Abstract
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.
Highlights
Genetic engineering of microbes has diverse applications in biotechnology, enabling improved and valuable products for human consumption, industrial applications and therapeutic solutions
The parent strain JC0190 secreting β-glucosidase was mutagenized with ethyl methanesulfonate (EMS) and by the end of a 3-h treatment, 0.32% of the cells survived (Figure 1)
We chose the library after 3-h EMS mutagenesis for the further experiment, as an approximately 99% killing rate would be eligible for droplet screening (Chen et al, 2017)
Summary
Genetic engineering of microbes has diverse applications in biotechnology, enabling improved and valuable products for human consumption, industrial applications and therapeutic solutions. The integration of systems biology and synthetic biology has allowed for the development of high performing cell factories. The production of proteins and chemicals have proven to be economical and environmentally sustainable by endowing desirable properties on microorganisms by rerouting existing metabolic pathways or introducing entirely new ones (Lee et al, 2012). In order to exploit cell factories for efficient heterologous expression, optimization of the properties of the strains and enabling secretion of proteins into the culture media at high efficiency are important. It is critical to understand the complex molecular mechanisms of the cells in order to fully harness the technology in delivering the desired phenotypes and products
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