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WO2019035955A1 - SYSTÈME À HAUT RENDEMENT UTILISANT UN SYSTÈME D'EXPRESSION ACELLULAIRE ET UN SÉQUENÇAGE IN SITU <i /> - Google Patents

SYSTÈME À HAUT RENDEMENT UTILISANT UN SYSTÈME D'EXPRESSION ACELLULAIRE ET UN SÉQUENÇAGE IN SITU <i /> Download PDF

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WO2019035955A1
WO2019035955A1 PCT/US2018/000230 US2018000230W WO2019035955A1 WO 2019035955 A1 WO2019035955 A1 WO 2019035955A1 US 2018000230 W US2018000230 W US 2018000230W WO 2019035955 A1 WO2019035955 A1 WO 2019035955A1
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cell
nucleic acid
free
template nucleic
template
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PCT/US2018/000230
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Daniel Jordan WIEGAND
Nili OSTROV
George M. Church
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B20/00—Methods specially adapted for identifying library members
    • C40B20/08—Direct analysis of the library members per se by physical methods, e.g. spectroscopy

Definitions

  • Embodiments of the present disclosure are directed to a method of screening a plurality of template nucleic acid sequences for small molecule or protein production including adding a template nucleic acid sequence from the plurality to each reaction volume within a plurality of reaction volumes, wherein each reaction volume includes a cell-free expression system under conditions of transcription and translation; detecting generation of a small molecule in a target reaction volume; and sequencing of the template nucleic acid sequence of the target reaction volume.
  • Figs. 1A-1C are directed to optimization of cell extract assay.
  • Fig 1A The Mg- glutamate concentration was optimized for the S. lividans cell extract used to produce sfGFP in a cell free reaction vessel. The optimal final concentration of Mg-glutamate was found to be 4 mM, however each concentration produced GFP over the course of 4 hours in comparison to the blank (35% cell extract and no energy buffer).
  • Fig I B A non-denaturing 16% Tris-Glycine protein gel was used to verify the weight of the GFP produced from the in vitro reactions.
  • Fig. 1C The K-Glutamate concentration was calibrated to the S.
  • lividans cell extract after determining the optimal Mg-Glutamate concentration of 4 niM.
  • Fig. 2 is a spectral scan of lycopene in 35% cell extract diluted in hexane - blank corrected.
  • Fig. 3 is a schematic of a screening process using a metagenomic DNA library.
  • Figs. 4A-4I depict data from various experiments directed to a V. natriegens cell free system.
  • Figs 5A-5F depict data from various experiments directed to a Pseudomonas aeruginosa cell-free expression system.
  • Figs 6A-6B depict -data from various experiments directed to temperature calibration of a Pseudomonas aeruginosa cell-free expression system.
  • Fig. 7 depicts data directed to cyclic voltammetry electrochemical analysis of Pseudomonas aeruginosa cell-free reactions.
  • Fig. 8 depicts data directed to square wave voltammetry electrochemical analysis of Pseudomonas aeruginosa cell-free reactions.
  • Figs. 9A-9E depict data directed to square wave voltammetry electrochemical analysis of Streptomyces spps. cell-free reactions.
  • Figs. 10A-10D depict data directed to square wave voltammetry electrochemical analysis of Vibrio natriegens cell-free reactions (effect of optical density).
  • Figs. 1 1 A-1 1G depict data directed to square wave voltammetry electrochemical analysis of Vibrio natriegens cell-free reactions (effect of growth media).
  • Fig. 12 depicts data directed to cell-free protein synthesis from mixtures of E. coli and V. natriegens crude extracts.
  • Embodiments of the present disclosure are directed to high throughput screening assays using cell-free systems for the screening of small molecule production.
  • a plurality of individual reaction volumes of a cell-free expression system is used to screen nucleic acid templates for the production of small molecules.
  • a single nucleic acid template is provided to or is contained within a single reaction volume of the plurality of reaction volumes of the cell-free expression system under conditions favoring expression of the nucleic acid template into one or more proteins or enzymes.
  • each reaction volume of the plurality of reaction volumes of the cell-free expression system includes a nucleic acid template, such as a single nucleic acid template, under conditions favorable for nucleic acid expression of one or more proteins or enzymes.
  • the reaction volume also includes factors for manufacture of the small molecule by the one or more proteins or enzymes.
  • the reaction volume coordinates in vitro co- expression of multiple enzymes required for synthesis of the small molecule.
  • Each reaction volume is analyzed to determine whether a small molecule has been generated in the reaction volume using a detection method, such as a color detection method should the small molecule exhibit a color as a feature of the small molecule.
  • a detection method such as a color detection method should the small molecule exhibit a color as a feature of the small molecule.
  • Other small molecule detection methods known to those of skill in the art, such as Liquid Chromatography - Mass Spectrometry (LC-MS), Nuclear Magnetic Resonance (NMR), X- Ray Crystallography, electrochemical sensing methods or other small molecule or protein or enzyme detection methods can be used for detection and characterization of the small molecule, protein or enzyme.
  • the reaction volume is subjected to nucleic acid sequencing, such as in situ nucleic acid sequencing, to determine the sequence of the template nucleic acid sequence and therefore the identity of the one or more proteins or enzymes used to make the small molecule.
  • nucleic acid sequencing such as in situ nucleic acid sequencing
  • the small molecule may be characterized by methods known to those of skill in the art to determine features, such as structure or other characteristics of the small molecule.
  • aspects of the present disclosure are directed to a method for high throughput screening of biomolecules, such as proteins, small molecules and natural products, using a cell-free system.
  • Methods described herein enable rapid identification of products in cell-free extracts, such that large libraries of metabolic pathways can be screened, for example metagenomic fragments.
  • a cell-free system including cell extract is used for expression of proteins and small molecules within a reaction volume. Since the present disclosure contemplate high throughput methods, a plurality of reaction volumes are used. Each reaction volume includes cell extract in an amount, for example, of about 10 uL or less.
  • a nucleic acid template, encoding a single gene or an entire metabolic pathway, is added to the cell extract in the reaction volume to initiate transcription and translation of proteins or enzymes required for production of product.
  • each reaction volume of the plurality of reaction volumes within the high throughput system includes a different nucleic acid template.
  • Libraries of nucleic acid templates are known and methods of incorporating a single nucleic acid template from a library of nucleic acid templates into an individual reaction volume are known to those of skill in the art, for example, by dilution of pooled libraries or using microfluidic device to compartmentalize single template in single droplet.
  • an array of cell-free reactions is utilized for high throughput screening of proteins, enzymes or small molecules using detection methods, such as spectrophotometric or fiuorimetric methods or other detection methods known to those of skill in the art.
  • aspects of the present disclosure are directed to an in vitro Streptomyces cell-free expression or reaction system capable of producing colored small molecules for detection.
  • Such a system can be used to determine the ability of the in vitro Streptomyces cell-free reaction system to produce a target molecule.
  • Such an in vitro Streptomyces cell-free reaction system capable of producing colored small molecules has utility in methods of high- throughput screening and optimization of biosynthesis of biomolecules.
  • aspects of the present disclosure are directed to a cell-free system including the cellular machinery of Streptomyces or other cell source, such as bacterial, yeast, plant, insect, or mammalian cell extract, for a cell-free transcription and translation system utilized in a high throughput screening system to identify template nucleic acids that produce small molecules.
  • a cell-free system including the cellular machinery of Streptomyces or other cell source, such as bacterial, yeast, plant, insect, or mammalian cell extract, for a cell-free transcription and translation system utilized in a high throughput screening system to identify template nucleic acids that produce small molecules.
  • Embodiments of the present disclosure are directed to a cell-free expression system including cell extract from Streptomyces for the expression of proteins and small molecules.
  • a reaction volume of cell extract from Streptomyces in the range of from about 1 ⁇ to about 10 ⁇ is provided or generated in a reaction system.
  • a plurality of a reaction volumes of cell extract from Streptomyces in the range of from about 1 ⁇ to about 10 ⁇ is provided or generated in a reaction system.
  • a nucleic acid template is within the reaction volume of cell extract from Streptomyces under conditions to initiate transcription and translation of the nucleic acid template.
  • the nucleic acid template may be a DNA or RNA template, such as a DNA template encoding a single gene, one or more genes or a metabolic pathway.
  • Useful templates are those as are known in the art and can be readily identified by those of skill based on the present disclosure.
  • the template can be linear (PCR product) or circular (plasmid) nucleic acid.
  • the DNA template may be added to the extract to initiate transcription and translation of proteins or enzymes required for production of a target protein or target small molecule. Each reaction volume is analyzed for the small molecule generation. If a small molecule is detected, then the template nucleic acid is sequenced to identify the proteins or enzymes or biosynthetic pathway used to make the small molecule.
  • the small molecule may be identified using characterization methods know to those of skill in the art, such as LC-MS, NMR, X-Ray Crystallography or electrochemical sensing methods.
  • aspects of the present disclosure integrate or combine an in vitro cell-free reaction system with a high-throughput screening method.
  • a high throughput screening method may utilize traditional well plates, such as a 96- or 384-well plate, or depressions or wells on a slide to form in vitro compartments or may utilize droplets, microdroplets or emulsion microdroplets for the reaction volume which may then sorted based on positive hits, deposition on the surface, or in a gel. See, for example, Zinchenko, A. et al., 2014. One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.
  • aspects of the present disclosure are directed to detecting and/or identifying small molecules produced by a template nucleic acid sequence in a cell-free expression system.
  • the system and methods described herein can also be used to screen for regulators of biosynthetic pathways which might enhance expression of cryptic pathways for production of novel molecules. Additional methods include identification of unknown biomolecules in metagenomic or other libraries; screening for in vitro production of synthetic natural products analogs; screening for optimized conditions for cell-free production of natural products; screening for optimized conditions for cell-free production of synthetic biomolecules; or screening for global regulators (transcription factors) that can enhance expression of cryptic pathways in host cell-extract system.
  • aspects of the present disclosure utilize emulsion droplet methods for compartmentalization of reaction volumes. Such droplet methods can be scaled up for biosynthetic libraries and desired biomolecules.
  • the emulsion droplet reaction volumes can be integrated into systems for primary readout of the emulsion droplets. FACS and cell sorting methods can be adapted to the emulsion droplet reaction volumes.
  • Embodiments of the present disclosure utilize automated systems and devices for reagent delivery and/or mixing in reagent volumes, reagent volume immobilization such as in a gel (a polyacrylamide gel for example) or three dimensional compartment and in situ sequencing. Screening of library components, such as metagenomic fragments or biosynthetic pathways, can be multiplexed, i.e. massively parallel screening, wherein library components can reach on the order of 10 10 members.
  • CELL-FREE SYSTEMS CELL-FREE SYSTEMS
  • the screening method utilizes a cell-free expression system to screen for expression of a template nucleic acid sequence and production of a small molecule. It is to be understood that one of skill will readily be able to identify useful cell- free expressions known in the art based on the present disclosure. According to one aspect, methods are provided for using an active cell free system to rapidly screen for production of valuable molecules or proteins. According to one aspect, one or more, two or more or a plurality of different genus or species of cells, such as those described herein, can be used for making cell-free systems as described herein. Accordingly, a library of cell free systems is provided to carry out the methods described herein.
  • Cell-free expression systems are useful in the present embodiments as they greatly reduce the complexity of cellular assays by using cell extract rather than live cells.
  • cell lysis is followed by removal of cellular debris and chromosomal DNA.
  • the remaining cell extract is added with energy substrates, such as NTPs, PEP (phosphoenolpyruvic acid) or 3-PGA (3-phosphoglyceric acid) or other energy source, cofactors, salts, amino acids and deoxynucleotides in sufficient amounts and under sufficient conditions to be able to transcribe and translate a temple nucleic acid sequence.
  • the desired template DNA or RNA is added for transcription and translation of desired proteins or biomolecules. See Carlson, E.D. et al., 2012. Cell-free protein synthesis: applications come of age. Biotechnology advances, 30(5), pp.1 185-1 194 hereby incorporated by reference in its entirety.
  • a cell for providing or producing a cell free extract is selected based on the number and type of readily detectable natural products, which may be readily detectable, and subsequently, available precursor compounds.
  • Such cells are selected for preparing extracts, crude or otherwise, for cell-free expression as such extracts are advantageous as a host for screening biosynthesis pathways in a high-throughput manner.
  • extracts and cell-free expression systems can be used in the analysis of the underlying biosynthetic pathways involved in breakdown and/or production of chemical compounds, such as hydrocarbons or other biopolymers, and in resistance studies, such as antibiotic resistance.
  • Exemplary cells may have one or more of clinical relevance, bioenergy relevance, environmental relevance, soil dwelling relevance, natural product relevance, radiation resistance relevance; high temperature relevance, extreme growth conditions relevance, thermostable enzyme relevance and the like.
  • cell-free expression systems are based on extracts of the Staphylococcus genus.
  • Exemplary species include Staphylococcus aureus or Staphylococcus epidermidis and the like.
  • cell-free expression systems are based on extracts of the Pseudomonas genus.
  • Exemplary species include P. aeruginosa, P. fluorescens, and P. putida.
  • cell-free expression systems are based on extracts of the Streptomyces genus.
  • Exemplary species include Streptomyces coelicolor, S. lividans, S. albicans, S. griseus, and S. plicatosporus and the like.
  • cell-free expression systems are based on extracts of the Flavobacterium genus.
  • Exemplary species include F. columnare, F. psychrophilum, F. branchiophilum, F. aquatile; F. ferrugineum; F. johnsoniae; F. limicola; F. micromati; and F. psychrolimnae and the like.
  • cell-free expression systems are based on extracts of the Bacillus genus.
  • Exemplary species include Bacillus cereus, Bacillus halodurans, Bacillus insolitus, Bacillus pumilis, and Bacillus subtilis and the like.
  • cell-free expression systems are based on extracts of the Deinococcus genus.
  • Exemplary species include D. geothermalis, D. grandis, D. indicus, D. murrayi, D. p oteolyticus, and D. radiodurans and the like.
  • cell-free expression systems are based on extracts of the Thermus genus.
  • Exemplary species include T. antranikianii, T. aquaticus, T. brockianus, T. caldophilus, T. filiformis, T. igniterrae, T. kawarayuensis, T. nonproteolyticus, T. oshimai, T. rehai, T. scotoductus, and T. thermophilus and the like.
  • cell-free expression systems are based on extracts of the Escherichia genus.
  • Exemplary species include E. coli and the like.
  • cell-free expression systems are based on extracts of the Vibrio genus.
  • Exemplary species include V. cholerae, V. natriegens, V. parahaemolyticus and the like.
  • cell-free expression systems are based on extracts of plants.
  • Exemplary species include Nicotiana tabacum, Arabidopsis thaliana, Artemisia annua, and the like.
  • cell-free expression systems are based on extracts of fungal cells.
  • Exemplary species include Aspergillus oryzae, Saccharomyces rouxii, Aspergillus terreus, Aspergillus griseus, Penicillium notatum, S. cerevisiae, S. pombe and the like.
  • methods are provided that utilize mixtures of various cell-free crude extracts to express template nucleic acids such as in a method to screen biosynthesis pathways that may be active in the presence of the respective combination or mixture.
  • a mixture of Streptomyces violaceruber and Pseudomonas aeruginosa is contemplated to yield natural products from otherwise inaccessible pathways not common naturally.
  • Any mixture of cell-free extracts from different genus or species of cells is contemplated.
  • cell-free expression systems are based on extracts of one or more, two or more or a plurality of cells of the following genera: Staphylococcus, Pseudomonas, Streptomyces, Flavobacterium, Bacillus, Deinococcus, Thermus, Escherichia or Vibrio.
  • cell-free expression systems are based on extracts of two or more different genus of cells, such as is described herein.
  • cell- free expression systems are based on extracts of two or more different species of cells, such as is described herein.
  • cell-free expression systems are based on a combination of extracts from two or more different genera or species of cells.
  • cell-free expression systems are based on a combination of extracts from two or more different genus or species of cells, for example, cells selected from two or more genera among Staphylococcus, Pseudomonas, Streptomyces, Flavobacterium, Bacillus, Deinococcus, Thermus, Escherichia or Vibrio or the species described herein.
  • Cell-free expression systems have been based on extracts of E. coli. See Dudley, Q.M., Karim, A.S. & Jewett, M.C., 2015. Cell-free metabolic engineering: biomanufacturing beyond the cell. Biotechnology journal, 10(1), pp.69-82 and Dudley, Q.M., Anderson, K.C. & Jewett, M.C., 2016. Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis. ACS synthetic biology, 5(12), pp.1578-1588 each of which is hereby incorporated by reference in its entirety. Cell-free expression systems have been based on yeast, plant and several eukaryotic cell systems.
  • Cell-free systems are also commercially available such as Thermo-fisher ⁇ -step IVT kits' (product AM1200: Rabbit reticulocyte, product 88881 : Human HeLa cell lysate, product 88893: CHO (chinese hamster ovary) lysate), New England biolabs E.coli IVT system (product E6800S), Promega Wheat germ extract (L4330).
  • Exemplary cell free systems for protein synthesis include those a cell free system in E. coli (see Kim H-C, Kim T-W, Kim D-M. Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source. Process Biochem.
  • Rabbit reticulocyte see Hancock JF. [7] Reticulocyte lysate assay for in Vitro translation and posttranslational modification of Ras proteins. Methods Enzymol. 1995;255: 60-65; Gibbs PE, Zouzias DC, Freedberg IM. Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system. Biochim Biophys Acta.
  • Embodiments of the present disclosure are directed to a cell-free expression system based on the Streptomyces genus.
  • Streptomyces includes the genetic components and cellular machinery to produce antibiotics such as Streptomycin and Tetracyclin. See Kieser, T. et al., Practical Streptomyces Genetics. 2000. Norwich: John Innes Foundation Google Scholar hereby incorporated by reference in its entirety.
  • Streptomyces also includes the genetic components and cellular machinery to produce bioactive compounds for immunosuppression, antifungal agents, antiparasitic drugs, and antitumoral medications. See Avignone-Rossa, C, Kierzek, A.M. & Bushell, M.E., 2013.
  • Streptomyces have been genome sequenced and metabolic pathways are known to those of skill in the art. See, for example, the complete genome sequencing of S. coelicolor A3(2) and other species as described in Bentley, S.D. et al., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature, 417(6885), pp.141— 147 hereby incorporated by reference in its entirety.
  • Streptomyces species S. lividans Li, J. et al., 2017. Establishing a high yielding streptomyces-based cell-free protein synthesis system. Biotechnology and bioengineering, 1 14(6), pp.1343— 1353 hereby incorporated by reference in its entirety
  • S. venezuelae Moore, S.J. et al., 2017. Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool. Biotechnology journal, 12(4).
  • Certain cell-free protein expression systems based on the genus Pseudomonas find use in the present disclosure.
  • a particularly exemplary species includes Pseudomonas aeruginosa which is known to produce naturally occurring products such as the phenazines class of compounds, which are primarily utilized by Pseudomonas aeruginosa in quorum sensing, virulence, and other important biological processes.
  • Important phenazine compounds produced by Pseudomonas aeruginosa include, but are not limited to: pyocyanin, pyoverdine, pyorubin, pyomelanin, fluorescein, 1 -hydroxyphenzaine, phenazine-l-carboxamide, 5- methylphenazine-l-carboxylic acid.
  • a defining characteristic of these compounds is that many are redox-active colored pigments which can be readily detected at low concentrations using high-throughput UV-Vis spectral analysis or electrochemical methods such as cyclic voltammetry, square wave voltammetry, and the like.
  • V. natrigens which can be used for providing extracts, crude or otherwise for use as cell free system for expression of template nucleic acids.
  • Exemplary template nucleic acids include libraries of genes or metabolic pathways, such as libraries generated from genomic fragments, from metagenomic fragments and other genetic source material.
  • Such template nucleic acids may include biosynthetic pathways, fragments containing a biosynthetic pathway, a library of biosynthetic pathways.
  • the source of such biosynthetic pathways may include natural genomic DNA from bacteria, plants, mammals or any other source of natural biosynthetic pathways.
  • the biosynthetic pathways may be synthetic, i.e. engineered, insofar as they do not exist in nature but are created by combining particular genes to create a particular small molecule.
  • a DNA library for screening can be rationally designed, or can be combinatorial enzyme libraries, or can be derived from natural pathways derived from unknown metagenomic sequences.
  • DNA template for cell-free system can be linear or circular, and prepared from natural or synthetic DNA.
  • DNA is extracted from unknown or unculturable environmental organisms to make metagenomic libraries.
  • Natural metagenomic libraries are constructed by extraction and cloning of large DNA fragments into bacterial artificial chromosome (BAC) or other vectors. The pooled library is then maintained and amplified in E.coli for further screening (see for example Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR, et al.
  • synthetic libraries can also be constructed by mutagenesis of natural genes such that the resulting proteins promote biosynthesis of novel molecules. Methods for producing synthetic genes are known to those of skill in the art and include de novo DNA synthesis, directed evolution, PCR mutagenesis, recombination or chimeragenesis).
  • one aspect of the present disclosure is a screening method or assay and so the template nucleic acid within a particular reaction volume is unknown. If small molecule generation is detected, then the template nucleic acid is subject to sequencing to determine its sequence, such as by in situ sequencing.
  • aspects of the present disclosure are directed to the production of a protein, enzyme or small molecule using a cell-free expression system. It is to be understood that one aspect of the present disclosure is a screening method or assay and so the small molecule that may be generated is unknown. Likewise, the nucleic acid template is unknown.
  • Small molecules within the scope of the present disclosure include natural products, synthetic products and the like.
  • Important natural product groups include, but are not limited to, terpenoids, anthocyanins, batalains, flavonoids and polyketides (see Delgado- Vargas F, Jimenez AR, Paredes-Lopez O.
  • Synthetic products include natural products analogs and synthetic small molecules.
  • aspects of the present disclosure are directed to a system or method for creating or using a plurality of individual reaction volumes including a cell-free expression system for the screening of small molecule production from a template nucleic acid.
  • Methods and apparatuses for creating a plurality of reaction volumes for high throughput methods are known to those of skill in the art and are adaptable to the present disclosure.
  • cell-free expression systems are advantageous insofar as they utilize a minimal reaction volume which promotes low cost of critical reagents, and are easily integratable into a high- throughput assay scheme using high-capacity well-plates, microarrays, emulsion microdroplets, or mechanical automation. See Sun, Z.Z. et al., 2013.
  • reaction volumes may be present within wells of a well plate, on slides, on microarrays, within reaction chambers of a microfluidic device, within microdroplets of an emulsion and the like. See Guo MT, Rotem A, Heyman JA, Weitz DA. Droplet microfluidics for high-throughput biological assays. Lab Chip. 2012; 12: 2146-2155; Zinchenko A, Devenish SRA, Kintses B, Colin P-Y, Fischlechner M, Hollfelder F.
  • each reaction volume within a plurality of reaction volumes can contain a single nucleic acid template or one or more or multiple or a plurality of nucleic acid templates encoding one or more or a plurality of genes of a biosynthetic pathway for making a small molecule.
  • a single reaction volume can contain one or more or multiple or a plurality of nucleic acid templates. It is to be understood that each reaction volume is not limited to a single nucleic acid template.
  • a single nucleic acid template may be used when the single nucleic acid template encodes all gene form a biosynthetic pathway. However, multiple separate nucleic acid templates can be used which express one or more genes for one or more biosynthetic pathways that together synthesize a small molecule.
  • Exemplary reaction volumes are within the range of about 1 ul to about 10 ul for high capacity plates, such as a 384-well plate and within the range of approximately 100 pL to 10 nL for microdroplets.
  • Microdroplets are generated using a liquid pump system and microfluidic channels/array from commercial available vendors such as Dolomite Microfluidics, where aqueous droplets containing the reagents for the cell-free reaction are formed within a flowing organic phase. Additional components such as small beads can also be incorporated into this design in order to deliver sensitive payloads like those that could be degraded by endogenous nucleases or proteases as well as to increase temporal control of the cell-free reaction system.
  • Microdroplet formation can be viewed and evaluated for efficiency using a mounted magnifying scope, CCD camera and computer-aided detection software.
  • microdroplets containing the cell free reaction are singular in nature, however the preferred embodiment would be a double-emulsion in which microdroplets containing the cell-free reaction are again encapsulated in another droplet of larger size formed in a directly connecting channel/array.
  • the double emulsion cell-free system allows for microdroplets to be screened in a high-throughput manner with Fluorescence-activated cell sorting (FACS), general colorimetric based cell sorting, surface deposition on a microarray, or in situ sequencing without prematurely bursting or lose of microdroplet contents.
  • FACS Fluorescence-activated cell sorting
  • aspects of the present disclosure are directed to the detection of a small molecule, protein or enzyme using any suitable detection methods, such as detection of color as a feature of the small molecule, protein or enzyme.
  • the screening of desired products can be done by colorimetric, fluorescent or electrochemical methods. Electrochemical screening is exemplary for redox-based detection in several samples.
  • Exemplary methods for detecting a small molecule exhibiting a color include spectrophotometric methods, fiuorimetric methods and the like. Individual reaction volumes are analyzed to detect whether a small molecule has been produced. Where the reaction volumes are droplets, methods known to those of skill in the art such as FACS or flow cell sorting or other sorting methods may be used to identify the presence of a small molecule in a droplet and then separate out and isolate the droplet for further analysis.
  • the cell-free reactions in 384-well plates are scanned using an instrument with the capacity for scanning the absorbance in the UV and visible wavelengths or an instrument using another detection method.
  • a positive hit in which a biosynthesis pathway has produced a small molecule, protein or enzyme from a known or unknown pathway from the starting template, is indicated by the formation of distinct peaks on the spectrogram upon the cell-free reaction reaching its termination after a pre-defined number of hours.
  • high capacity plates include a set number of wells containing only cell-free extract without template or reaction buffer to be used as a reference blank. These wells do not produce a colorimetric or fluorescent signal beyond background noise.
  • Biomolecules or small molecules extracted from positive hits are then further analyzed using the aforementioned instrumental analysis methods such Liquid Chromatography-Mass Spectrometry (LC-MS), Nuclear Magnetic Resonance (NMR), X-Ray Crystallography to determine molecular structure as well as function with additional assays.
  • the template within the positive hit well is then sequenced with traditional sequencing methods (such as Sanger sequencing), next-generation gene sequencing, or in situ sequencing.
  • This general method for identifying and screening for biomolecules or small molecules can be applied to the aforementioned microdroplet emulsion (singular or double) system established in the previous section in combination with flow cytometry or microarraying methods.
  • methods are provided for detection of expression products using electrochemical sensing, such as where the expressed product is redox-active. Accordingly, methods are provided for screening redox-active products, such as natural products, in cell-free systems as described herein using electrochemical sensing. For example, methods are provided for the electrochemical detection of natural products in V.natriegens, P. aeruginosa and several Streptomyces spp. IN SITU AMPLIFICATION AND SEQUENCING
  • sequencing is used to identify the coding DNA once a desired product is detected. Sequencing facilitates testing of libraries of unknown metabolic pathways.
  • the template nucleic acid sequence can be amplified using methods known to those of skill in the art.
  • Methods of amplifying nucleic acids include rolling circle amplification in situ.
  • methods of amplifying nucleic acids involves the use of PCR, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91 :360-364; incorporated herein by reference in their entirety for all purposes).
  • LCR ligation chain reaction
  • Alternative amplification methods include: self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874, incorporated herein by reference in its entirety for all purposes), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. US. 86: 1 173, incorporated herein by reference in its entirety for all purposes), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6: 1 197, incorporated herein by reference in its entirety for all purposes), recursive PCR (Jaffe et al. (2000) J. Biol. Chem.
  • the template nucleic acid within a given reaction volume can be sequenced using methods known to those of skill in the art such as in situ sequencing.
  • General sequencing methods known in the art such as sequencing by extension with reversible terminators, fluorescent in situ sequencing (FISSEQ), pyrosequencing, massively parallel signature sequencing (MPSS) and the like (described in Shendure et al. (2004) Nat. Rev. 5:335, incorporated herein by reference in its entirety), are suitable for use with the matrix in which the nucleic acids are present.
  • Reversible termination methods use step-wise sequencing-by- synthesis biochemistry that coupled with reversible termination and removable fluorescence (Shendure et al. supra and U.S. Patent Nos.
  • FISSEQ is a method whereby DNA is extended by adding a single type of fluorescently-labelled nucleotide triphosphate to the reaction, washing away unincorporated nucleotide, detecting incorporation of the nucleotide by measuring fluorescence, and repeating the cycle. At each cycle, the fluorescence from previous cycles is bleached or digitally subtracted or the fluorophore is cleaved from the nucleotide and washed away.
  • FISSEQ is described further in Mitra et al. (2003) Anal. Biochem. 320:55, incorporated herein by reference in its entirety for all purposes.
  • Pyrosequencing is a method in which the pyrophosphate (PPi) released during each nucleotide incorporation event (i.e., when a nucleotide is added to a growing polynucleotide sequence).
  • the PPi released in the DNA polymerase-catalyzed reaction is detected by ATP sulfurylase and luciferase in a coupled reaction which can be visibly detected.
  • the added nucleotides are continuously degraded by a nucleotide-degrading enzyme. After the first added nucleotide has been degraded, the next nucleotide can be added. As this procedure is repeated, longer stretches of the template sequence are deduced. Pyrosequencing is described further in Ronaghi et al.
  • MPSS utilizes ligation-based DNA sequencing simultaneously on microbeads. A mixture of labelled adaptors comprising all possible overhangs is annealed to a target sequence of four nucleotides. The label is detected upon successful ligation of an adaptor. A restriction enzyme is then used to cleave the DNA template to expose the next four bases. MPSS is described further in Brenner et al. (2000) Nat. Biotech. 18:630, incorporated herein by reference in its entirety for all purposes.
  • a Streptomyces optimized cell free system for in vitro protein expression was developed using an enhanced Transcription-Translation (TX-TL) preparation protocol, based on previously described methods.
  • TX-TL Transcription-Translation
  • S. lividans with strain designation 3213 was purchased from ATCC as a lyophilized spore stock was used to optimize an enhanced TX-TL protocol for expression and quantification of Superfolder GFP (sfGFP).
  • 10 mL of Yeast Extract-Malt Extract (YEME) medium was inoculated in a 100 mL baffled flask with spores from S. lividans lyophilized stock and incubated at 30°C, rotating at 250 RPM for 72 hours.
  • cell suspensions were diluted at 1 :500 ratio in 50 mL of fresh YEME medium in a 500 mL baffled flask and incubated for 8 hours under the same conditions.
  • the resulting cell-suspensions were then transferred to 50 mL Falcon tubes and pelleted via centrifugation at 3000 x G for 25 minutes at 4°C.
  • the supernatant of the resulting pellets were suctioned off and the pellets were washed with fresh S30A buffer containing 2 mM DTT twice before being resuspended in 1 mL of S30A buffer in a 2 mL Eppendorf tube for cell lysis via sonication.
  • Concentrated cells-suspensions were then pulse sonicated (Qsonica Sonicator Model CI- 18) for 40 sec with 59 sec stops x4 times in an ice water bath within a cold-room at 4°C.
  • the resulting cell extracts were spun at 15,000 x G for 30 minutes at 4°C and the supernatants were passed through a microcentrifuge column polyethylene filter with a 30 um pore size (Thermo Scientific) at 1000 x G for 5 minutes at 4°C to remove any remaining cell debris.
  • the cell extracts' total protein concentration was quantified with a Qubit Fluorometer Protein Kit (Invitrogen) before being flash frozen in liquid nitrogen and stored until needed at -80°C.
  • Amino Acid Master Mix Solutions of each amino acid was obtained from BiotechRabbit (P/N: BR1401801) at a concentration of 168 mM (Lysine at 140 mM). At 4X master mix was made up at a final concentration of 8 mM in this following order: (ALA, ARG, ASN, ASP, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, PRO, SER, THR, VAL, TRP, TYR, LEU, CYS) and flash frozen in liquid nitrogen and stored until needed at -80°C.
  • a 10X master mix of the energy solution was prepared at a total volume of 125 uL. This contained: 500 mM HEPES (pH 8.0 adjusted with 5M KOH), 15 mM ATP, 15 mM GTP, 9 mM CTP, 9 mM UTP, 2 mg/mL E. coli tRNA (MRE100), 2.6 mM CoA, 3.3 mM NAD, 7.5 mM cAMP, 0.70 mM folinic acid, 10 mMspermidine, and 300 mM 3-Phosphoglyceric Acid (Santa Cruz) added in this order.
  • sfGFP signal was measured every 5 minutes over 4 hours on a BioTek Synergy HT plate reader, ex: 485/20 nm & em: 528/20 nm.
  • K-Glutamate Concentration Using the best results from the Mg-Glutamate concentration calibration (4 mM), the K- Glutamate concentration for TX-TL reactions was also calibrated using the reagents as previously described above, with the exception of variable K-Glutamate.
  • GFP Quantification A recombinant GFP (Roche) standard curve was made up in 35% S. lividans cell extract and phosphate buffer saline (PBS) (dynamic range 250 ug/mL - 3.9 ug/mL). After 4 hours of incubation, the TX-TL reaction was stopped by placing plate on ice and then GFP standard curve was added to the 384-well plate. Samples were quantified by comparing their signal at 485/20 nm & em: 528/20 nm to the linear regression generated by the GFP standards. The resulting concentrations were adjusted for background signal with a blank.
  • PBS phosphate buffer saline
  • Example I Demonstrate biosynthesis of known biomolecule: To test for cell free production of small molecules, the system of Example I was used for expression of lycopene, an isoprenoid pigment naturally produced in bacteria and plants.
  • the lycopene biosynthetic pathway previously heterologously expressed in E.coli and S. cerevisiae, is a simple 3-enzyme pathway widely used in microbial metabolic engineering. See Farmer, W.R. & Liao, J.C., 2000. Improving lycopene production in Escherichia coli by engineering metabolic control. Nature biotechnology, 18(5), pp.533-537; Yamano, S. et al., 1994.
  • UV-Vis spectroscopy is used to demonstrate the production of lycopene in the Streptomyces cell-free reaction system by first performing a spectral scan from 300 nm to 700 nm with 10 nm increments and comparing these to a reference standard of lycopene.
  • a standard curve of lycopene (Sigma) (See Fig. 2) is used to generate a linear regression in which the biosynthesized lycopene can be compared.
  • concentration and rate of production of lycopene can be determined.
  • Lycopene further can be characterized with LC-MS, NMR, X-Ray Crystallography or any additional instrumental analysis methods. It is to be understood that the methods described herein for in vitro production and detection of lycopene with instrumental analysis methods can be applied to any other molecule produced in a cell-free reaction environment, for optimization of in vitro conditions and for screening for in vitro production of analogs of natural products.
  • a library of unknown biosynthetic pathways for example metagenomic fragments, can be screened for production of desired molecules exhibiting color.
  • Extremely small volume (100 pL to 10 nL) transcription - translation reactions can be carried out in 384- well plate or in emulsion droplets generated with a microfluidic chip (t-junction or y- junction) and biphasic (organic/aqueous) pump system. Each microdroplet contains a single barcoded plasmid encoding a biosynthesis pathway of interest.
  • Pathway enzymes can be controlled by T7 promoter or a species-specific promoter, provided the appropriate polymerase is included in the extract.
  • the emulsion droplets can be encapsulated in a secondary droplet. The mix is screened by flow sorting, or via spectral and fluorescent plate scan.
  • Exemplary method steps include growing cells and creating cell lysate.
  • the cell lysate is aliquoted (l Oul) in 384-well format with additional assay components.
  • a single template from a DNA library is added to each well to induce small molecule production.
  • High throughput screening is carried out using a plate reader to detect color change.
  • the identity of the small molecule is determined, for example using LC-MS.
  • DNA sequencing is carried out to identify the template nucleic acid sequence for the underlying biosynthetic pathway.
  • Fig. 3 depicts a schematic of a screening process using a metagenomic DNA library.
  • Cell extracts are produced by optimized protocol (described in Examples I and II).
  • Cell-free extract is aliquoted into array, for example 348-well plate, glass slide or droplets.
  • DNA library is added as template for a transcription-translation system (TX-TL system) (for example metagenomic library carrying unknown or cryptic pathways), such that each reaction mix carries a single template.
  • TX-TL system transcription-translation system
  • DNA template initiates transcription and translation of desired pathway, followed by production of biomolecules.
  • the array is then screened for color compounds, for example by UV-vis or fluorimeter. Positive spots are analyzed to detect the product of interest by LC-MS, NMR, X-Ray crystallography and the like and to identify the underlying DNA pathway by NGS (or in situ) sequencing.
  • embodiments include a method of screening a plurality of template nucleic acid sequences for small molecule production including adding a template nucleic acid sequence from the plurality to each reaction volume within a plurality of reaction volumes, wherein each reaction volume includes a cell-free expression system under conditions of transcription and translation; detecting generation of a small molecule in a target reaction volume; and sequencing of the template nucleic acid sequence of the target reaction volume.
  • the cell-free expression system includes cell lysate from bacterial cells, yeast cells, plant cells, insect cells, or mammalian cells.
  • the cell-free expression system includes cell lysate from bacterial cells.
  • the cell-free expression system includes cell lysate from Streptomyces, Escherichia or Bacillus. According to one aspect, the cell-free expression system includes cell lysate from Streptomyces.
  • the template nucleic acid sequence is a DNA template or an RNA template. According to one aspect, the template nucleic acid sequence is a DNA template encoding one or more genes for making the small molecule. According to one aspect, the template nucleic acid sequence is a DNA template encoding a metabolic pathway for the small molecule. According to one aspect, the template nucleic acid sequence is a linear DNA template or circular DNA template. According to one aspect, each reaction volume includes a single template nucleic acid sequence from the plurality.
  • the method includes detecting generation of small molecules in a plurality of reaction volumes and sequencing of the template nucleic acid sequence of the plurality of reaction volumes.
  • detecting generation of the small molecule includes detecting color of the small molecule.
  • the template nucleic acid sequence is sequenced using in situ sequencing.
  • the template nucleic acid sequence is sequenced using fluorescent in situ sequencing.
  • the plurality of reaction volumes is a plurality of wells in a well plate.
  • the plurality of reaction volumes is a plurality of droplets.
  • the plurality of reaction volumes is a plurality of droplets within an emulsion.
  • each reaction volume includes one or more energy substrates, one or more cofactors, one or more salts, one or more amino acids and one or more nucleotides.
  • the plurality of template nucleic acid sequences is a library of template nucleic acid sequences.
  • the plurality of template nucleic acid sequences is a subset of a library of template nucleic acid sequences.
  • the plurality of template nucleic acid sequences is a subset of a library of biosynthetic pathways.
  • the plurality of template nucleic acid sequences is a subset of a library of fragments comprising a biosynthetic pathway.
  • the plurality of template nucleic acid sequences comprises natural genomic DNA. According to one aspect, the plurality of template nucleic acid sequences comprises natural biosynthetic pathways. According to one aspect, the plurality of template nucleic acid sequences comprises engineered biosynthetic pathways. According to one aspect, the plurality of template nucleic acid sequences is comprised of metagenomic sequences. According to one aspect, the plurality of template nucleic acid sequences is comprised of unknown metagenomic sequences.
  • FIG. 4A depicts the effect of culture growth media on cell-free extract protein yield.
  • V. natriegens cell-free systems derived from crude extract grown in each of these media diversifies the library of cell-free reactions to perform high-throughput screens.
  • Fig. 4B depicts the generation time and sfGFP yield for each media type tested. This indicates the general activity level in terms of cell-free protein expression for each of the media types.
  • Fig. 4D depicts results of a study of cell-free incubation temperature.
  • the temperature of cell-free incubation affects the expression activity of the cell-free system. This can vary depending on the application, for example, 26°C is exemplary for protein expression in V. natriegens cell-free reactions.
  • Fig. 4E depicts results of a study of supplemented potassium ions in reaction buffer.
  • concentration of supplemented potassium ions also affects the activity of the cell-free system. This can vary depending on the application, for example, 80 mM K+ is exemplary for protein expression in V. natriegens cell-free reactions.
  • Fig. 4F depicts results of a study of supplemented magnesium ions in reaction buffer.
  • concentration of supplemented magnesium ions also affects the activity of the cell-free system. This can vary depending on the application, for example, 3.5 mM Mg 2+ is exemplary for protein expression in V. nulriegem cell-free reactions.
  • Fig. 4G depicts percent of cell free extract relative to total reaction volume. Yield and rate of reaction are shown. The percent of cell free extract relative to the total reaction volume affects the activity of the cell-free system.
  • Fig. 4H depicts the template DNA used for expression of sfGFP.
  • Equimolar amount of circular plasmid pJLl, grey
  • linear DNA PCR product, black solid line
  • linear DNA with two phosphorothioated bonds on each end PCR product, black dotted line
  • the use of linear template over circular plasmid in cell-free reactions allows for template derived from PCR amplicons to be directly expressed in the cell-free reaction. This increases the accessibility testable sequences for high-throughput screening.
  • FIG. 5A and 5B depict sfGFP production measured at varying potassium and magnesium ion concentrations that was used to perform an initial calibration of T7 RNA Polymerase mediated protein synthesis by the cell-free expression system.
  • Figs. 5C and 5D depict natural product production at varying potassium and magnesium ion concentrations as measuring by UV/Vis analysis.
  • High absorbances at 340-390 nm indicate a natural product accumulating in Pseudomonas aeruginosa cell-free expression system. Please describe the importance of the data.
  • Fig. 11A and 11B indicate cell-free reaction activity by plotting kinetic expression of sfGFP.
  • Fig. 1 1C depicts generation time of Vibrio natriegens in several media types. Black arrows indicate detected peaks corresponding to redox-active compounds in cell-free reactions derived from cells grown in LB3 (Fig. 1 1D), Brain Heart Infusion + Ocean Salts (Fig. HE), LB + Ocean Salts (Fig. 1 IF), and Marine Broth (Fig. 1 1G). All electrochemical measurements were baseline corrected.
  • Fig. 12 depicts from left to right: optimized control reaction for V. natriegens at the preferred incubation temperature of 26C, optimized control reactions for E. coli strain A19 at the preferred incubation temperature of 37C, mixture of crude extracts incubated at V. natriegens preferred incubation temperature and optimal conditions, mixture of crude extracts at E. coli strain A19 preferred incubation temperature and V. natriegens optimal conditions, mixture of crude extracts at V. natriegens preferred incubation temperature and E.
  • Keasling JD Manufacturing molecules through metabolic engineering. Science. 2010;330: 1355-1358.

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L'invention concerne également un procédé de criblage à haut rendement de voies de biosynthèse à l'aide d'un système acellulaire.
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WO2023152518A1 (fr) * 2022-02-14 2023-08-17 Nuclera Ltd Procédés pour optimiser des réactifs de synthèse de protéines acellulaires

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CN114018964A (zh) * 2021-10-15 2022-02-08 华东师范大学 一种基于原位高场核磁共振技术检测四环素类有机污染物废水降解的方法
WO2023152518A1 (fr) * 2022-02-14 2023-08-17 Nuclera Ltd Procédés pour optimiser des réactifs de synthèse de protéines acellulaires
WO2023152519A3 (fr) * 2022-02-14 2023-09-28 Nuclera Ltd Réactifs d'expression de protéines pour modifications post-traductionnelles

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