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Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137953/ https://www.ncbi.nlm.nih.gov/pubmed/34026744 http://dx.doi.org/10.3389/fbioe.2021.668513 |
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author | Balasubramanian, Suvasini Chen, Jun Wigneswaran, Vinoth Bang-Berthelsen, Claus Heiner Jensen, Peter Ruhdal |
author_facet | Balasubramanian, Suvasini Chen, Jun Wigneswaran, Vinoth Bang-Berthelsen, Claus Heiner Jensen, Peter Ruhdal |
author_sort | Balasubramanian, Suvasini |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8137953 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-81379532021-05-22 Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion Balasubramanian, Suvasini Chen, Jun Wigneswaran, Vinoth Bang-Berthelsen, Claus Heiner Jensen, Peter Ruhdal Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2021-05-07 /pmc/articles/PMC8137953/ /pubmed/34026744 http://dx.doi.org/10.3389/fbioe.2021.668513 Text en Copyright © 2021 Balasubramanian, Chen, Wigneswaran, Bang-Berthelsen and Jensen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Balasubramanian, Suvasini Chen, Jun Wigneswaran, Vinoth Bang-Berthelsen, Claus Heiner Jensen, Peter Ruhdal Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title | Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title_full | Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title_fullStr | Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title_full_unstemmed | Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title_short | Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion |
title_sort | droplet-based microfluidic high throughput screening of corynebacterium glutamicum for efficient heterologous protein production and secretion |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137953/ https://www.ncbi.nlm.nih.gov/pubmed/34026744 http://dx.doi.org/10.3389/fbioe.2021.668513 |
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