1.
Engineering a High-Activity Photosensitive Synthase for Optogenetic Control of c-di-GMP and Biofilm Dynamics.
Abstract:
Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a crucial role in bacterial signaling pathways, allowing bacterial cells to respond to various environmental stimuli. The prevalence of c-di-GMP and its potential applications underscore the necessity for developing tools and methods to regulate intracellular c-di-GMP levels. Optogenetic control of c-di-GMP dynamics is particularly attractive because it enables tunable and spatiotemporal regulation of c-di-GMP metabolism. The development of sensitive optogenetic control systems requires highly active, light-responsive c-di-GMP synthases. Here, we report an engineered, highly active photosensitive c-di-GMP synthase, BphS-13. This engineered c-di-GMP synthase was developed from a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase, BphS, using a three-step directed evolution process that included error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis. After two rounds of this directed evolution strategy, we generated a BphS variant with 13 mutations, referred to as BphS-13. The diguanylate cyclase (DGC) activity of BphS-13 was approximately 13 times higher than that of the original BphS, and it exhibited tightly regulated DGC activity in response to NIR light with minimal leakage in the dark. We then demonstrated the effectiveness of BphS-13 in controlling biofilm dynamics. Overall, this study highlights BphS-13 as a highly active and photosensitive tool for optogenetic applications in biotechnology and suggests its future potential application in mammalian systems for precise control of gene expression, particularly given the lack of native c-di-GMP signaling pathways in mammalian cells.
2.
Optogenetic modulation of a catalytic biofilm for biotransformation of indole into tryptophan.
Abstract:
In green chemical synthesis, biofilms as biocatalysts have shown great promise. Efficient biofilm-mediated biocatalysis requires the modulation of biofilm formation. Optogenetic tools are ideal for controlling biofilms, as light is non-invasive, easily controllable and cost-efficient. In this study, we employed a near infrared (NIR) light-responsive gene circuit to modulate the cellular level of c-di-GMP, a central regulator of the prokaryote biofilm lifestyle, which allows us to regulate biofilm formation using NIR light. By applying the engineered biofilm to catalyze the biotransformation of indole into tryptophan in submerged biofilm reactors, we showed that NIR light enhanced biofilm formation to result in ~ 30% increase in tryptophan yield, which demonstrates the feasibility of applying light to modulate the formation and performance of catalytic biofilms for chemical production. The c-di-GMP targeted optogenetic approach for modulating catalytic biofilm we have demonstrated here would allow the wide application for further biofilm-mediated biocatalysis.
3.
Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.
Abstract:
Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. 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 such as biofilm-based biocatalysis.
