CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

BackgroundThe circular economy closes loops in industrial manufacturing processes and minimizes waste. A bio-based economy aims to replace fossil-based resources and processes by sustainable alternatives which exploits renewable biomass for the generation of products used in our daily live. A current trend in fungal biotechnology—the production of fungal-based biomaterials—will contribute to both.ResultsThis study gives an overview of various trends and development applications in which fungal mycelium is used as new and sustainable biomaterial. A patent survey covering the last decade (2009–2018) yielded 47 patents and patent applications claiming fungal biomass or fungal composite materials for new applications in the packaging, textile, leather and automotive industries. Furthermore, fungal-based materials are envisaged for thermal insulation and as fire protection materials. Most patents and patent applications describe the use of different lignin- and cellulose-containing waste biomass as substrate for fungal cultivations, covering 27 different fungal species in total. Our search uncovered that most patent activities are on-going in the United States and in China.ConclusionCurrent patent developments in the field suggest that fungal bio-based materials will considerable shape the future of material sciences and material applications. Fungal materials can be considered as an excellent renewable and degradable material alternative with a high innovation potential and have the potential to replace current petroleum-based materials.

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.

Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.