CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Glarea lozoyensis and Its Application in Manipulating gloF

Abstract

Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B 0 , which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans-3-hydroxy-L-proline to trans-4-hydroxy-L-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C 0 to (pneumocandin B 0 + pneumocandin C 0 ) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C 0 . However, the gene replacement has been hampered by traditional gene tools since gloF and gloG, two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF, and results showed that the knock-in strain retained the ability to produce pneumocandin B 0 , but the production of pneumocandin C 0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B 0 . In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

“…Establishing efficient genetic operating systems in endophytic fungi is crucial to further in-depth research. The CRISPR/Cas9 system has been developed into a set of powerful tools for manipulating the filamentous fungi genome [24][25][26][27][28]. In this study, a CRISPR/Cas9-dependent genome editing system was successfully developed in the endophytic fungus P. fici.…”

“…The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been established in some filamentous fungi, such as Trichoderma reesei [24], Penicillium chrysogenum [25], A. fumigatus [26], A. niger [27] and Glarea lozoyensis [28]. Most commonly used is the CRISPR/Cas9 system from Streptococcus pyogenes, which consists of Cas9 protein and two RNAs-CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) [29,30].…”

Fungal Biofilms as a Valuable Target for the Discovery of Natural Products That Cope with the Resistance of Medically Important Fungi—Latest Findings

“…For example, the polyenes nystatin and amphotericin B were isolated from Streptomyces spp. [ 105 ]; the spiro-diketone griseofulvin, was obtained from Penicillum griseofulvum [ 106 ]; the echinocandins caspofungin and anidulafungin are semisynthetic derivatives from the natural pneumocandin B, which was isolated from the fungus Glarea lozoyensis [ 107 ]. In turn, micafungin derived from the lipopeptide FR901379 was isolated from the fungus Coleophoma empetri [ 107 ], a plant pathogen associated with postharvest fruit rot in cranberries [ 108 ].…”

“…[ 105 ]; the spiro-diketone griseofulvin, was obtained from Penicillum griseofulvum [ 106 ]; the echinocandins caspofungin and anidulafungin are semisynthetic derivatives from the natural pneumocandin B, which was isolated from the fungus Glarea lozoyensis [ 107 ]. In turn, micafungin derived from the lipopeptide FR901379 was isolated from the fungus Coleophoma empetri [ 107 ], a plant pathogen associated with postharvest fruit rot in cranberries [ 108 ]. Under this scenario, it is clear that nature has provided a robust platform for finding novel scaffolds for the discovery of antifungal drugs.…”

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

“…Disrupting the nonhomologous end-joining (NHEJ) pathways is a general way to improve the frequency of gene-targeting in filamentous fungi ( Ninomiya et al, 2004 ). Even in the CRISPR/Cas9 genome editing system, the chassis cell of NHEJ deficient is also inevitable ( Wei et al, 2020 ). In addition, the elements of promoter and selectable marker are also indispensable, especially for the sequential metabolic engineering of industrial strains ( Blumhoff et al, 2013 ).…”