基于遗传密码子拓展技术搭建生物催化剂 定向进化高效筛选平台

生物催化剂的定向进化技术能有效改善酶的催化活性、反应选择性、稳定性、底物识别范围等性能，促进了生物催化剂在有机合成领域的广泛应用。使用液相色谱、质谱或酶标仪等基于筛选的酶活测试方法需要逐一测试突变体的酶活，筛选通量有限。而基于选择的方法能够将酶的活性与筛选宿主的生存进行关联，使得通量相比基于筛选的方法有着数量级的提升。然而，将生物催化剂的活性与宿主的“生死”进行关联这一策略仍存在着较大的挑战，目前已知能够实现这类关联的酶催化反应很少，普适性也较差。
本文提出了一种利用遗传密码子拓展技术将酶活与宿主细胞生死联系起来的策略。以非天然氨基酸为纽带，一方面将非天然氨基酸设计作为酶促反应的产物，其产量与酶活正相关；另一方面，插入非天然氨基酸作为控制宿主关键基因（抗性基因）表达的元件，其生产与宿主的生存直接关联。该策略在实际运用中可简单地通过抗生素的添加来挑选所需的酶，人为地构建了生物催化剂的酶活与宿主细胞的生存表型间的联系。本文选择酶促反 应产物 为 L-色氨酸类似物 的色氨酸合成 酶 （ tryptophan synthase，TrpS）的 β 亚基 ( TrpB )作为实例，主要研究成果如下：（1）证明了 TrpB催化反应产物（L-色氨酸衍生物）能够通过遗传密码子拓展技术与细胞荧光及存活表型呈现正相关关系，达到理想的从突变体文库中消除无义变体、富集得到具有更高活性的突变体的目的。（2）应用本研究所搭建的基于宿主细胞生死的高效选择平台，最快能够在两轮的选择实验中将有义突变体在文库中的分布频率富集 100 倍。（3）最终，作者应用该平台对 TrpB 突变体文库进行了挑选，得到了 1 个催化效率高于野生型酶的突变体，使之催化反应产率提高了 15%。综上所述，本研究所提出的策略从一种新角度构建了基于生死的选择平台，为酶的定向进化研究提供了一种强有力的工具，理论上适用于所有以非天然氨基酸为酶促反应产物的酶。

[1] TAMAKI F K. Directed evolution of enzymes [J]. Emerging Topics in Life Sciences,2020, 4(2): 119-27.
[2] WANG Y, XUE P, CAO M, et al. Directed Evolution: Methodologies and Applications[J]. Chem Rev, 2021, 121(20): 12384-444.
[3] KACIAN D L, MILLS D R, KRAMER F R, et al. A Replicating RNA Molecule Suitablefor a Detailed Analysis of Extracellular Evolution and Replication [J]. Proceedings ofthe National Academy of Sciences, 1972, 69(10): 3038-42.
[4] LEVISOHN R, SPIEGELMAN S. Further extracellular Darwinian experiments withreplicating RNA molecules: diverse variants isolated under different selectiveconditions [J]. Proceedings of the National Academy of Sciences, 1969, 63(3): 805-11.
[5] MILLS D R, PETERSON R L, SPIEGELMAN S. An extracellular Darwinianexperiment with a self-duplicating nucleic acid molecule [J]. Proceedings of theNational Academy of Sciences, 1967, 58(1): 217-24.
[6] SMITH G P. Filamentous Fusion Phage: Novel Expression Vectors That DisplayCloned Antigens on the Virion Surface [J]. Science, 1985, 228(4705): 1315-7.
[7] EIGEN M, GARDINER W. Evolutionary molecular engineering based on RNAreplication [J]. Pure and Applied Chemistry, 1984, 56(8): 967-78.
[8] CHEN K, ARNOLD F H. Tuning the activity of an enzyme for unusual environments:sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide [J].Proceedings of the National Academy of Sciences, 1993, 90(12): 5618-22.
[9] ELLINGTON A D, SZOSTAK J W. In vitro selection of RNA molecules that bindspecific ligands [J]. Nature, 1990, 346(6287): 818-22.
[10] LIAO H, MCKENZIE T, HAGEMAN R. Isolation of a thermostable enzyme variant bycloning and selection in a thermophile [J]. Proceedings of the National Academy ofSciences, 1986, 83(3): 576-80.
[11] HALL B G. Experimental evolution of a new enzymatic function. II. Evolution ofmultiple functions for ebg enzyme in E. coli [J]. Genetics, 1978, 89(3): 453-65.
[12] CARLSON J C, BADRAN A H, GUGGIANA-NILO D A, et al. Negative selection andstringency modulation in phage-assisted continuous evolution [J]. Nature ChemicalBiology, 2014, 10(3): 216-22.
[13] RAVIKUMAR A, ARRIETA A, LIU C C. An orthogonal DNA replication system inyeast [J]. Nature Chemical Biology, 2014, 10(3): 175-7.
[14] TIAN R, ZHAO R, GUO H, et al. Engineered bacterial orthogonal DNA replicationsystem for continuous evolution [J]. Nature Chemical Biology, 2023, 19(12): 1504-12.
[15] ARNOLD F H. Innovation by Evolution: Bringing New Chemistry to Life (NobelLecture) [J]. Angewandte Chemie International Edition, 2019, 58(41): 14420-6.
[16] PACKER M S, LIU D R. Methods for the directed evolution of proteins [J]. NatureReviews Genetics, 2015, 16(7): 379-94.
[17] FUSHIMI T, MIURA N, SHINTANI H, et al. Mutant firefly luciferases with improvedspecific activity and dATP discrimination constructed by yeast cell surface engineering[J]. Applied Microbiology and Biotechnology, 2013, 97(9): 4003-11.
[18] ZHANG K, NELSON KATHRYN M, BHURIPANYO K, et al. Engineering theSubstrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display[J]. Chemistry & Biology, 2013, 20(1): 92-101.
[19] WHITE K A, ZEGELBONE P M. Directed Evolution of a Probe Ligase with Activityin the Secretory Pathway and Application to Imaging Intercellular Protein–ProteinInteractions [J]. Biochemistry, 2013, 52(21): 3728-39.
[20] YI L, GEBHARD M C, LI Q, et al. Engineering of TEV protease variants by yeast ERsequestration screening (YESS) of combinatorial libraries [J]. Proceedings of theNational Academy of Sciences, 2013, 110(18): 7229-34.
[21] CHERF G M, COCHRAN J R. Applications of Yeast Surface Display for ProteinEngineering [M]//LIU B. Yeast Surface Display: Methods, Protocols, and Applications.New York, NY; Springer New York. 2015: 155-75.
[22] MILLER B G, RAINES R T. Identifying Latent Enzyme Activities:  SubstrateAmbiguity within Modern Bacterial Sugar Kinases [J]. Biochemistry, 2004, 43(21):6387-92.
[23] BOERSMA Y L, DRÖGE M J, VAN DER SLOOT A M, et al. A Novel Genetic SelectionSystem for Improved Enantioselectivity of Bacillus subtilis Lipase A [J].ChemBioChem, 2008, 9(7): 1110-5.
[24] TARAN F. Enzyme Assays: High-Throughput Screening, Genetic Selection andFingerprinting. Edited by Jean-Louis Reymond [J]. ChemBioChem, 2006, 7(9): 1457-.
[25] WU S, XIANG C, ZHOU Y, et al. A growth selection system for the directed evolutionof amine-forming or converting enzymes [J]. Nature Communications, 2022, 13(1):7458.
[26] VAN SINT FIET S, VAN BEILEN J B, WITHOLT B. Selection of biocatalysts forchemical synthesis [J]. Proceedings of the National Academy of Sciences, 2006, 103(6):1693-8.
[27] MICHENER J K, SMOLKE C D. High-throughput enzyme evolution in Saccharomycescerevisiae using a synthetic RNA switch [J]. Metabolic Engineering, 2012, 14(4): 306-16.
[28] TANG S-Y, CIRINO P C. Design and Application of a Mevalonate-ResponsiveRegulatory Protein [J]. Angewandte Chemie International Edition, 2011, 50(5): 1084-6.
[29] LIN H, TAO H, CORNISH V W. Directed Evolution of a Glycosynthase via ChemicalComplementation [J]. Journal of the American Chemical Society, 2004, 126(46):15051-9.
[30] XIE J, SCHULTZ P G. A chemical toolkit for proteins — an expanded genetic code [J].Nature Reviews Molecular Cell Biology, 2006, 7(10): 775-82.
[31] XIE J, SCHULTZ P G. An expanding genetic code [J]. Methods, 2005, 36(3): 227-38.
[32] BOYLE J. Molecular biology of the cell, 5th edition by B. Alberts, A. Johnson, J.Lewis, M. Raff, K. Roberts, and P. Walter [J]. Biochemistry and Molecular BiologyEducation, 2008, 36(4): 317-8.
[33] ALFF-STEINBERGER C, EPSTEIN R. Codon Preference in the Terminal Region ofE. coli Genes and Evolution of Stop Codon Usage [J]. Journal of Theoretical Biology,1994, 168(4): 461-3.
[34] NOREN C J, ANTHONY-CAHILL S J, GRIFFITH M C, et al. A General Method forSite-specific Incorporation of Unnatural Amino Acids into Proteins [J]. Science, 1989,244(4901): 182-8.
[35] DRIENOVSKÁ I, ROELFES G. Expanding the enzyme universe with geneticallyencoded unnatural amino acids [J]. Nature Catalysis, 2020, 3(3): 193-202.
[36] WANG L, MAGLIERY T J, LIU D R, et al. A New Functional SuppressortRNA/Aminoacyl−tRNA Synthetase Pair for the in Vivo Incorporation of UnnaturalAmino Acids into Proteins [J]. Journal of the American Chemical Society, 2000,122(20): 5010-1.
[37] WANG L, BROCK A, HERBERICH B, et al. Expanding the Genetic Code ofEscherichia coli [J]. Science, 2001, 292(5516): 498-500.
[38] PASTRNAK M, SCHULTZ P G. Phage selection for site-specific incorporation ofunnatural amino acids into proteins in vivo [J]. Bioorganic & Medicinal Chemistry,2001, 9(9): 2373-9.
[39] DAVIS L, CHIN J W. Designer proteins: applications of genetic code expansion in cellbiology [J]. Nature Reviews Molecular Cell Biology, 2012, 13(3): 168-82.
[40] DUMAS A, LERCHER L, SPICER C D, et al. Designing logical codon reassignment– Expanding the chemistry in biology [J]. Chemical Science, 2015, 6(1): 50-69.
[41] NEUMANN H, WANG K, DAVIS L, et al. Encoding multiple unnatural amino acidsvia evolution of a quadruplet-decoding ribosome [J]. Nature, 2010, 464(7287): 441-4.
[42] ZÜRCHER J F, ROBERTSON W E, KAPPES T, et al. Refactored genetic codes enablebidirectional genetic isolation [J]. Science, 2022, 378(6619): 516-23.
[43] HANCOCK S M, UPRETY R, DEITERS A, et al. Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNASynthetase/tRNA Pair [J]. Journal of the American Chemical Society, 2010, 132(42):14819-24.
[44] MUKAI T, KOBAYASHI T, HINO N, et al. Adding l-lysine derivatives to the geneticcode of mammalian cells with engineered pyrrolysyl-tRNA synthetases [J].Biochemical and Biophysical Research Communications, 2008, 371(4): 818-22.
[45] XI Z, DAVIS L, BAXTER K, et al. Using a quadruplet codon to expand the geneticcode of an animal [J]. Nucleic Acids Research, 2021, 50(9): 4801-12.
[46] BRAIG D, BÄR C, THUMFART J-O, et al. Two Cooperating Helices Constitute theLipid-binding Domain of the Bacterial SRP Receptor [J]. Journal of Molecular Biology,2009, 390(3): 401-13.
[47] OKUDA S, TOKUDA H. Model of mouth-to-mouth transfer of bacterial lipoproteinsthrough inner membrane LolC, periplasmic LolA, and outer membrane LolB [J].Proceedings of the National Academy of Sciences, 2009, 106(14): 5877-82.
[48] VIRDEE S, KAPADNIS P B, ELLIOTT T, et al. Traceless and Site-SpecificUbiquitination of Recombinant Proteins [J]. Journal of the American Chemical Society,2011, 133(28): 10708-11.
[49] ZHANG X, HUANG H, LIU Y, et al. Optical Control of Protein Functions viaGenetically Encoded Photocaged Aspartic Acids [J]. Journal of the American ChemicalSociety, 2023, 145(35): 19218-24.
[50] KOH M, YAO A, GLEASON P R, et al. A General Strategy for EngineeringNoncanonical Amino Acid Dependent Bacterial Growth [J]. Journal of the AmericanChemical Society, 2019, 141(41): 16213-6.
[51] CHANG T, DING W, YAN S, et al. A robust yeast biocontainment system with twolayered regulation switch dependent on unnatural amino acid [J]. NatureCommunications, 2023, 14(1): 6487.
[52] TACK D S, ELLEFSON J W, THYER R, et al. Addicting diverse bacteria to anoncanonical amino acid [J]. Nature Chemical Biology, 2016, 12(3): 138-40.
[53] RUBINI R, MAYER C. Addicting Escherichia coli to New-to-Nature Reactions [J].ACS Chemical Biology, 2020, 15(12): 3093-8.
[54] RUBINI R, IVANOV I, MAYER C. A Screening Platform to Identify and TailorBiocompatible Small-Molecule Catalysts [J]. Chemistry – A European Journal, 2019,25(70): 16017-21.
[55] RUBINI R, JANSEN S C, BEEKHUIS H, et al. Selecting Better Biocatalysts byComplementing Recoded Bacteria** [J]. Angewandte Chemie International Edition,2023, 62(2): e202213942.
[56] KOH M, NASERTORABI F, HAN G W, et al. Generation of an Orthogonal Protein–Protein Interface with a Noncanonical Amino Acid [J]. Journal of the AmericanChemical Society, 2017, 139(16): 5728-31.
[57] MANDELL D J, LAJOIE M J, MEE M T, et al. Biocontainment of genetically modifiedorganisms by synthetic protein design [J]. Nature, 2015, 518(7537): 55-60.
[58] XUAN W, SCHULTZ P G. A Strategy for Creating Organisms Dependent onNoncanonical Amino Acids [J]. Angewandte Chemie International Edition, 2017,56(31): 9170-3.
[59] GAN F, LIU R, WANG F, et al. Functional Replacement of Histidine in Proteins ToGenerate Noncanonical Amino Acid Dependent Organisms [J]. Journal of the AmericanChemical Society, 2018, 140(11): 3829-32.
[60] WATKINS-DULANEY E, STRAATHOF S, ARNOLD F. Tryptophan Synthase:Biocatalyst Extraordinaire [J]. ChemBioChem, 2021, 22(1): 5-16.
[61] RIX G, WATKINS-DULANEY E J, ALMHJELL P J, et al. Scalable continuousevolution for the generation of diverse enzyme variants encompassing promiscuousactivities [J]. Nature Communications, 2020, 11(1): 5644.
[62] DING W, ZHAO H, CHEN Y, et al. Chimeric design of pyrrolysyl-tRNAsynthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion[J]. Nature Communications, 2020, 11(1): 3154.
[63] ZHAO H, DING W, ZANG J, et al. Directed-evolution of translation system forefficient unnatural amino acids incorporation and generalizable synthetic auxotrophconstruction [J]. Nature Communications, 2021, 12(1): 7039.
[64] PEDELACQ J-D, CABANTOUS S. Development and Applications of Superfolder andSplit Fluorescent Protein Detection Systems in Biology [J]. International Journal ofMolecular Sciences, 2019, 20(14): 3479.
[65] HIRAKAWA H, INAZUMI Y, MASAKI T, et al. Indole induces the expression ofmultidrug exporter genes in Escherichia coli [J]. Molecular Microbiology, 2005, 55(4):1113-26.
[66] WANG Y, TIAN T, ZHANG J, et al. Indole Reverses Intrinsic Antibiotic Resistance byActivating a Novel Dual-Function Importer [J]. mBio, 2019, 10(3):10.1128/mbio.00676-19.
[67] CHIMEREL C, FIELD C M, PIÑERO-FERNANDEZ S, et al. Indole preventsEscherichia coli cell division by modulating membrane potential [J]. Biochimica etBiophysica Acta (BBA) - Biomembranes, 2012, 1818(7): 1590-4.
[68] LEE J-H, CHO M H, LEE J. 3-Indolylacetonitrile Decreases Escherichia coli O157:H7Biofilm Formation and Pseudomonas aeruginosa Virulence [J]. EnvironmentalMicrobiology, 2011, 13(1): 62-73.
[69] BUNDERS C A, MINVIELLE M J, WORTHINGTON R J, et al. Intercepting BacterialIndole Signaling with Flustramine Derivatives [J]. Journal of the American ChemicalSociety, 2011, 133(50): 20160-3.
[70] PEREZ J G, CARLSON E D, WEISSER O, et al. Improving genomically recodedEscherichia coli to produce proteins containing non-canonical amino acids [J].Biotechnology Journal, 2022, 17(4): 2100330.
[71] ROMNEY D K, MURCIANO-CALLES J, WEHRMÜLLER J E, et al. UnlockingReactivity of TrpB: A General Biocatalytic Platform for Synthesis of TryptophanAnalogues [J]. Journal of the American Chemical Society, 2017, 139(31): 10769-76.
[72] FUJITA H, DOU J, MATSUMOTO N, et al. Enzymatically triggered chromogeniccross-linking agents under physiological conditions [J]. New Journal of Chemistry,2020, 44(3): 719-43.
[73] REBELO S L H, LINHARES M, SIMÕES M M Q, et al. Indigo dye production byenzymatic mimicking based on an iron(III)porphyrin [J]. Journal of Catalysis, 2014,315: 33-40.
[74] LIU L, LIU Y, ZHANG G, et al. Genetically Encoded Chemical Decaging in Living Bacteria [J]. Biochemistry, 2018, 57(4): 446-50.
[75] DENNIS LO Y M. The Amplification Refractory Mutation System [M]//LO Y M D. Clinical Applications of PCR. Totowa, NJ; Humana Press. 1998: 61-9.
[76] BULLER A R, BRINKMANN-CHEN S, ROMNEY D K, et al. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation [J]. Proceedings of the National Academy of Sciences, 2015, 112(47):14599-604.
[77] BAO Z, SUN S, LI J, et al. Direct Identification of Tryptophan in a Mixture of AminoAcids by the Naked Eye [J]. Angewandte Chemie International Edition, 2006, 45(40):6723-5.
[78] ZHANG H, WANG Y, LI J, et al. Biosynthetic energy cost for amino acids decreases in cancer evolution [J]. Nature Communications, 2018, 9(1): 4124