基于液滴微流控技术的超高通量筛选体系及其在合成生物学中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2017.01.009

摘要/Abstract

摘要： 构建高效的合成生物学体系经常需要进行大量的筛选工作来优化人工体系的运行效率。液滴微流控（droplet microfluidics）技术将常规筛选体系微型化，在只有皮升（pL）级体积、大小均一的反应器中对酶反应或目标代谢产物进行单细胞水平的检测和筛选，筛选速度可高达108克隆/d，从而极大地提升了人们对催化元件乃至工程菌株优化的能力。综述了液滴微流控技术在合成生物学应用中的技术背景，重点阐述了目标酶反应及产物检测的荧光信号偶联策略，进一步介绍了液滴微流控技术在酶催化元件以及代谢通路的筛选改造方面的研究进展，并展望了该领域的发展趋势。

关键词: 液滴微流控, 合成生物学, 代谢途径, 酶, 高通量筛选

Abstract: The construction of a highly efficient synthetic biological system requires laborious screening work to optimize the performance of artificial system. Droplet microfluidic techniques miniaturize traditional screening systems,in a uniform pico-liter micro-reactors,the detection of enzymatic reactions and metabolites at single-cell level were measured and screened,thus it can screen genes/cells at a speed up to >108 events per day,which greatly improves the optimizing capability of catalytic parts and engineered strains. Herein,the technical backgrounds while applying droplet microfluidics in synthetic biology system are summarized,focusing on the fluorescence-coupling strategies in the detection of target enzyme reactions and metabolites. Moreover,recent progress on employing droplet microfluidics in the screening and engineering of catalytic parts and metabolic pathways is reviewed,the developing trends of this field are discussed.

Key words: droplet microfluidics, synthetic biology, metabolic pathway, enzyme, high-throughput screening

马富强，杨广宇. 基于液滴微流控技术的超高通量筛选体系及其在合成生物学中的应用[J]. 生物技术通报, 2017, 33(1): 83-92.

MA Fu-qiang, YANG Guang-yu. Ultra-high-throughput Screening System Based on Droplet Microfluidics and Its Applications in Synthetic Biology[J]. Biotechnology Bulletin, 2017, 33(1): 83-92.

参考文献

[1] Lartigue C, Glass JI, Alperovich N, et al. Genome transplantation in bacteria：Changing one species to another[J]. Science, 2007, 317（5838）：632-638.
[2] Annaluru N, Muller H, Mitchell LA, et al. Total synthesis of a functional designer eukaryotic chromosome[J]. Science, 2014, 344（6179）：55-58.
[3] Ro DK, Paradise EM, Ouellet M, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast[J]. Nature, 2006, 440（7086）：940-943.
[4] 陈大明, 刘晓, 毛开云, 等. 合成生物学应用产品开发现状与趋势[J]. 中国生物工程杂志, 2016, 7：117-126.
[5] Manyika J, Chui M, Bughin J, et al. Disruptive technologies：Advances that will transform life, business, and the global economy[M]. McKinsey Global Institute San Francisco, CA, 2013.
[6] Denard CA, Ren H, Zhao H. Improving and repurposing biocatalysts via directed evolution[J]. Current Opinion in Chemical Biology, 2015, 25：55-64.
[7] Currin A, Swainston N, Day PJ, et al. Synthetic biology for the directed evolution of protein biocatalysts：Navigating sequence space intelligently[J]. Chemical Society Reviews, 2014, 44（5）：1172-239.
[8] Tizei PAG, Csibra E, Torres L, et al. Selection platforms for directed evolution in synthetic biology[J]. Biochemical Society Transactions, 2016, 44（4）：1165-1175.
[9] Williams TC, Pretorius IS, Paulsen IT. Synthetic evolution of metabolic productivity using biosensors[J]. Trends in biotechnology, 2016, 34（5）：371-381.
[10] 林炳承, 秦建华. 图解微流控芯片实验室[M]. 北京：科学出版社, 2008.
[11] Kintses B, Vliet LDV, Devenish SR, et al. Microfluidic droplets：New integrated workflows for biological experiments[J]. Current Opinion in Chemical Biology, 2010, 14（5）：548-555.
[12] Wen N, Zhao Z, Fan B, et al. Development of droplet microfluidics enabling high-throughput single-cell analysis[J]. Molecules, 2016, 21（7）：E881.
[13] Kaminski TS, Scheler O, Garstecki P. Droplet microfluidics for microbiology：Techniques, applications and challenges[J]. Lab on A Chip, 2016, 16（12）：2168-2187.
[14] Chou WL, Lee PY, Yang CL, et al. Recent advances in applications of droplet microfluidics[J]. Micromachines, 2015, 6（9）：1249-1271.
[15] Mastrobattista E, Taly V, Chanudet E, et al. High-throughput screening of enzyme libraries：In vitro evolution of a β-galactosidase by fluorescence-activated sorting of double emulsions[J]. Chemistry & Biology, 2005, 12（12）：1291-1300.
[16] Hardiman E, Gibbs M, Reeves R, et al. Directed evolution of a thermophilic β-glucosidase for cellulosic bioethanol production[J]. Applied Biochemistry and Biotechnology, 2010, 161（1-8）：301-312.
[17] Ma F, Xie Y, Huang C, et al. An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization[J]. PLoS One, 2014, 9（2）：e89785.
[18] Tu R, Martinez R, Prodanovic R, et al. A flow cytometry-based screening system for directed evolution of proteases[J]. Journal of Biomolecular Screening, 2011, 16（3）：285-294.
[19] Agresti JJ, Antipov E, Abate AR, et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution[J]. Proceedings of the National Academy of Sciences, 2010, 107（9）：4004-4009.
[20] Aharoni A, Amitai G, Bernath K, et al. High-throughput screening of enzyme libraries：Thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments[J]. Chemistry & Biology, 2005, 12（12）：1281-1289.
[21] Varadarajan N, Gam J, Olsen MJ, et al. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102（19）：6855-6860.
[22] Varadarajan N, Rodriguez S, Hwang BY, et al. Highly active and selective endopeptidases with programmed substrate specificities[J]. Nature Chemical Biology, 2008, 4（5）：290-294.
[23] Varadarajan N, Georgiou G, Iverson BL, et al. An engineered protease that cleaves specifically after sulfated tyrosine[J]. Angewandte Chemie, 2007, 47（41）：7861-7863.
[24] Yang Y, Babiak P, Reymond JL. Low background fret-substrates for lipases and esterases suitable for high-throughput screening under basic（pH 11）conditions[J]. Organic & Biomolecular Chemistry, 2006, 4（9）：1746-1754.
[25] Mizukami S, Watanabe S, Hori Y, et al. Covalent protein labeling based on noncatalytic β-lactamase and a designed fret substrate[J]. Journal of the American Chemical Society, 2009, 131（14）：5016-5017.
[26] Yang GY, Li C, Fischer M, et al. A fret probe for cell-based imaging of ganglioside-processing enzyme activity and high-throughput screening[J]. Angewandte Chemie International Edition in English, 2015, 54（18）：5389-5893.
[27] Bjerregaard S, Pedersen H, Vedstesen H, et al. Parenteral water/oil emulsions containing hydrophilic compounds with enhanced in vivo retention：Formulation, rheological characterisation and study of in vivo fate using whole body gamma-scintigraphy[J]. International Journal of Pharmaceutics, 2001, 215（1）：13-27.
[28] Pays K, Giermanska-Kahn J, Pouligny B, et al. Double emulsions：How does release occur?[J]. Journal of Controlled Release, 2002, 79（1）：193-205.
[29] Cheng J, Chen JF, Zhao M, et al. Transport of ions through the oil phase of w 1/o/w 2 double emulsions[J]. Journal of Colloid and Interface Science, 2007, 305（1）：175-182.
[30] Wen L, Papadopoulos KD. Visualization of water transport in w 1/o/w 2 emulsions[J]. Colloids and surfaces A：Physicochemical and Engineering Aspects, 2000, 174（1）：159-167.
[31] Prodanovic R, Ostafe R, Blanusa M, et al. Vanadium bromoperoxi-dase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions[J]. Analytical and Bioanalytical Chemistry, 2012, 404（5）：1439-1447.
[32] Woronoff G, El Harrak A, Mayot E, et al. New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications[J]. Analytical Chemistry, 2011, 83（8）：2852-2857.
[33] Najah M, Mayot E, Mahendra-Wijaya IP, et al. New glycosidase substrates for droplet-based microfluidic screening[J]. Analytical Chemistry, 2013, 85（20）：9807-9814.
[34] Ma F, Fischer M, Han Y, et al. Substrate engineering enabling fluorescence droplet entrapment for ivc-facs based ultrahigh-throughput screening[J]. Analytical Chemistry, 2016, 88（17）：8587-8595.
[35] Hammar P, Angermayr SA, Sjostrom SL, et al. Single-cell screening of photosynthetic growth and lactate production by cyanobacteria[J]. Biotechnology for Biofuels, 2015, 8（1）：193-201.
[36] Wang BL, Ghaderi A, Zhou H, et al. Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption[J]. Nature Biotechnology, 2014, 32（5）：473-478.
[37] Abalde-Cela S, Gould A, Liu X, et al. High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform[J]. Journal of The Royal Society Interface, 2015, 12（106）：20150216.
[38] Ostafe R, Prodanovic R, Nazor J, et al. Ultra-high-throughput screening method for the directed evolution of glucose oxidase[J]. Chemistry & Biology, 2014, 21（3）：414-421.
[39] Ostafe R, Prodanovic R, Ung WL, et al. A high-throughput cellulase screening system based on droplet microfluidics[J]. Biomicrofluidics, 2014, 8（4）：041102.
[40] The lac operon：A short history of a genetic paradigm[M]. Walter de Gruyter, 1996.
[41] Gallegos MT, Schleif R, Bairoch A, et al. Arac/xyls family of transcriptional regulators[J]. Microbiology & Molecular Biology Reviews Mmbr, 1997, 61（4）：393-410.
[42] Ramos JL, Martínezbueno M, Molinahenares AJ, et al. The tetr family of transcriptional repressors[J]. Microbiology & Molecular Biology Reviews, 2005, 69（2）：326-356.
[43] Tropel D, Jr VDM. Bacterial transcriptional regulators for degradation pathways of aromatic compounds[J]. Microbiology & Molecular Biology Reviews, 2004, 68（3）：474-500.
[44] Link KH, Breaker RR. Engineering ligand-responsive gene-control elements：Lessons learned from natural riboswitches[J]. Gene Therapy, 2009, 16（10）：1189-1201.
[45] Sudarsan N, Wickiser JK, Nakamura S, et al. An mrna structure in bacteria that controls gene expression by binding lysine[J]. Genes & Development, 2003, 17（21）：2688-2697.
[46] Cheng F, Kardashliev T, Pitzler C, et al. A competitive flow cytometry screening system for directed evolution of therapeutic enzyme[J]. Acs SyntheticBiology, 2015, 4（7）：768-775.
[47] Siedler S, Schendzielorz G, Binder S, et al. Soxr as a single-cell biosensor for nadph-consuming enzymes in Escherichia coli[J]. Acs Synthetic Biology, 2014, 3（1）：41-47.
[48] Schallmey M, Frunzke J, Eggeling L, et al. Looking for the pick of the bunch：High-throughput screening of producing microorganisms with biosensors[J]. Current Opinion in Biotechnology, 2014, 26C（26C）：148-154.
[49] Binder S, Schendzielorz G, Stäbler N, et al. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level[J]. Genome Biology, 2012, 13（5）：405-413.
[50] Fang M, Wang T, Chong Z, et al. Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli[J]. Metabolic Engineering, 2015, 33：41-51.
[51] Pitzler C, Wirtz G, Vojcic L, et al. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes[J]. Chemistry & Biology, 2014, 21（12）：1733-1742.
[52] Larsen AC, Dunn MR, Hatch A, et al. A general strategy for expanding polymerase function by droplet microfluidics[J]. Nature Communications, 2016, 7：11235.
[53] Fischlechner M, Schaerli Y, Mohamed MF, et al. Evolution of enzyme catalysts caged in biomimetic gel-shell beads[J]. Nature Chemistry, 2014, 6（9）：791-796.
[54] Woronoff G, Ryckelynck M, Wessel J, et al. Activity-fed translation（aft）assay：A new high-throughput screening strategy for enzymes in droplets[J]. Chembiochem, 2015, 16（9）：1343-1349.
[55] 韩云宾, 黄琛, 冯雁. 催化元件和途径的人工设计与组装[J]. 生命科学, 2011, 23（9）：869-874.
[56] Kintses B, Hein C, Mohamed MF, et al. Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution[J]. Chemistry & Biology, 2012, 19（8）：1001-1009.
[57] Hosokawa M, Hoshino Y, Nishikawa Y, et al. Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes[J]. Biosensors and Bioelectronics, 2015, 67：379-385.
[58] Colin PY, Kintses B, Gielen F, et al. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics[J]. Nature Communications, 2015, 6：10008.
[59] Stapleton JA, Swartz JR. Development of an in vitro compartmentalization screen for high-throughput directed evolution of[FeFe]hydrogenases[J]. PLoS One, 2010, 5（12）：e15275.
[60] Ryckelynck M, Baudrey S, Rick C, et al. Using droplet-based microfluidics to improve the catalytic properties of rna under multiple-turnover conditions[J]. RNA, 2015, 21（3）：458-469.
[61] Obexer R, Pott M, Zeymer C, et al. Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting[J]. Protein Engineering Design and Selection, 2016, 29（9）：355-366.
[62] Sjostrom SL, Bai Y, Huang M, et al. High-throughput screening for industrial enzyme production hosts by droplet microfluidics[J]. Lab on a Chip, 2014, 14（4）：806-813.
[63] Huang M, Bai Y, Sjostrom SL, et al. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast[J]. Proceedings of the National Academy of Sciences, 2015, 112（34）：E4689-E4696.
[64] Jang S, Lee B, Jeong HH, et al. On-chip analysis, indexing and screening for chemical producing bacteria in microfluidic static droplet array[J]. Lab on a Chip, 2016, 16（10）：1909-1916.
[65] Smith CA, Li X, Mize TH, et al. Sensitive, high throughput detection of proteins in individual, surfactant-stabilized picoliter droplets using nanoelectrospray ionization mass spectrometry[J]. Analytical Chemistry, 2013, 85（8）：3812-3816.
[66] Sun S, Kennedy RT. Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors[J]. Analytical Chemistry, 2014, 86（18）：9309-9314.
[67] Meier TA, Beulig R, Klinge E, et al. On-chip monitoring of chemical syntheses in microdroplets via surface-enhanced raman spectroscopy[J]. Chemical Communications, 2015, 51（41）：8588-8591.
[68] Stehle R, Goerigk G, Wallacher D, et al. Small-angle x-ray scattering in droplet-based microfluidics[J]. Lab on a Chip, 2013, 13（8）：1529-1537.
[69] Cheow LF, Viswanathan R, Chin CS, et al. Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform[J]. Analytical Chemistry, 2014, 86（19）：9901-9908.
[70] Choi JW, Min KM, Hengoju S, et al. A droplet-based microfluidic immunosensor for high efficiency melamine analysis[J]. Biosensors & Bioelectronics, 2015, 80：182-186.
[71] Niu X, Pereira F, Edel JB, et al. Droplet-interfaced microchip and capillary electrophoretic separations[J]. Analytical Chemistry, 2013, 85（18）：8654-8660.