Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion
| Autor: | Vinoth Wigneswaran, Suvasini Balasubramanian, Jun Chen, Peter Ruhdal Jensen, Claus Heiner Bang-Berthelsen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Histology Mutant Biomedical Engineering Mutagenesis (molecular biology technique) Heterologous Bioengineering 01 natural sciences Corynebacterium glutamicum 03 medical and health sciences Plasmid 010608 biotechnology Protein biosynthesis Secretion Gene Original Research Chemistry droplet-based microfluidics Bioengineering and Biotechnology heterologous protein production α-amylase 030104 developmental biology Biochemistry β-glucosidase TP248.13-248.65 high throughput screening Biotechnology |
| Zdroj: | Balasubramanian, S, Chen, J, Wigneswaran, V, Bang-Berthelsen, C H & Jensen, P R 2021, ' Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion ', Frontiers in Bioengineering and Biotechnology, vol. 9, 668513 . https://doi.org/10.3389/fbioe.2021.668513 Frontiers in Bioengineering and Biotechnology, Vol 9 (2021) Frontiers in Bioengineering and Biotechnology |
| DOI: | 10.3389/fbioe.2021.668513 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|