基于荧光检测的高通量筛选技术和装备助力细胞工厂构建

孙梦楚; 陆亮宇; 申晓林; 孙新晓; 王佳; 袁其朋

doi:10.12211/2096-8280.2023-017

您当前的位置：

首页 >

文章列表页 >

基于荧光检测的高通量筛选技术和装备助力细胞工厂构建

特约评述 | 更新时间：2025-03-20

- 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建
- Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction
- 合成生物学 2023年4卷第5期页码：947-965
- 作者机构：
  
  北京化工大学化工资源有效利用国家重点实验室，北京软物质科学与工程高精尖创新中心，北京 100029
- 作者简介：
  
  [ "孙梦楚（1992—），女，博士研究生。研究方向为代谢工程及合成生物学。E-mail：xcs624816@163.com" ]
  [ "王佳（1989—），女，博士，副教授。研究方向为代谢工程及合成生物学。E-mail：wangjia@mail.buct.edu.cn" ]
  [ "袁其朋（1969—），男，博士，教授。研究方向为生物化工，代谢工程及微生物合成生物学。E-mail：yuanqp@mail.buct.edu.cn" ]
- 基金信息：
  
  国家自然科学基金(21908003);国家重点研发计划(2018YFA0903000)
- DOI：10.12211/2096-8280.2023-017
  中图分类号： Q812
- 收稿：2023-02-28，
  
  修回：2023-04-17，
  
  纸质出版：2023-10-31
- 稿件说明：
移动端阅览
孙梦楚，陆亮宇，申晓林，孙新晓，王佳，袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建［J］. 合成生物学， 2023， 4（5）： 947-965

SUN Mengchu， LU Liangyu， SHEN Xiaolin， SUN Xinxiao， WANG Jia， YUAN Qipeng. Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction［J］. Synthetic Biology Journal， 2023， 4（5）： 947-965
孙梦楚，陆亮宇，申晓林，孙新晓，王佳，袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建［J］. 合成生物学， 2023， 4（5）： 947-965 DOI： 10.12211/2096-8280.2023-017.

SUN Mengchu， LU Liangyu， SHEN Xiaolin， SUN Xinxiao， WANG Jia， YUAN Qipeng. Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction［J］. Synthetic Biology Journal， 2023， 4（5）： 947-965 DOI： 10.12211/2096-8280.2023-017.

摘要

微生物工业制造是以微生物细胞工厂为核心，利用低成本、可再生资源为原料，实现高附加值化合物的绿色生产。依赖于“设计-构建-测试-学习”（DBTL）循环的微生物细胞工厂开发过程中“测试”阶段已成为制约合成生物学和代谢工程发展的瓶颈之一。基于微量滴定板（MTP）高通量自动化筛选平台极大降低了高通量筛选过程的劳动强度，流式细胞术和液滴微流控技术的发展大幅度提高了筛选通量。尤其是荧光激活液滴分选（FADS）高通量筛选技术的开发为自动化、高通量和低消耗筛选工作提供了新的解决方案。本文综述了不同高通量筛选技术在合成生物学和代谢工程领域应用的主要进展，重点介绍了近几年荧光激活细胞分选技术（FACS）和FADS在微生物细胞工厂和酶定向进化方面的应用实例，关注了待测分子与荧光信号偶联的常用策略，并简单介绍目前国内外基于液滴微流控技术高通量筛选装备的研发情况。

Abstract

Microbial industrial manufacturing focuses on c

onstruction of microbial cell factories using low-cost

renewable resources as materials to achieve sustainable production of value-added compounds. The "test" stage in the development of microbial factories that relies on the "Design-Built-Test-Learn" cycle has quickly become one of the bottlenecks restricting the development of synthetic biology and metabolic engineering. To accelerate DBTL cycling

high-throughput screening techniques need to match the size of the library during the testing phase. Microtiter plates (MTP)

as a traditional screening method

uses the optical changes of metabolites in microliters of culture medium for detection and analysis

which can meet the repeated detection and accurate determination of mutants in the library

and also have the ability to screen high-yield strains of extracellular metabolites. However

this screening method is time-consuming and has low throughput. The automated platform is a good solution to the limitations of low screening throughput of microplates. However

the high cost of automation equipment and equipment maintenance makes this method not universal. At present

the main method of high-throughput screening is fluorescence-activated cell sorting (FACS)

which can reach a screening throughput of about 100 000 cells per second. However

FACS is limited to detecting intracellular fluorescence signals associated with target products or metabolite fluorescence signals bound to membranes. This problem is well solved by droplet microfluidic technology

which embeds and cultures single cells in monodisperse and picoliter droplets; each droplet acts as a separate microreactor to achieve genotype and phenotype coupling. In the process of screening of huge mutant libraries by droplet microfluidic technology

the screening throughput can reach 10

per day

which effectively improves the work efficiency

and also shows great advantages in experimental cost

realizing the development of microbial cell factories with high screening throughput and low cost an

d the screening of highly active enzyme variants. In conclusion

based on the high-throughput automated screening platform using microtiter plates

the human labor investment of the high-throughput screening process is greatly reduced

and the development of FACS and droplet microfluidic technology further improves the throughput. In particular

the development of fluorescence-activated droplet sorting (FADS) high-throughput screening technology opens up the possibility for automated

high-throughput

and low-consumption screening. This paper reviews the main progress of the application of different high-throughput screening techniques in the field of synthetic biology. Emphasis will be put on the application of fluorescence-activated cell sorting and FADS in microbial cell factories and enzyme directed evolution in recent years

especially the common strategies of coupling the molecules to be tested with fluorescence signals. We also briefly introduce the current research and development of high-throughput screening equipment based on droplet microfluidic technology.

关键词

Keywords

references

LEGGIERI P A , LIU Y Y , HAYES M , et al . Integrating systems and synthetic biology to understand and engineer microbiomes [J ] . Annual Review of Biomedical Engineering , 2021 , 23 : 169 - 201 .

NAKAMURA C E , WHITED G M . Metabolic engineering for the microbial production of 1,3-propanediol [J ] . Current Opinion in Biotechnology , 2003 , 14 ( 5 ): 454 - 459 .

LUO X Z , REITER M A , D'ESPAUX L , et al . Complete biosynthesis of cannabinoids and their unnatural analogues in yeast [J ] . Nature , 2019 , 567 ( 7746 ): 123 - 126 .

LIU Y Z , CRUZ-MORALES P , ZARGAR A , et al . Biofuels for a sustainable future [J ] . Cell , 2021 , 184 ( 6 ): 1636 - 1647 .

ZARGAR A , BAILEY C B , HAUSHALTER R W , et al . Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels [J ] . Current Opinion in Biotechnology , 2017 , 45 : 156 - 163 .

ZHANG J , HANSEN L G , GUDICH O , et al . A microbial supply chain for production of the anti-cancer drug vinblastine [J ] . Nature , 2022 , 609 ( 7926 ): 341 - 347 .

LAWSON C E , HARCOMBE W R , HATZENPICHLER R , et al . Common principles and best practices for engineering microbiomes [J ] . Nature Reviews Microbiology , 2019 , 17 ( 12 ): 725 - 741 .

LOK C . Mining the microbial dark matter [J ] . Nature , 2015 , 522 ( 7556 ): 270 - 273 .

CHOI K R , JANG W D , YANG D S , et al . Systems metabolic engineering strategies: integrating systems and synthetic biology with metabolic engineering [J ] . Trends in Biotechnology , 2019 , 37 ( 8 ): 817 - 837 .

SALESKI T E , KERNER A R , CHUNG M T , et al . Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries [J ] . Metabolic Engineering , 2019 , 54 : 232 - 243 .

MICHAEL S , AULD D , KLUMPP C , et al . A robotic platform for quantitative high-throughput screening [J ] . ASSAY and Drug Development Technologies , 2008 , 6 ( 5 ): 637 - 657 .

LONG Q , LIU X X , YANG Y K , et al . The development and application of high throughput cultivation technology in bioprocess development [J ] . Journal of Biotechnology , 2014 , 192 Pt B: 323- 338 .

MAYR L M , FUERST P . The future of high-throughput screening [J ] . Journal of Biomolecular Screening , 2008 , 13 ( 6 ): 443 - 448 .

MAYR L M , BOJANIC D . Novel trends in high-throughput screening [J ] . Current Opinion in Pharmacology , 2009 , 9 ( 5 ): 580 - 588 .

WANG B L , 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 .

WAGNER J M , LIU L Q , YUAN S F , et al . A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica [J ] . Metabolic Engineering , 2018 , 47 : 346 - 356 .

JOENSSON H N , ANDERSSON SVAHN H . Droplet microfluidics—a tool for single-cell analysis [J ] . Angewandte Chemie International Edition , 2012 , 51 ( 49 ): 12176 - 12192 .

STUCKI A , VALLAPURACKAL J , WARD T R , et al . Droplet microfluidics and directed evolution of enzymes: an intertwined journey [J ] . Angewandte Chemie International Edition , 2021 , 60 ( 46 ): 24368 - 24387 .

ZENG W Z , GUO L K , XU S , et al . High-throughput screening technology in industrial biotechnology [J ] . Trends in Biotechnology , 2020 , 38 ( 8 ): 888 - 906 .

SUN G Y , QU L S , AZI F , et al . Recent progress in high-throughput droplet screening and sorting for bioanalysis [J ] . Biosensors and Bioelectronics , 2023 , 225 : 115107 .

CIRINO P C , QIAN S . Chapter 2 - Protein engineering as an enabling tool for synthetic biology [M/OL ] // Synthetic biology . Boston : Academic Press , 2013 : 23 - 42 [2023-02-01] . https://www.sciencedirect.com/science/article/abs/pii/B97801239443 06000029?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/B9780123944306000029?via%3Dihub .

ZENG W Z , DU G C , CHEN J , et al . A high-throughput screening procedure for enhancing α-ketoglutaric acid production in Yarrowia lipolytica by random mutagenesis [J ] . Process Biochemistry , 2015 , 50 ( 10 ): 1516 - 1522 .

SUN L , ZHANG H , YUAN H , et al . A double-enzyme-coupled assay for high-throughput screening of succinic acid-producing strains [J ] . Journal of Applied Microbiology , 2013 , 114 ( 6 ): 1696 - 1701 .

ZHANG P P , HU S , MEI L H , et al . Improving the activity of cytochrome P 450 BM- 3 catalyzing indole hydroxylation by directed evolution [J ] . Applied Biochemistry and Biotechnology , 2013 , 171 ( 1 ): 93 - 103 .

TU R , LV T , SUN L , et al . Development of a simple colorimetric assay for determination of the isoamyl alcohol-producing strain [J ] . Applied Biochemistry and Biotechnology , 2020 , 192 ( 2 ): 632 - 642 .

ADAN A , ALIZADA G , KIRAZ Y , et al . Flow cytometry: basic principles and applications [J ] . Critical Reviews in Biotechnology , 2017 , 37 ( 2 ): 163 - 176 .

MCKINNON K M . Flow cytometry: an overview [J ] . Current Protocols in Immunology , 2018 , 120 : 5 .1.-5. 1 . 11 .

RADCLIFF G , JAROSZESKI M J . Basics of flow cytometry [M/OL ] // Flow Cytometry Protocols- Methods in molecular biology . New Jersey: Humana Press, 2003 : 1 - 24 [2023-02-01] . https://link.springer.com/protocol/10.1385/0-89603-354-6:1 https://link.springer.com/protocol/10.1385/0-89603-354-6:1 .

BARET J C , MILLER O J , TALY V , et al . Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity [J ] . Lab on a Chip , 2009 , 9 ( 13 ): 1850 - 1858 .

MARKEL U , ESSANI K D , BESIRLIOGLU V , et al . Advances in ultrahigh-throughput screening for directed enzyme evolution [J ] . Chemical Society Reviews , 2020 , 49 ( 1 ): 233 - 262 .

GAO J S , DU M H , ZHAO J H , et al . Design of a genetically encoded biosensor to establish a high-throughput screening platform for L-cysteine overproduction [J ] . Metabolic Engineering , 2022 , 73 : 144 - 157 .

SAVITSKAYA J , PROTZKO R J , LI F Z , et al . Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production [J ] . Scientific Reports , 2019 , 9 : 5815 .

TEREKHOV S S , SMIRNOV I V , MALAKHOVA M V , et al . Ultrahigh-throughput functional profiling of microbiota communities [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2018 , 115 ( 38 ): 9551 - 9556 .

LIU M M , ZHANG J , LIU X Q , et al . Rapid gene target tracking for enhancing β-carotene production using flow cytometry-based high-throughput screening in Yarrowia lipolytica [J ] . Applied and Environmental Microbiology , 2022 , 88 ( 19 ): e0114922 .

WANG G K , JIA W D , CHEN N , et al . A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi [J ] . Biotechnology for Biofuels , 2018 , 11 : 232 .

ZHU X D , SHI X , WANG S W , et al . High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting [J ] . RSC Advances , 2019 , 9 ( 8 ): 4507 - 4513 .

ZHOU S H , YUAN S F , NAIR P H , et al . Development of a growth coupled and multi-layered dynamic regulation network balancing malonyl-CoA node to enhance (2 S )-naringenin biosynthesis in Escherichia coli [J ] . Metabolic Engineering , 2021 , 67 : 41 - 52 .

MA C X , TAN Z L , LIN Y , et al . Gel microdroplet-based high-throughput screening for directed evolution of xylanase-producing Pichia pastoris [J ] . Journal of Bioscience and Bioengineering , 2019 , 128 ( 6 ): 662 - 668 .

GUO L K , ZENG W Z , XU S , et al . Fluorescence-activated droplet sorting for enhanced pyruvic acid accumulation by Candida glabrata [J ] . Bioresource Technology , 2020 , 318 : 124258 .

KORTMANN M , MACK C , BAUMGART M , et al . Pyruvate carboxylase variants enabling improved lysine production from glucose identified by biosensor-based high-throughput fluorescence-activated cell sorting screening [J ] . ACS Synthetic Biology , 2019 , 8 ( 2 ): 274 - 281 .

LIN A E , LIN Q . Rapid identification of functional pyrrolysyl-tRNA synthetases via fluorescence-activated cell sorting [J ] . International Journal of Molecular Sciences , 2018 , 20 ( 1 ): 29 .

TAN Y M , ZHANG Y , HAN Y B , et al . Directed evolution of an α1, 3-fucosyltransferase using a single-cell ultrahigh-throughput screening method [J ] . Science Advances , 2019 , 5 ( 10 ): eaaw8451 .

SADLER J C , CURRIN A , KELL D B . Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting [J ] . Analyst , 2018 , 143 ( 19 ): 4747 - 4755 .

MA F Q , GUO T J , ZHANG Y F , et al . An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries [J ] . Environmental Microbiology , 2021 , 23 ( 2 ): 996 - 1008 .

LIU H , HOU Y H , WANG Y , et al . Enhancement of sulfur conversion rate in the production of l-cysteine by engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2020 , 68 ( 1 ): 250 - 257 .

WENDISCH V F . Metabolic engineering advances and prospects for amino acid production [J ] . Metabolic Engineering , 2020 , 58 : 17 - 34 .

WOOLSTON B M , ROTH T , KOHALE I , et al . Development of a formaldehyde biosensor with application to synthetic methylotrophy [J ] . Biotechnology and Bioengineering , 2018 , 115 ( 1 ): 206 - 215 .

TU R , ZHANG Y , HUA E B , et al . Droplet-based microfluidic platform for high-throughput screening of Streptomyces [J ] . Communications Biology , 2021 , 4 : 647 .

WHITESIDES G M . The origins and the future of microfluidics [J ] . Nature , 2006 , 442 ( 7101 ): 368 - 373 .

SEEMANN R , BRINKMANN M , PFOHL T , et al . Droplet based microfluidics [J ] . Reports on Progress in Physics , 2012 , 75 ( 1 ): 016601 .

GIELEN F , HOURS R , EMOND S , et al . Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS) [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2016 , 113 ( 47 ): E7383 - E7389 .

MAZUTIS L , GILBERT J , UNG W L , et al . Single-cell analysis and sorting using droplet-based microfluidics [J ] . Nature Protocols , 2013 , 8 ( 5 ): 870 - 891 .

ALKAYYALI T , CAMERON T , HALTLI B , et al . Microfluidic and cross-linking methods for encapsulation of living cells and bacteria-a review [J ] . Analytica Chimica Acta , 2019 , 1053 : 1 - 21 .

MASHAGHI S , ABBASPOURRAD A , WEITZ D A , et al . Droplet microfluidics: a tool for biology, chemistry and nanotechnology [J ] . TrAC Trends in Analytical Chemistry , 2016 , 82 : 118 - 125 .

MARUO S , NAKAMURA O , KAWATA S . Three-dimensional microfabrication with two-photon-absorbed photopolymerization [J ] . Optics Letters , 1997 , 22 ( 2 ): 132 - 134 .

CHURSKI K , NOWACKI M , KORCZYK P M , et al . Simple modular systems for generation of droplets on demand [J ] . Lab on a Chip , 2013 , 13 ( 18 ): 3689 - 3697 .

ANNA S L , BONTOUX N , STONE H A . Formation of dispersions using "flow focusing" in microchannels [J ] . Applied Physics Letters , 2003 , 82 ( 3 ): 364 - 366 .

LIAN Z , CHAN Y , LUO Y , et al . Microfluidic formation of highly monodispersed multiple cored droplets using needle-based system in parallel mode [J ] . Electrophoresis , 2020 , 41 ( 10/11 ): 891 - 901 .

ZHU P G , WANG L Q . Passive and active droplet generation with microfluidics: a review [J ] . Lab on a Chip , 2017 , 17 ( 1 ): 34 - 75 .

BOITARD L , COTTINET D , KLEINSCHMITT C , et al . Monitoring single-cell bioenergetics via the coarsening of emulsion droplets [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2012 , 109 ( 19 ): 7181 - 7186 .

ZIMMERMANN H F , ANDERLEI T , BÜCHS J , et al . Oxygen limitation is a pitfall during screening for industrial strains [J ] . Applied Microbiology and Biotechnology , 2006 , 72 ( 6 ): 1157 - 1160 .

SUEA-NGAM A , HOWES P D , SRISA-ART M , et al . Droplet microfluidics: from proof-of-concept to real-world utility? [J ] . Chemical Communications , 2019 , 55 ( 67 ): 9895 - 9903 .

HOLTZE C , ROWAT A C , AGRESTI J J , et al . Biocompatible surfactants for water-in-fluorocarbon emulsions [J ] . Lab on a Chip , 2008 , 8 ( 10 ): 1632 - 1639 .

THEBERGE A B , COURTOIS F , SCHAERLI Y , et al . Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology [J ] . Angewandte Chemie International Edition , 2010 , 49 ( 34 ): 5846 - 5868 .

CHUNG M T , NÚñEZ D , CAI D W , et al . Deterministic droplet-based co-encapsulation and pairing of microparticles via active sorting and downstream merging [J ] . Lab on a Chip , 2017 , 17 ( 21 ): 3664 - 3671 .

BRAGHERI F , MARTÍNEZ VÁZQUEZ R , OSELLAME R . Microfluidics [M/OL ] // Three-Dimensional Microfabrication Using Two-Photon Polymerization . Amsterdam : Elsevier , 2020 : 493 - 526 [2023-02-01] . https://www.sciencedirect.com/science/article/abs/pii/B9780128178270000576?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/B9780128178270000576?via%3Dihub .

DEBON A , POTT M , OBEXER R , et al . Ultrahigh-throughput screening enables efficient single-round oxidase remodelling [J ] . Nature Catalysis , 2019 , 2 ( 9 ): 740 - 747 .

HASAN S , BLAHA M E , PIENDL S K , et al . Two-photon fluorescence lifetime for label-free microfluidic droplet sorting [J ] . Analytical and Bioanalytical Chemistry , 2022 , 414 ( 1 ): 721 - 730 .

DUNCOMBE T A , PONTI A , SEEBECK F P , et al . UV-vis spectra-activated droplet sorting for label-free chemical identification and collection of droplets [J ] . Analytical Chemistry , 2021 , 93 ( 38 ): 13008 - 13013 .

WANG X X , XIN Y , REN L H , et al . Positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo [J ] . Science Advances , 2020 , 6 ( 32 ): eabb3521 .

HOLLAND-MORITZ D A , WISMER M K , MANN B F , et al . Mass activated droplet sorting (MADS) enables high-throughput screening of enzymatic reactions at nanoliter scale [J ] . Angewandte Chemie International Edition , 2020 , 59 ( 11 ): 4470 - 4477 .

GIRAULT M , KIM H , ARAKAWA H , et al . An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution [J ] . Scientific Reports , 2017 , 7 : 40072 .

SCIAMBI A , ABATE A R . Accurate microfluidic sorting of droplets at 30 kHz [J ] . Lab on a Chip , 2015 , 15 ( 1 ): 47 - 51 .

CLARK I C , THAKUR R , ABATE A R . Concentric electrodes improve microfluidic droplet sorting [J ] . Lab on a Chip , 2018 , 18 ( 5 ): 710 - 713 .

QIAO Y X , HU R , CHEN D W , et al . Fluorescence-activated droplet sorting of PET degrading microorganisms [J ] . Journal of Hazardous Materials , 2022 , 424 Pt B: 127417.

LIU Y F , YUAN H L , DING D Q , et al . Establishment of a biosensor-based high-throughput screening platform for tryptophan overproduction [J ] . ACS Synthetic Biology , 2021 , 10 ( 6 ): 1373 - 1383 .

NIKOOMANZAR A , VALLEJO D , CHAPUT J C . Elucidating the determinants of polymerase specificity by microfluidic-based deep mutational scanning [J ] . ACS Synthetic Biology , 2019 , 8 ( 6 ): 1421 - 1429 .

HE R L , DING R H , HEYMAN J A , et al . Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei [J ] . Journal of Industrial Microbiology and Biotechnology , 2019 , 46 ( 11 ): 1603 - 1610 .

TU R , LI L P , YUAN H L , et al . Biosensor-enabled droplet microfluidic system for the rapid screening of 3-dehydroshikimic acid produced in Escherichia coli [J ] . Journal of Industrial Microbiology and Biotechnology , 2020 , 47 ( 12 ): 1155 - 1160 .

YU X Y , LI S , FENG H B , et al . CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion [J ] . Metabolic Engineering , 2023 , 75 : 192 - 204 .

VAN LOO B , HEBERLEIN M , MAIR P , et al . High-throughput, lysis-free screening for sulfatase activity using Escherichia coli autodisplay in microdroplets [J ] . ACS Synthetic Biology , 2019 , 8 ( 12 ): 2690 - 2700 .

JANG S , LEE B , JEONG H H , et al . On-chip analysis, indexing and screening for chemical producing bacteria in a microfluidic static droplet array [J ] . Lab on a Chip , 2016 , 16 ( 10 ): 1909 - 1916 .

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 .

JIAN X J , GUO X J , CAI Z S , et al . Single-cell microliter-droplet screening system (MISS Cell): an integrated platform for automated high-throughput microbial monoclonal cultivation and picking [J ] . Biotechnology and Bioengineering , 2023 , 120 ( 3 ): 778 - 792 .

LI S , LIAO X H , YU X Y , et al . Combining genetically encoded biosensors with droplet microfluidic system for enhanced glutaminase production by Bacillus amyloliquefaciens [J ] . Biochemical Engineering Journal , 2022 , 186 : 108586 .

LI C , GAO X , QI H B , et al . Substantial improvement of an epimerase for the synthesis of D-allulose by biosensor-based high-throughput microdroplet screening [J ] . Angewandte Chemie International Edition , 2023 , 62 ( 10 ): e202216721 .

HUA E B , ZHANG Y , YUN K Y , et al . Whole-cell biosensor and producer Co-cultivation-based microfludic platform for screening Saccharopolyspora erythraea with hyper erythromycin production [J ] . ACS Synthetic Biology , 2022 , 11 ( 8 ): 2697 - 2708 .

BEST R J , LYCZAKOWSKI J J , ABALDE-CELA S , et al . Label-free analysis and sorting of microalgae and cyanobacteria in microdroplets by intrinsic chlorophyll fluorescence for the identification of fast growing strains [J ] . Analytical Chemistry , 2016 , 88 ( 21 ): 10445 - 10451 .

YUAN H L , TU R , TONG X W , et al . Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system [J ] . Journal of Industrial Microbiology and Biotechnology , 2022 , 49 ( 3 ): kuac007 .

CHEN J , VESTERGAARD M , JENSEN T G , et al . Finding the needle in the haystack—the use of microfluidic droplet technology to identify vitamin-secreting lactic acid bacteria [J ] . mBio , 2017 , 8 ( 3 ): e00526-17 .

AHARONI A , THIEME K , CHIU C P C , et al . High-throughput screening methodology for the directed evolution of glycosyltransferases [J ] . Nature Methods , 2006 , 3 ( 8 ): 609 - 614 .

KALIDASAN K , SU Y , WU X Y , et al . Fluorescence-activated cell sorting and directed evolution of α- N -acetylgalactosaminidases using a quenched activity-based probe ( q ABP) [J ] . Chemical Communications , 2013 , 49 ( 65 ): 7237 - 7239 .

DENNIG A , MARIENHAGEN J , RUFF A J , et al . Directed evolution of P 450 BM 3 into a p-xylene hydroxylase [J ] . ChemCatChem , 2012 , 4 ( 6 ): 771 - 773 .

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 ( 1/2 ): 13 - 27 .

PRODANOVIC R , OSTAFE R , BLANUSA M , et al . Vanadium bromoperoxidase-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 .

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 .

CHENG F , KARDASHLIEV T , PITZLER C , et al . A competitive flow cytometry screening system for directed evolution of therapeutic enzyme [J ] . ACS Synthetic Biology , 2015 , 4 ( 7 ): 768 - 775 .

BARAHONA E , ISIDRO E S , SIERRA-HERAS L , et al . A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus [J ] . Frontiers in Microbiology , 2022 , 13 : 991123 .

BARAHONA E , JIMÉNEZ-VICENTE E , RUBIO L M . Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening [J ] . Scientific Reports , 2016 , 6 : 38291 .

KASEY C M , ZERRAD M , LI Y W , et al . Development of transcription factor-based designer macrolide biosensors for metabolic engineering and synthetic biology [J ] . ACS Synthetic Biology , 2018 , 7 ( 1 ): 227 - 239 .

ZHENG J T , SAGAR V , SMOLINSKY A , et al . Structure and function of the macrolide biosensor protein, MphR(a), with and without erythromycin [J ] . Journal of Molecular Biology , 2009 , 387 ( 5 ): 1250 - 1260 .

QIU X L , XU P , ZHAO X R , et al . Combining genetically-encoded biosens ors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica [J ] . Metabolic Engineering , 2020 , 60 : 66 - 76 .

MEYER A , PELLAUX R , POTOT S , et al . Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors [J ] . Nature Chemistry , 2015 , 7 ( 8 ): 673 - 678 .

JOSEPHIDES D , DAVOLI S , WHITLEY W , et al . Cyto-mine: an integrated, picodroplet system for high-throughput single-cell analysis, sorting, dispensing, and monoclonality assurance [J ] . SLAS Technology , 2020 , 25 ( 2 ): 177 - 189 .

GAA R , MENANG-NDI E , PRATAPA S , et al . Versatile and rapid microfluidics-assisted antibody discovery [J ] . mAbs , 2021 , 13 ( 1 ): 1978130 .

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

暂无数据