Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

Background [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. Methodology/Principal Findings Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. Conclusions/Significance [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.

[1]  K Schulten,et al.  Molecular dynamics and experimental investigation of H(2) and O(2) diffusion in [Fe]-hydrogenase. , 2005, Biochemical Society transactions.

[2]  James A. Stapleton,et al.  A Cell-Free Microtiter Plate Screen for Improved [FeFe] Hydrogenases , 2010, PloS one.

[3]  Michael Seibert,et al.  Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase* , 2004, Journal of Biological Chemistry.

[4]  Hideo Nakano,et al.  High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression. , 2002, Journal of molecular biology.

[5]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[6]  Christos Stathopoulos,et al.  Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines , 1997, Nature Biotechnology.

[7]  H. Nakano,et al.  Microbeads display of proteins using emulsion PCR and cell‐free protein synthesis , 2008, Biotechnology progress.

[8]  Chaoyong James Yang,et al.  High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets. , 2008, Analytical chemistry.

[9]  Andrew D Griffiths,et al.  Miniaturizing chemistry and biology in microdroplets. , 2007, Chemical communications.

[10]  Wang Jing-lin,et al.  In vitro selection and evolution of functional proteins by using ribosome display , 2003 .

[11]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[12]  Dmitry Pushkarev,et al.  Single-molecule sequencing of an individual human genome , 2009, Nature Biotechnology.

[13]  A. Griffiths,et al.  High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions. , 2005, Chemistry & biology.

[14]  M. Pirrung,et al.  A general method for the spatially defined immobilization of biomolecules on glass surfaces using "caged" biotin. , 1996, Bioconjugate chemistry.

[15]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[16]  J. Shendure,et al.  Materials and Methods Som Text Figs. S1 and S2 Tables S1 to S4 References Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome , 2022 .

[17]  J. Stapleton,et al.  Cell‐free synthesis and maturation of [FeFe] hydrogenases , 2008, Biotechnology and bioengineering.

[18]  H. Gest,et al.  A NEW PROCEDURE FOR ASSAY OF BACTERIAL HYDROGENASES , 1956, Journal of bacteriology.

[19]  A. Griffiths,et al.  Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  B J Lemon,et al.  X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. , 1998, Science.

[21]  D. Whitcombe,et al.  The elimination of primer-dimer accumulation in PCR. , 1997, Nucleic acids research.

[22]  H. Stone,et al.  Formation of dispersions using “flow focusing” in microchannels , 2003 .

[23]  J. S. Johnson,et al.  Biocompatible surfactants for water-in-fluorocarbon emulsions. , 2008, Lab on a chip.

[24]  Dan S. Tawfik,et al.  In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting. , 2004, Analytical biochemistry.

[25]  Dan S. Tawfik,et al.  Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry , 2002, FEBS letters.

[26]  Frank Diehl,et al.  BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions , 2006, Nature Methods.

[27]  Liang-Yin Chu,et al.  Designer emulsions using microfluidics , 2008 .

[28]  Dan S. Tawfik,et al.  Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery. , 2005, Journal of molecular biology.

[29]  F. Ghadessy,et al.  A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system. , 2004, Protein engineering, design & selection : PEDS.

[30]  J W Szostak,et al.  RNA-peptide fusions for the in vitro selection of peptides and proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Mehmet Toner,et al.  Controlled encapsulation of single-cells into monodisperse picolitre drops. , 2008, Lab on a chip.

[32]  M. Levy,et al.  Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions , 2009, Current protocols in molecular biology.

[33]  Ie-Ming Shih,et al.  Principle and applications of digital PCR , 2004, Expert review of molecular diagnostics.

[34]  Dan S. Tawfik,et al.  Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization , 2003, The EMBO journal.

[35]  M. Adams,et al.  The structure and mechanism of iron-hydrogenases. , 1990, Biochimica et biophysica acta.

[36]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K D Wittrup,et al.  Yeast surface display for directed evolution of protein expression, affinity, and stability. , 2000, Methods in enzymology.

[38]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[39]  J. Stapleton,et al.  Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation , 2009, PloS one.

[40]  Viktor Stein,et al.  Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets. , 2009, Analytical chemistry.

[41]  Andrew D Griffiths,et al.  Microfluidic production of droplet pairs. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[42]  Dan S. Tawfik,et al.  High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. , 2005, Chemistry & biology.

[43]  D. Weitz,et al.  Geometrically mediated breakup of drops in microfluidic devices. , 2003, Physical review letters.

[44]  J. Rothberg,et al.  Overview: methods and applications for droplet compartmentalization of biology , 2006, Nature Methods.