Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii

Area of Science:

• Biotechnology
• Molecular Biology
• Mycology

Background:

• Thermophilic fungi offer advantages for industrial protein production.
• Paecilomyces variotii is a thermophilic fungus with potential as a protein production host.
• Developing efficient gene editing tools is crucial for optimizing fungal hosts.

Purpose of the Study:

• To establish a gene editing system in Paecilomyces variotii for heterologous protein production.
• To create a conidia-deficient strain for safe handling and environmental containment.
• To evaluate different promoter strengths and the effect of signal sequences on protein expression.

Main Methods:

• Isolation and characterization of a thermophilic Paecilomyces variotii strain (MR1).
• UV mutagenesis to generate a conidia-deficient strain (UM7).
• Cas9-gRNA ribonucleoprotein (RNP)-mediated gene editing with homology-directed repair (HDR) using pyrG as a selection marker.
• Co-transformation of protoplasts with RNP and HR donor DNA containing various promoter sequences and eGFP.
• Sequence analysis to confirm successful HDR and evaluation of promoter efficiency.

Main Results:

• Successful generation of a conidia-deficient strain (UM7) of P. variotii.
• Demonstration of RNP-mediated gene editing and HDR in P. variotii, yielding 16 pyrG-disrupted mutants with integrated promoter-eGFP constructs.
• Identification of PgpdA as the strongest promoter for intracellular GFP production.
• Observation that the signal peptide negatively impacted extracellular GFP expression.
• Enhanced GFP expression in both mycelial cells and culture broth in a newly edited strain lacking the signal sequence.

Conclusions:

• This study reports the first successful RNP-mediated gene editing in Paecilomyces variotii.
• The developed gene editing system enables the optimization of P. variotii as a host for heterologous protein production.
• Eliminating the signal peptide significantly improved protein expression and secretion, highlighting its role in optimizing fungal protein production platforms.