Development Of Artificial Naringenin-Resposive Riboswich-based Fluorescent Biosensor Module For Co-culture

Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing.However,productivity and titer of current recombinant strains still do not meet the requirements of industrial processes.Currently,few protein sensor-regulators for flavonoids exist.Unlike the protein-based trans-regulating controllers,riboswitches can respond to their ligands faster and eliminate off-target effects.Here,we developed artificial riboswitches that activate gene expression in response to naringenin,an important flavonoid.RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library.Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches.The selected riboswitches were responsive to naringenin and activated gene expression up to 2.77-fold.Operational ranges of the riboswitches were distinguished based on their selection route.A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches(M1,M2,M3,O,L and H3).A quantitative structure-property relationship approach was employed to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter.Robust models of riboswitches M1,M2 and O that had good predictive power were constructed with descriptors selected for their high correlation.Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches.Hydroxyl groups at the C-3'and C-4' positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2.Riboswitches O and L preferred multi-hydroxylated flavones as ligands.Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process.O-glycosylated derivatives were not recognized by any of the riboswitches,presumably due to steric hindrances.The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains.The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering.Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters,and demonstrate a high-throughput flow cytometry based screening method for identification of naringenin over producing Escherichia coli strains in co-culture.Our efforts helped identify a number of key operating parameters that affect biosensor performance,including the selection of promoter and linker elements within the sensor-actuator domain,and the effect of host strain,fermentation time,and growth medium on sensor dynamic range.The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin Abstract duced by the second member of the synthetic co-culture system.Additionally,the riboswitches applicability as dynamic regulators were confirmed.Despite the challenges of detecting RNA conformational change after ligand binding,the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified.The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design.Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for reoptimization of the producer module when the biosensor is replaced or removed.Collectively,the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.