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

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.

Keywords: Corynebacterium glutamicum; droplet-based microfluidics; heterologous protein production; high throughput screening; α-amylase; β-glucosidase.

FIGURE 1

Survival rate after EMS treatment. The survival percentage of the cells by the end of the third hour was 0.315.

FIGURE 2

Schematic overview of development and screening of the mutant library: A whole genome mutated library was generated with EMS chemical mutagenesis on shake flask culture and were recovered on an agar as shown in (A). Steps B₁–B₅ represents the microfluidic process—The recovered mutants were diluted to 10⁷ mutant libraries mixed with fluorogenic substrate (B₁). The droplets generated incubates for the cells to accumulate secretion (B₂). Post incubation, droplets are injected into the sorting chip and sorted based on the fluorescence intensity and collected in an Eppendorf tube. The sorted cells are then released and injected into the device for a second round of droplet generation and screening (B₅). The sorted cells are then released and cultivated on agar plates. The outgrown single colonies are picked for downstream characterization, e.g., shake flask fermentation and protein secretion tests.

FIGURE 3

Growth curves of the parent and mutant strains carried out in BioLector. The growth of the strains were monitored in a 48-well flower plate (culture volume: 1,000 μL, temperature: 30∘C, agitation: 800 rpm) by measuring scattered light (ex: 620 nm, Gain: 20).

FIGURE 4

SDS PAGE analysis of the supernatant of the selected mutants. The arrow points the band β-glucosidase. The lanes from left to right are the wild-type strain with empty plasmid, and the reference strain followed by the mutants. 15 μL of acetone precipitated protein samples were loaded on the wells.

FIGURE 5

Protein secretion capacity of selected mutant strains: (A) β-glucosidase activity: fold increase in the relative fluorescence activity of the mutant strains secreting β-glucosidase in comparison to the reference strain JC0190. (B) Total protein: total protein content of the reference and mutant strains in the supernatants. The protein content of mutant 4 is around 4 μg and hence negligible in the graph. (C) α-Amylase secretion: fold increase in the α-amylase enzyme units of the strains SB024 and SB025 derived from mutants JCMT1 and JCMT8, respectively, compared to the reference strain SB023.

FIGURE 6

The gene clusters enriching mutations in GO and KEGG categories by DAVID.

FIGURE 7

Tricarboxylic acid cycle with genes enriching mutations. The red stars represent the genes with presence of mutations.

FIGURE 8

Design of microfluidic chips. (A) The droplet generation chip for cell encapsulation consists of an oil phase inlet (1), and two aqueous phase inlets (2 and 3). The fluid resistors (4) dampen fluctuations arising from the mechanical instability of syringe pumps and PDMS device. Due to the surface tension of the water–oil interface, the aqueous phase breaks up into droplets at the flow-focusing junction. Droplets are collected into a syringe or an Eppendorf tube from the outlet (5). (B) The droplet-sorting chip consists of two inlets for droplets (1) and spacing oil (2). The flow rate ratio of the oil and injected emulsions are varied to control the distance between adjacent droplets. The droplets at the sorting junction are sorted based on the fluorescence intensity. Downstream to the detection, electrodes—ground (3, 4), high voltage (5) are located. Next to the electrodes, the channel is split into two outlets for collecting sorted droplets (6) and unsorted/waste cells (7).