Engineering thickness-controllable catalytic biofilms through synthetic biology approaches

Biofilm represents the prevalent mode of microbial growth in natural, engineered and medical settings. Because of their natural immobilization and high tolerance to physicochemical stresses, biofilms have been demonstrated in recent studies as promising biocatalysts. Biofilm thickness is an important factor influencing solute diffusion and biocatalytic activity. It plays an important role in determining the overall performance of biofilm-mediated bioprocesses. Although biofilm development can be modulated through adjusting physicochemical parameters in biofilm-based bioreactors, it is highly challenging to maintain biofilm thickness in an optimal range allowing for sufficient activity and efficient mass transfer. The objective of this study is to develop novel strategies to control the thickness of biofilms though synthetic biology approaches for various biotechnological applications. Biofilm development is often regulated by sophisticated intracellular signaling networks that modulate the levels of small molecules. Among them, Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a prevalent intracellular messenger that regulates biofilm formation. Recent studies modulated biofilm formation by constitutive or chemical-inducible (over)expression of c-di-GMP synthases or hydrolases. However, this may result in an overly thick or thin biofilm due to the irreversibility of constitutively expressing enzymes as well as limited spatial and temporal resolution of chemicals as inducers. Therefore, optogenetic tools are ideal for controlling biofilms, as light is non-invasive, easily controllable and cost-efficient. In Chapter 3 of this thesis, I constructed and tested a near-infrared (NIR) light responsive c-di-GMP module for the modulation of an electroactive biofilm development by expressing a NIR responsive c-di-GMP synthase, BphS, in Shewanella oneidensis. In the study, NIR light increased c-di-GMP levels and enhanced biofilm formation on electrodes, resulting in increased bioelectricity generation in microbial fuel cells. In Chapter 4 of this thesis, I explored the use of optogenetic modulation of a catalytic biofilm to enhance biotransformation of indole into tryptophan by co-transforming the NIR light responsive c-di-GMP module and a tryptophan synthase gene circuit into Escherichia coli. In submerged biofilm reactors, NIR light improved biofilm formation to result in a ~ 30% increase in tryptophan yield. These results demonstrated the feasibility of applying light to “guide” the biofilm formation and improve catalytic performance in bioreactors. As a constantly high c-di-GMP level could result in an undesirably thick biofilm, I subsequently introduced a blue light activated c-di-GMP hydrolase gene, eb1, into the NIR light responsive c-di-GMP module to bi-directionally control c-di-GMP levels and applied this dichromatic optogenetic c-di-GMP gene circuit to mitigate biofouling on water purification membranes in Chapter 5. In this study, quorum quenching activity was used to show the effectiveness of the light-responsive gene circuit to modulate biofilm thickness while exhibiting desirable functions. Taken together, this thesis work exemplifies the potential for translational research from biofilm biology to biofilm engineering for applied and environmental biotechnology.Doctor of Philosoph