm formation, and a blue light sensory domain (BLUF domain) which binds to flavin chromophores. Under blue light, conformational change of EB1 leads to the activation of its PDE activity, resulting in a decreased c-di-GMP level and subsequently inducing biofilm dispersal. Because of the spectral compatibility of NIR light (660 nm) and blue light (450 nm), c-di-GMP can be synthesized from GTP by BphS in the presence of NIR light, whereas it can be degraded to pGpG by EB1 in the presence of blue light. Thus, bidirectional control of intracellular c-di-GMP level can be achieved by NIR and blue lights. Integrating this gene circuit into <em>E. coli</em>, we showed that the thickness of the engineered biofilm could be adjusted using NIR and blue lights through modulating the intracellular c-di-GMP concentration. We further demonstrated the potential application of the engineered light-responsive biofilm in mitigating biofouling of water purification forward osmosis membranes by integrating quorum quenching activity into the biofilm. The c-di-GMP targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications.</p>