Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

Abstract

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene manipulating tools. In this study, two different Cas9s from mesophilic and thermophilic bacteria, with in vivo sgRNA expression under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii. We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii. The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50-86%) than the thermophilic CRISPR/Cas9 system (40-67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent PKS-NRPS biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from that in the heterologous host Aspergillus. Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative and cell-wall-perturbing agents. IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. duponti remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. duponti. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. duponti, but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Acetylation of Sesquiterpenyl Epoxy-Cyclohexenoids Regulates Fungal Growth, Stress Resistance, Endocytosis, and Pathogenicity of Nematode-Trapping Fungus Arthrobotrys oligospora via Metabolism and Transcription

“…To investigate the function of 273, a mutant strain with 273 depletion was constructed through a modified homologous recombination . A previous study suggested that SEC metabolites were predominantly accumulated in rich nutrient medium .…”

“…A modified homologous recombination method , was applied to construct the vector for disruption gene 273 and mutant Δ273. All primers used in this study are provided in Table S1.…”

“…Then, the 3′ flank DNA fragment was inserted into the specific enzyme-digested PUC19-1300-D-HYB-5′ vector to generate the PUC19-1300-D-HYB-5′–3′ vector. The homologous fragment was amplified and purified as described previously. , …”

“…Our previous studies on potent nematicidal thermolides from thermophilic fungus Thermomyces dupontii suggested that thermolides A–B with acetylation displayed potent nematicidal activity while thermolide D without acetylation did not . Further study exhibited that acetylation of thermolide D was the final step in the biosynthesis of thermolides A–B . Phenotype bioassay suggested that mutant with disruption of the acetylation gene displayed varied fungal mycelia compared with wild-type (WT) strain .…”

“…14 Further study exhibited that acetylation of thermolide D was the final step in the biosynthesis of thermolides A−B. 15 Phenotype bioassay suggested that mutant with disruption of the acetylation gene displayed varied fungal mycelia compared with wild-type (WT) strain. 15 Thus, we were inspired to investigate whether acetylation of the SECs in A. oligospora might be involved in different biological activities and in regulating mycelia trap formation.…”