Customer Article | Core–Shell Droplet-Based Microfluidic Screening System for Filamentous Fungi
Spherical fungi have long been considered ideal hosts for the production of compounds and enzymes due to their excellent extracellular protein production and secretion capabilities [1]. However, the lack of a clear genetic background and effective genetic manipulation tools has significantly restricted the development and improvement of spherical fungi [2]. The combination of biotechnological tools with high-throughput screening (HTS) techniques to improve and select superior hosts can promote the application of spherical fungi in the fields of chemical engineering, food, and medicine [3]. Nonetheless, there is still a lack of rapid, universal screening systems for spherical fungi, severely limiting the identification of highly active/newly active enzymes and high-yield strains. Developing efficient, low-cost, high-throughput screening technology to easily, quickly, and sensitively detect and isolate spherical fungi is crucial for the industrial application of high-quality spherical fungal hosts.
?
In August 2023, Professor Jingwen Zhou's team from Jiangnan University published an article titled "Core–Shell Droplet-Based Microfluidic Screening System for Filamentous Fungi" in the journal "ACS Sensors". They designed a novel biocompatible core-shell droplet microfluidic (REPID) system for encapsulating, cultivating, and screening filamentous fungi to address the aforementioned issues. Using the REPID system, they obtained 19 mutant strains with high secretion functionality. Among these, the most efficient strain showed a 2.02-fold increase in secretion capability. This REPID system can be applied in strain engineering and rapid screening of filamentous fungi, aiding in the identification of improved hosts for large-scale protein production [4].
?
"The relevant genes used in this study were synthesized and cloned into the pUC57 vector by GENCEFE BIOTECH ?for expression in Aspergillus niger AG11."
Research Findings
1. Preparation of Core-Shell Droplets
The spread of mycelium and prolonged incubation times impose certain requirements on the robust structure and stability of the droplet system. This study devised a biocompatible core-shell droplet system for encapsulating, incubating, and screening filamentous fungi. The structure of the core-shell droplets comprises three components: an outer oil phase, a shell of Gelatin Methacrylate (Gel-MA), and an inner aqueous phase (Figure 1).
2. Cultivation and Detection of Aspergillus niger in Core-Shell Droplets
The designed core-shell droplets were used to encapsulate and cultivate Aspergillus niger spores. The mycelium maintained stable growth within the droplets (Figure 2A), while spores encapsulated in normal droplets ruptured the droplets after 15 hours of incubation (Figure 2B). This demonstrated the suitability of core-shell droplets for encapsulation and incubation of filamentous fungi. Spores of strain AG11-eGFP, expressing enhanced green fluorescent protein, were encapsulated in core-shell droplets, and fluorescent signals within the droplets were observed at different stages (Figure 2C). Signals were detectable after 8 hours of incubation, gradually intensifying over the following 28 hours, and began to diminish after 48 hours (Figure 2D). This indicates the accurate detection of mycelial fluorescence within the core-shell droplets over a relatively extended cultivation period.
3. Characterization of Fluorescent Signals in Core-Shell Droplets
Five different naturally occurring constitutive promoters, including PmbfA, PcoxA, PsrpB, PtvdA, and PmanB, with varying transcriptional strengths in Aspergillus niger AG11, were selected from the genome. After 48 hours of cultivation in a 24-well plate, the strain containing PmbfA exhibited the strongest fluorescent signal (Figure 3A). At 36 hours of incubation at 30°C, all strains showed robust growth and displayed green fluorescence within the droplets (Figure 3B). The fluorescence data within the droplets corresponded consistently with the fluorescence data from the 24-well plate, indicating the reliability and accuracy of the Aspergillus niger microfluidic analysis based on core-shell droplets.
4. Optimization of Core-Shell Droplet-Based Screening Process
After 36 hours of incubation at 30°C, 1% of the mixed droplets exhibited high fluorescence, while the rest did not fluoresce (Figure 4A). Upon completion of droplet sorting, the collected droplets from the collection channel were placed on an observation chip for fluorescence imaging (Figure 4B). At deviation voltages of 800V, 1000V, and 1200V, the positivity rates were 86.7%, 85.7%, and 92.1%, respectively (Figure 4C). Beyond 1200V, the droplets were pulled and deformed. Therefore, subsequent experiments opted for 1200V. Current experiments indicate that the throughput of the REPID system reaches up to 106 droplets per hour, several hundred times higher than traditional microtiter plate-based methods.
领英推荐
5. Using the Split GFP System to Screen Mutants with High Secretion Capability
To assess the stability of GFP1?10, it was co-incubated with the GFP11-lacking strain AG11-α. The results indicated that after 36 hours of co-incubation with AG11-α, the concentration of GFP1?10 decreased by approximately 60%, with no fluorescence signal observed (Figure 5C). However, co-incubation with the GFP11-tagged strain AG11-αG resulted in detectable fluorescence (Figures 5B, 5D). These experiments estimated the optimal addition of GFP1?10 to be 100 μM, an excessive yet entirely sufficient concentration for completing fluorescence complementation.
6. Screening Strains with Enhanced Amylase Secretion Using the REPID System
A mutant library of AG11-αG spores was constructed via ARTP mutagenesis and co-encapsulated with 100 μM GFP1?10 in core-shell droplets for incubation and high-throughput screening using the REPID system. Post-incubation, highly secreted target proteins could facilitate Split GFP self-assembly, resulting in stronger fluorescence. Extracellular protein content assays revealed that among the 21 screened mutant strains, 19 (>90%) exhibited higher α-amylase secretion than the original AG11-αG strain. Among these, strain M5 exhibited the highest amylase production, at 2.02 times that of AG11-αG (Figure 6C). Sequencing analysis did not detect any mutations in the α-amylase expression box of the strains exhibiting increased secretion capacity. The increase in secretion capability might be attributed to genomic changes regulating α-amylase secretion.
This study introduces a novel high-throughput screening method for filamentous fungi, termed the 'REPID system,' utilizing core-shell droplets and the Split GFP system. It successfully identified an Aspergillus niger mutant strain with a 2.02-fold increase in α-amylase secretion. This method accurately characterizes the fermentation process of mycelial morphology, screens for extracellular secretions, and significantly enhances the efficiency of filamentous fungi screening. The entire process from encapsulation to screening can be completed within 3 days, with a screening throughput of 106/h. This bears substantial value and significance for utilizing filamentous fungi in the production of pharmaceuticals, compounds, proteins, and more. Furthermore, this REPID system holds promise for the modification and screening of enzymes in various industrial applications involving filamentous fungi, indicating its potential for significant contributions.
?
?
?
[References]
[1]?Wosten, H. A. B. Filamentous fungi for the production of enzymes, chemicals and materials. Curr. Opin. Biotechnol. 2019, 59, 65?70.
[2] Li, Q.; Lu, J.; Zhang, G.; Liu, S.; Zhou, J.; Du, G.; Chen, J. Recent advances in the development of Aspergillus for protein production. Bioresour. Technol. 2022, 348, 126768.
[3] Strong, P. J.; Self, R.; Allikian, K.; Szewczyk, E.; Speight, R.; O’Hara, I.; Harrison, M. D. Filamentous fungi for future functional food and feed. Curr. Opin. Biotechnol. 2022, 76, 102729.
[4] Zhang C, Wu X, Song F, et al. Core–Shell Droplet-Based Microfluidic Screening System for Filamentous Fungi[J]. ACS sensors, 2023, 8(9): 3468-3477.
?