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. 2021 Nov 19;10(11):2850-2861.
doi: 10.1021/acssynbio.1c00260. Epub 2021 Nov 2.

Modular Synthetic Biology Toolkit for Filamentous Fungi

László Mózsik  1 , Carsten Pohl  2 , Vera Meyer  2 , Roel A L Bovenberg  3   4 , Yvonne Nygård  5 , Arnold J M Driessen  1
Affiliations

Modular Synthetic Biology Toolkit for Filamentous Fungi

László Mózsik et al. ACS Synth Biol. .

Abstract

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.

Keywords: Modular Cloning; filamentous fungi; hybrid transcription factor; inducible promoter; synthetic biology toolkit; transcriptional regulation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
List of vectors in the Fungal Modular Cloning Toolkit. (a) Location of genetic parts in a transcription unit with their corresponding linker sequences. (b) List of parts of the toolkit, containing promoters (P1), UASs (P2), UAS-compatible core promoters (P3), coding sequences with various fusion possibilities (CDS1–5), terminators (T1), complete transcription units (TUs), and additional vectors (sgRNA transcription units, flanking sequences, and AMA1 vectors). Abbreviations (Pc, An, Ania, Ao, Sc) indicate the origin of the template (P. rubens, A. niger, A. nidulans, A. oryzae, S. cerevisiae, respectively).
Figure 2
Figure 2
Transcription unit construction using the MoClo system and delivery platforms. A schematic representation of the recombination and assembly of the MoClo entry vectors into transcription units is shown. Transcription units can be assembled into (a) fungal shuttle vectors or (b, c) multigene constructs that can be delivered (b) as AMA1-based episomal vectors or (c) via genomic integration by homologous recombination.
Figure 3
Figure 3
Transcription unit assembly from MoClo entry vectors on a pLM-AMA002 fungal shuttle vector and delivery to filamentous fungi. (a) Schematic representation of the assembly of MoClo entry vectors into a single transcription unit delivered to P. rubens on the pLM-AMA002 fungal shuttle vector. (b) Fluorescence microscopy imaging of filaments of a P. rubens strain carrying pLM-AMA002 with the dSpCas9–eGFP-NLS transcription unit, showing protein expression of the fluorescently labeled gene product. Scale bars represent 20 μm.
See this image and copyright information in PMC

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