CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

BackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

First Report of Fusarium venenatum causing foot and root rot of wheat (Triticum aestivum) in Germany

Field experiments were established in the 2018/19 and 2019/20 growing seasons at the experimental station in Neu-Eichenberg, Hessen, Germany to examine the suitability of multiple wheat cultivars for intercropping with pea and the effect of mixtures on diseases. Approximately 600 wheat tillers (BBCH 49-61, depending on the year and cultivar) were sampled in each year and assessed for severity of foot (lower stem) rot symptoms. Fungi from plants with Fusarium-like symptoms exhibiting reddish-brown discolorations on the stems were isolated following the methods described in Šišić et al. (2018). Surface disinfected (3% NaOCl for 10s) wheat stems and roots were cut into three 1 cm long pieces and placed on COONS agar (Coons, 1916). Following 7 – 12 days of incubation under constant blacklight blue fluorescent light, pure cultures were generated on potato dextrose and synthetic nutrient-poor agar (Nirenberg, 1976) using the hyphal tip transfer technique, and the resulting colonies examined microscopically. Based on morphology ca 15% of all Fusarium isolates recovered belonged to a distinct taxonomic unit and were initially identified as F. sambucinum-like (Leslie and Summerell, 2006). The identity of 16 randomly selected isolates (GenBank accession numbers MW085924 - MW085939) was confirmed by sequencing a portion of the translation elongation factor 1-alpha gene region (O’Donnell et al., 1998). Blast analysis in the FUSARIUM-ID (Geiser et al., 2004) and the NCBI databases revealed >99 to 100% identity match with the Fusarium venenatum accession numbers NRRL 22196, FRC R-09186 and MRC 2394. Pathogenicity tests were conducted on wheat cultivar Torborzo using six F. venenatum isolates. Inoculum was prepared using a sterile sand-millet mix infested with six agar plugs of each of the isolates. Once fully colonized, the inoculum was mixed with sterilized sand in a 1:7 ratio (by volume), transferred to 300 ml pots and 4 surface sterilized wheat seeds (5 min 70% alcohol) were sown in each pot. Non-inoculated controls were amended with sterilized inoculum. The experiment was conducted in a greenhouse in a completely randomized design with 5 replicates. Disease symptoms were assessed after four weeks. All isolates induced reddish-brown discolorations on the lower stems similar to those observed in the field grown plants. In addition, the infected plants developed dark brown to black discolorations on the crowns and roots and showed clear signs of stunted root growth. These symptoms were further accompanied by chlorosis (yellowing) of the lower leaves starting from the leaf tip. All isolates were successfully reisolated from the infected wheat plants but not from the controls. To the best of our knowledge, this is the first report of F. venenatum causing foot and root rot of wheat in Germany. Results from field and greenhouse inoculation experiments indicate that F. venenatum may be an important pathogen of wheat in Germany. Further studies on distribution and relative abundance of the species in the Fusarium foot and root rot complex of wheat in Germany are warranted. In addition, it is important to note that our results contrast the results from previous studies (Farr and Rossman, 2021) which reported F. venenatum primarily as a soil saprophyte and presumably non-pathogenic fungus in its nature. Our findings thus, also warrant the need to further investigate pathogenic potential of this species and the role it may play on other common rotational crops in Germany.

Bioconversion of pretreated wheat straw to ethanol by Monascus purpureus CBS 109.07 and Fusarium venenatum ATCC 20334 using simultaneous saccharification and fermentation

Yudianto D, Nainggolan EA, Millati R, Hidayat C, Lennartsson P, Taherzadeh MJ, Niklasson C. 2019. Bioconversion of pretreated wheat straw to ethanol by Monascus purpureus CBS 109.07 and Fusarium venenatum ATCC 20334 using simultaneous saccharification and fermentation. 20: 2229-2235. Fractions of sulfuric acid-pretreated wheat straw, i.e. solid, liquid, and a mixture of liquid and solid were used as substrates in simultaneous saccharification and fermentation (SSF) process to produce ethanol. The bioconversion was performed by Monascus purpureus CBS 109.07 and Fusarium venenatum ATCC 20334. The highest ethanol yields from solid, liquid and a mixture of solid and liquid fractions by M. purpureus CBS 109.07 were 0.36, 0.41, and 0.37 g/g glucose, respectively. The corresponding values by F. venenatum ATCC 20334 were 0.21, 0.54, 0.35 g/g glucose, respectively.

Enhancing the Nutritional Value of Canola (Brassica napus) Meal Using a Submerged Fungal Incubation Process

The aim of this study was to determine the optimal fungal culture to increase the nutritional value of canola meal so it could be used at higher feed inclusion rates, and for a broad range of monogastrics, including fish. Submerged incubation conditions were used to evaluate the performance of seven fungal cultures in hexane extracted (HE) and cold pressed (CP) canola meal. Aureobasidium pullulans (Y-2311-1), Fusarium venenatum and Trichoderma reesei resulted in the greatest improvements in protein levels in HE canola meal, at 21.0, 23.8, and 34.8 %, respectively. These fungi reduced total glucosinolates (GLS) content to 2.7, 7.4, and 4.9 μM.g-1, respectively, while residual sugar levels ranged from 0.8 to 1.6 % (w/w). In trials with CP canola meal, the same three fungi increased protein levels by 24.6, 35.2, and 37.3 %, and final GLS levels to 6.5, 4.0, and 4.7 μM.g-1, respectively. Additionally, residual sugar levels were reduced to 0.3-1.0 % (w/w).