High‐throughput screening of amino acid producing Corynebacterium glutamicum strains with accelerated LC‐MS/MS

2020 ◽  
Vol 92 (9) ◽  
pp. 1337-1337
Author(s):  
A. Reiter ◽  
L. Herbst ◽  
W. Wiechert ◽  
M. Oldiges
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High throughput method development and optimised production of leaf protein concentrates with potential to support the agri-industry

2021 ◽  
Author(s):  
Ajay Iyer ◽  
Lisa Guerrier ◽  
Salomé Leveque ◽  
Charles S. Bestwick ◽  
Sylvia H. Duncan ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Extraction ◽  
Leaf Protein ◽  
Protein Recovery ◽  
Screening Methods ◽  
Extraction Technology ◽  
Protein Concentrates ◽  
Ethanol Precipitation

AbstractInvasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.

scholarly journals A Cell-Based Ultra-High-Throughput Screening Assay for Identifying Inhibitors of D-Amino Acid Oxidase

2006 ◽  
Vol 11 (5) ◽  
pp. 481-487 ◽  
Author(s):  
Philip E. Brandish ◽  
Chi-Sung Chiu ◽  
Jonathan Schneeweis ◽  
Nicholas J. Brandon ◽  
Clare L. Leech ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hormone Receptors ◽  
Chinese Hamster ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Compound Libraries ◽  
High Throughput Screening Assay ◽  
Artificial Environment

Enzymes are often considered less “druggable” targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve l-serine yield in Corynebacterium glutamicum

2018 ◽  
Vol 102 (14) ◽  
pp. 5939-5951 ◽  
Author(s):  
Xin Zhang ◽  
Xiaomei Zhang ◽  
Guoqiang Xu ◽  
Xiaojuan Zhang ◽  
Jinsong Shi ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Artp Mutagenesis

scholarly journals Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion

2021 ◽  
Vol 9 ◽  
Author(s):  
Suvasini Balasubramanian ◽  
Jun Chen ◽  
Vinoth Wigneswaran ◽  
Claus Heiner Bang-Berthelsen ◽  
Peter Ruhdal Jensen
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Production ◽  
Reference Strain ◽  
Functional Enrichment ◽  
Chemical Mutagenesis ◽  
Efficient Production ◽  
Food Ingredients ◽  
Synonymous Mutations

With emerging interests in heterologous production of proteins such as antibodies, growth factors, nanobodies, high-quality protein food ingredients, etc. the demand for efficient production hosts increases. Corynebacterium glutamicum is an attractive industrial host with great secretion capacity to produce therapeutics. It lacks extracellular protease and endotoxin activities and easily achieves high cell density. Therefore, this study focuses on improving protein production and secretion in C. glutamicum with the use of droplet-based microfluidic (DBM) high throughput screening. A library of C. glutamicum secreting β-glucosidase was generated using chemical mutagenesis coupled with DBM screening of 200,000 mutants in just 20 min. Among 100 recovered mutants, 16 mutants exhibited enhanced enzyme secretion capacity, 13 of which had unique mutation profiles. Whole-genome analysis showed that approximately 50–150 SNVs had occurred on the chromosome per mutant. Functional enrichment analysis of genes with non-synonymous mutations showed overrepresentation of genes involved in protein synthesis and secretion relevant biological processes, such as DNA and ribosome RNA synthesis, protein secretion and energy turnover. Two mutants JCMT1 and JCMT8 exhibited the highest secretion with a six and a fivefold increase in the β-glucosidase activity in the supernatant, respectively, relative to the reference strain JC0190. After plasmid curing, a new plasmid with the gene encoding α-amylase was cloned into these two mutants. The new strains SB024 and SB025 also exhibited a five and a sixfold increase in α-amylase activity in the supernatant, respectively, relative to the reference strain SB023. The results demonstrate how DBM screening can serve as a powerful development tool to improve cell factories for the production and secretion of heterologous proteins.

scholarly journals High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor

2015 ◽  
Vol 20 (10) ◽  
pp. 1218-1231 ◽  
Author(s):  
Ryan T. Terry-Lorenzo ◽  
Keiki Masuda ◽  
Kohtaroh Sugao ◽  
Q. Kevin Fang ◽  
Michael A. Orsini ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Association Studies ◽  
Therapeutic Drug ◽  
Human Populations ◽  
Acid Oxidase ◽  
Genome Wide Association Studies ◽  
Amino Acid Oxidase

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2 [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2 and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

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