Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae

Abstract

Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

“…In fungi, advanced CRISPR/Cas systems have been mainly established for Aspergillus spp. [31, 44, 45] and Ustilago maydis [21, 46]. They take advantage of autonomously replicating circular plasmids, namely the Aspergillus -derived AMA1 plasmid and pMS7 in U. maydis , for the delivery of Cas9 and sgRNA.…”

Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila

“…New approaches for marker recycling based on the powerful CRISPR-Cas9 system have been demonstrated recently in fungi, namely S. cerevisiae, C. albicans, A. oryzae and A. niger [36,37,61,62]. Most of these approaches combined Cas9 editing with the auxotrophic marker pryG/ura3 or recombinase-promoted excision.…”

“…Most of these approaches combined Cas9 editing with the auxotrophic marker pryG/ura3 or recombinase-promoted excision. These methods require additional steps for either constructing auxotrophic strains or autonomous replicating plasmids carrying the corresponding recombinase [36,37,62], which is complicated and time-consuming. Our results for generating the nonuple mutant M9 (Fig.…”

“…The counter-selection method requires auxotrophic strains that can be time-consuming and laborious to build, and therefore the Cre-loxP recombination system has been more widely adapted for marker rescue in fungi, such as Aspergillus nidulans, Neurospora crassa, Neotyphodium sp., Aspergillus oryzae, Cryphonectria parasitica, Metarhizium robertsii, Penicillium oxalicum, and Fusarium graminearum [29][30][31][32][33]. Recently, Katayama et al [36] established an efficient multiple genetic engineering technique in A. oryzae that was based on the CRISPR-Cas9 system and recycling of an AMA1based plasmid harboring the drug-resistance marker ptrA, allowing for repeatable genetic manipulation. More recently, Leynaud-Kieffer et al [37] reported a simple Cas9-based gene targeting method that provided selectable, iterative, and marker-free generation of genomic editing using the auxotrophic marker pyrG.…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…A. oryzae has also been used as a host for production of fungal secondary metabolites, e.g., cyclopiazonic acid [1] and 1,3,6,8-tetrahydroxynaphthalene [2], mainly because A. oryzae scarcely produces secondary metabolites that could otherwise confound the isolation of target compounds [3]. Many genetic tools have been developed for Aspergillus oryzae; e.g., constitutive and inducible promoters as described below, auxotrophic (pyrG [4], argB [5], niaD [6], sC [7] and adeA [8]) and dominant (amdS [9] and ptrA [10]) selective markers, a marker recycling system [11], a quadruple auxotrophic transformation system [12], and genome editing systems [13,14]. These tools facilitate simultaneous integration of several genes into the fungal genome, which is necessary for heterologous production of fungal secondary metabolites because usually several genes are involved in their biosynthesis.…”