Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase

Abstract β-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to β-alanine in bacteria. However, due to the low activity of ADC, industrial production of β-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of β-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of β-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of β-alanine via the biological conversion route in the future.

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A dual fluorescence reporter system for high throughput screening of effectors of Kiss1 gene expression

FEBS Open Bio ◽

10.1002/2211-5463.12476 ◽

2018 ◽

Vol 8 (8) ◽

pp. 1352-1363 ◽

Cited By ~ 2

Author(s):

Xiaoning Li ◽

Sijia Wang ◽

Yanhua Lu ◽

Huanhuan Yin ◽

Junhua Xiao ◽

...

Keyword(s):

Gene Expression ◽

High Throughput ◽

High Throughput Screening ◽

Dual Fluorescence ◽

Reporter System

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Biosensor-based multi-gene pathway optimization for enhancing the production of glycolate

Applied and Environmental Microbiology ◽

10.1128/aem.00113-21 ◽

2021 ◽

Author(s):

Shumin Xu ◽

Linpei Zhang ◽

Shenghu Zhou ◽

Yu Deng

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Metabolic Flux ◽

Agar Plate ◽

Expression Level ◽

Rapid Screening ◽

Screening Methods ◽

Synthetic Pathway ◽

Gene Pathway ◽

Pathway Optimization

Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies that is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multi-gene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short period of time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep well plate-scale high-throughput screening methods for rapid screening of superior glycolate producers from a large library. A total of 22 gradient strength promoter-5′-UTR complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6×105 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/L glycolate in a 5-L bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multi-gene pathway optimizations. Importance The use of strong promoters, such as pTrc and T7, to control gene expression not only need adding expensive inducers but also results in excessive protein expression that may be resulting in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of a high-throughput screening methods restricted the glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, then agar plate and 48-well plate scale high-throughput screening methods were developed for rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate producing strain with good biosynthetic performance, and the use of the expensive inducer IPTG was avoided, which broadens our understanding about the mechanism of glycolate synthesis.

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Non-invasive and high-throughput interrogation of exon-specific isoform expression

Nature Cell Biology ◽

10.1038/s41556-021-00678-x ◽

2021 ◽

Author(s):

Dong-Jiunn Jeffery Truong ◽

Teeradon Phlairaharn ◽

Bianca Eßwein ◽

Christoph Gruber ◽

Deniz Tümen ◽

...

Keyword(s):

Stem Cells ◽

High Throughput ◽

High Throughput Screening ◽

Embryonic Stem ◽

High Sensitivity ◽

Therapeutic Interventions ◽

Dominant Factor ◽

Reporter System ◽

Isoform Expression ◽

Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

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Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00601-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4825-4834 ◽

Cited By ~ 20

Author(s):

Kao-Lu Pan ◽

Jin-Ching Lee ◽

Hsing-Wen Sung ◽

Teng-Yuang Chang ◽

John T.-A. Hsu

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

High Throughput ◽

High Throughput Screening ◽

Reporter System ◽

Viral Protease ◽

Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

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A Novel Reporter System for Molecular Imaging and High-Throughput Screening of Anticancer Drugs

ChemBioChem ◽

10.1002/cbic.201300142 ◽

2013 ◽

Vol 14 (12) ◽

pp. 1494-1503 ◽

Cited By ~ 5

Author(s):

Jingping Xie ◽

Chunxia Wang ◽

John Virostko ◽

H. Charles Manning ◽

Wellington Pham ◽

...

Keyword(s):

Molecular Imaging ◽

Anticancer Drugs ◽

High Throughput ◽

High Throughput Screening ◽

Reporter System

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Application of QuantiGene™ Nucleic Acid Quantification Technology for High Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710000500506 ◽

2000 ◽

Vol 5 (5) ◽

pp. 343-351 ◽

Cited By ~ 22

Author(s):

Usha Warrior ◽

Yihong Fan ◽

Caroline A. David ◽

Julie A. Wilkins ◽

Evelyn M. McKeegan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

High Throughput Screening ◽

Molecular Probes ◽

Epithelial Cell Line ◽

Reporter System ◽

Screening Assay ◽

Human Lung Epithelial Cell ◽

High Throughput Screening Assay ◽

Time Required

To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the Quanti-Gene™ nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-la (IL-la) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.

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Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.600796 ◽

2020 ◽

Vol 7 ◽

Author(s):

Fuxiao Liu ◽

Qianqian Wang ◽

Yilan Huang ◽

Ning Wang ◽

Youming Zhang ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Canine Distemper Virus ◽

Reverse Genetics ◽

Rapid Screening ◽

Canine Distemper ◽

Virus Propagation ◽

The Family ◽

Conventional Methods

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

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Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

International Journal of Molecular Sciences ◽

10.3390/ijms21093034 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3034 ◽

Cited By ~ 2

Author(s):

Shella Gilbert-Girard ◽

Kirsi Savijoki ◽

Jari Yli-Kauhaluoma ◽

Adyary Fallarero

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

High Throughput ◽

High Throughput Screening ◽

Bacterial Infections ◽

Rapid Screening ◽

Bacterial Biofilms ◽

Main Concern ◽

Assay Optimization ◽

Screening Platform

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

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A Novel High-Throughput Screening Assay for Sickle Cell Disease Drug Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109333975 ◽

2009 ◽

Vol 14 (4) ◽

pp. 330-336 ◽

Cited By ~ 4

Author(s):

Eszter Pais ◽

John S. Cambridge ◽

Cage S. Johnson ◽

Herbert J. Meiselman ◽

Timothy C. Fisher ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

High Throughput ◽

High Throughput Screening ◽

Rapid Screening ◽

Cell Disease ◽

Screening Assay ◽

Drug Candidates ◽

Test Molecule ◽

High Throughput Screening Assay

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. ( Journal of Biomolecular Screening. 2009:330-336)

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