Evolving a generalist biosensor for bicyclic monoterpenes

Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically-encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida. Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the system’s signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.

[1]  J. Qiao,et al.  Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli. , 2021, ACS synthetic biology.

[2]  H. Alper,et al.  Using structurally fungible biosensors to evolve improved alkaloid biosyntheses , 2021, bioRxiv.

[3]  A. Ellington,et al.  Directed Evolution of an Improved Aminoacyl‐tRNA Synthetase for Incorporation of L‐3,4‐Dihydroxyphenylalanine (L‐DOPA) , 2021, Angewandte Chemie.

[4]  P. Santos,et al.  Current Advances in the Bacterial Toolbox for the Biotechnological Production of Monoterpene-Based Aroma Compounds , 2020, Molecules.

[5]  Fuzhong Zhang,et al.  Dynamic control in metabolic engineering: Theories, tools, and applications , 2020, Metabolic engineering.

[6]  Haiwei Song,et al.  Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps , 2020, Nature Communications.

[7]  Erik K. R. Hanko,et al.  A genome-wide approach for identification and characterisation of metabolite-inducible systems , 2020, Nature Communications.

[8]  E. Takano,et al.  An automated pipeline for the screening of diverse monoterpene synthase libraries , 2019, Scientific Reports.

[9]  Jay D. Keasling,et al.  Evolution-guided engineering of small-molecule biosensors , 2019, bioRxiv.

[10]  Vivek K. Mutalik,et al.  Jungle Express is a versatile repressor system for tight transcriptional control , 2018, Nature Communications.

[11]  M. Bennett,et al.  Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors , 2018, Nature Communications.

[12]  Ying Wang,et al.  Chassis and key enzymes engineering for monoterpenes production. , 2017, Biotechnology advances.

[13]  Mary J Dunlop,et al.  Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance. , 2017, ACS synthetic biology.

[14]  J. Keasling,et al.  Production of jet fuel precursor monoterpenoids from engineered Escherichia coli , 2017, Biotechnology and bioengineering.

[15]  Hal S Alper,et al.  Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications. , 2017, Biotechnology advances.

[16]  Dandan Xiong,et al.  Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor. , 2017, Metabolic engineering.

[17]  Marten Postma,et al.  mScarlet: a bright monomeric red fluorescent protein for cellular imaging , 2016, Nature Methods.

[18]  E. Takano,et al.  A ‘Plug and Play’ Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichia coli , 2016, ChemistrySelect.

[19]  Farren J. Isaacs,et al.  Engineering an allosteric transcription factor to respond to new ligands , 2015, Nature Methods.

[20]  Dan S. Tawfik,et al.  The robustness and innovability of protein folds. , 2014, Current opinion in structural biology.

[21]  Christopher A. Voigt,et al.  Ribozyme-based insulator parts buffer synthetic circuits from genetic context , 2012, Nature Biotechnology.

[22]  Christopher A. Voigt,et al.  Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca , 2012, Proceedings of the National Academy of Sciences.

[23]  Christopher A. Voigt,et al.  Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression , 2009, Nature Biotechnology.

[24]  Hossein Fazelinia,et al.  AraC regulatory protein mutants with altered effector specificity. , 2008, Journal of the American Chemical Society.

[25]  Jared R. Leadbetter,et al.  Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl‐homoserine lactones , 2004, Molecular microbiology.

[26]  S. Ghoshal,et al.  Characterization of a new solvent-responsive gene locus in Pseudomonas putida F1 and its functionalization as a versatile biosensor. , 2003, Environmental microbiology.

[27]  L. Serrano,et al.  Engineering stability in gene networks by autoregulation , 2000, Nature.

[28]  R. Eaton.  p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate , 1997, Journal of bacteriology.

[29]  H. Aramaki,et al.  Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli. , 1994, FEMS microbiology letters.

[30]  H. Aramaki,et al.  Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid , 1993, Journal of bacteriology.

[31]  H. Aramaki,et al.  Transcription of the cam operon and camR genes in Pseudomonas putida PpG1 , 1993, Journal of bacteriology.