scholarly journals Improvement of l-Valine Production by Atmospheric and Room Temperature Plasma Mutagenesis and High-Throughput Screening in Corynebacterium glutamicum

ACS Omega ◽  
2020 ◽  
Vol 5 (10) ◽  
pp. 4751-4758 ◽  
Author(s):  
Guoqiang Han ◽  
Ning Xu ◽  
Xieping Sun ◽  
Jinzhao Chen ◽  
Chun Chen ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Room Temperature ◽  
Temperature Plasma

scholarly journals Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

PLoS ONE ◽  
2017 ◽  
Vol 12 (5) ◽  
pp. e0176545 ◽  
Author(s):  
Xiaoguang Fan ◽  
Heyun Wu ◽  
Guoliang Li ◽  
Hui Yuan ◽  
Hongchao Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Room Temperature ◽  
Temperature Plasma

scholarly journals High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 1074-1085 ◽  
Author(s):  
Tadeo Moreno-Chicano ◽  
Ali Ebrahim ◽  
Danny Axford ◽  
Martin V. Appleby ◽  
John H. Beale ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Radiation Damage ◽  
High Throughput ◽  
High Throughput Screening ◽  
Room Temperature ◽  
Data Sets ◽  
Binding Studies ◽  
X Ray ◽  
Temperature Damage ◽  
Serial Femtosecond Crystallography

High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.

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 of Higher Α-Amylase Producing Bacillus Licheniformis by Fluorescence-Activated Droplet Sorting

2021 ◽  
Author(s):  
Huiling Yuan ◽  
Ran Tu ◽  
Xinwei Tong ◽  
Yuping Lin ◽  
Qinhong Wang
Keyword(s):  
Bacillus Licheniformis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Industrial Applications ◽  
Cell Factory ◽  
Temperature Plasma ◽  
Droplet Sorting

Abstract Backgroundα-Amylases is one of the most important starch degrading enzymes and has the widest range of industrial applications. Bacillus licheniformis has been widely used as a cell factory for industrial production of amylase. However, difficulties in genetic modification of B. licheniformis have limited its widespread use. Directed evolution, based on the combination of random mutagenesis and high throughput screening (HTS), has been proven an effective strategy in strain improvement for increasing the productivity, but it requires a suitable HTS system to screen the desired mutants. Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultra-high throughput, however, the accessibility of a droplet microfluidic HTS platform to users having no background in microfluidics is still an issue. ResultsHere, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting (FADS) technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing strains from a library of B. licheniformis strains (a strain already used at industrial-scale). The B. licheniformis mutant library was generated by atmospheric and room temperature plasma (ARTP) mutagenesis. The clones displaying over 50% improvement in α-amylases productivity compared to the wild-type were isolated.ConclusionsWe established an efficient droplet microfluidic platform which consisted of droplet generation, droplet incubation, and sorting of droplets with a throughput of up to 1 × 106 droplets per h. The screening platform was demonstrated by successfully identifying B. licheniformis clones with improved α-amylase production. We believe that the droplet platform could be extended to the development of other industrially valuable strains.

scholarly journals High-Throughput Screening of a Corynebacterium glutamicum Mutant Library on Genomic and Metabolic Level

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e86799 ◽  
Author(s):  
Lorenz C. Reimer ◽  
Jana Spura ◽  
Kerstin Schmidt-Hohagen ◽  
Dietmar Schomburg
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mutant Library
Sign in / Sign up

Export Citation Format

Share Document