•A tool, TAPE, for identification of efficient protospacers was implemented.•An efficient polymerase III promoter for production of sgRNA is presented.•tRNAs were used as ribozyme substrates to ...release sgRNA species.•Single-stranded oligonucleotides were used as repair templates for gene alterations.•Showing that the tools can be combined and used for multiplex experiments.
CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.
Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The ...catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.
Filamentous fungi are important producers of enzymes and bioactive secondary metabolites and are exploited for industrial purposes. Expression and characterization of biosynthetic pathways requires ...stable expression of multiple genes in the production host. Fungal promoters are indispensable for the accomplishment of this task, and libraries of promoters that show functionality across diverse fungal species facilitate synthetic biology approaches, pathway expression, and cell-factory construction.
In this study, we characterized the intergenic region between the genes encoding histones H4.1 and H3, from five phylogenetically diverse species of
, as bidirectional promoters (P
). By expression of the genes encoding fluorescent proteins mRFP1 and mCitrine, we show at the translational and transcriptional level that this region from diverse species is applicable as strong and constitutive bidirectional promoters in
. Bioinformatic analysis showed that the divergent gene orientation of
and
appears maintained among fungi, and that the P
display conserved DNA motifs among the investigated 85 Aspergilli. Two of the heterologous P
s were utilized for single-locus expression of four genes from the putative malformin producing pathway from
in
. Strikingly, heterologous expression of
encoding the non-ribosomal peptide synthetase is sufficient for biosynthesis of malformins in
, which indicates an iterative use of one adenylation domain in the enzyme. However, this resulted in highly stressed colonies, which was reverted to a healthy phenotype by co-expressing the residual four genes from the putative biosynthetic gene cluster.
Our study has documented that P
is a strong constitutive bidirectional promoter and a valuable new addition to the genetic toolbox of at least the genus
.
In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary ...metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that
Aspergillus homomorphus
turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (
1
) and B (
2
) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a
C
-methylation domain, which however seems to be promiscuous since only
2
is
C
-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi.
Key points
• Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA.
• CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.
•A biosynthetic gene cluster for the hybrid polyketide-nonribosomal peptide compound acurin A in Aspergillus aculeatus.•The PKS and NRPS responsible for acurin A production are two individually ...transcribed entities.•Phylogenetics suggest that the separated PKS and NRPS is a result from the fission of an ancestral PKS-NRPS hybrid.•The biosynthetic capacity of A. aculeatus is facilitated by the use of CRISPR/Cas9 gene editing.
This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.
Genome Editing: CRISPR-Cas9 Hoof, Jakob B; Nødvig, Christina S; Mortensen, Uffe H
Methods in molecular biology (Clifton, N.J.),
2018, Volume:
1775
Journal Article
In the present chapter, we present the protocols and guidelines to facilitate implementation of CRISPR-Cas9 technology in fungi where few or no genetic tools are in place. Hence, we firstly explain ...how to identify dominant markers for genetic transformation. Secondly, we provide a guide for construction of Cas9/sgRNA episomal expression vectors. Thirdly, we present how to mutagenize reporter genes to explore the efficiency of CRISPR-Cas9 in the relevant fungus and to ease subsequent CRISPR-mediated genetic engineering. Lastly, we describe how to make CRISPR-mediated marker-dependent and marker-free gene targeting.
Indoor fungi are a worldwide problem causing negative health effects for infected building's occupants and even deterioration of building structures. Different fungal species affect buildings and ...their inhabitants differently. Therefore, rapid and accurate identification of fungi to the species level is essential for health risk assessment and building remediation. This study focuses on molecular identification of two common indoor fungal genera: Stachybotrys and Chaetomium. This study proposes two new DNA barcode candidates for Stachybotrys and Chaetomium: the gene encoding mitogen activated protein kinase (hogA) and the intergenic region between histone 3 and histone 4 (h3-h4) as well as it introduces a rapid - 3.5h - protocol for direct Stachybotrys and Chaetomium species identification, which bypasses culture cultivation, DNA extraction and DNA sequencing.
•ITS is not suitable to delimit Chaetomium and Stachybotrys at the species level.•Two DNA regions, hogA and h3-h4, are suggested as novel DNA barcodes for Chaetomium and Stachybotrys spp.•Chaetomium and Stachybotrys spp. can be detected in 3.5 hoursh from indoor environments using a novel, rapid spore/tissue-PCR protocol.
Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the ...influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.
•S. chartarum and C. globosum, material penetration ability is dependent on the building material composition.•Water has bigger impact on structural changes of gypsum wallboard core but smaller on plywood than fungal growth.•Small size of the investigated microCT scan area can make the statistical analysis of porosity and S/V ratio biased.•MicroCT can become a tool to investigate the structural changes occurring in building materials during fungal infestation
The Photodetector Array Camera and Spectrometer (PACS) is one of the three science instruments on ESA's far infrared and submillimetre observatory. It employs two Ge:Ga photoconductor arrays ...(stressed and unstressed) with 16x25 pixels, each, and two filled silicon bolometer arrays with 16x32 and 32x64 pixels, respectively, to perform integral-field spectroscopy and imaging photometry in the 60-210\mu\ m wavelength regime. In photometry mode, it simultaneously images two bands, 60-85\mu\ m or 85-125\mu\m and 125-210\mu\ m, over a field of view of ~1.75'x3.5', with close to Nyquist beam sampling in each band. In spectroscopy mode, it images a field of 47"x47", resolved into 5x5 pixels, with an instantaneous spectral coverage of ~1500km/s and a spectral resolution of ~175km/s. We summarise the design of the instrument, describe observing modes, calibration, and data analysis methods, and present our current assessment of the in-orbit performance of the instrument based on the Performance Verification tests. PACS is fully operational, and the achieved performance is close to or better than the pre-launch predictions.
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