Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum

Nielsen, Mikkel Rank; Wollenberg, Rasmus Dam; Westphal, Klaus Ringsborg; Søndergaard, Teis Esben; Wimmer, Reinhard; Gardiner, Donald M.; Sørensen, Jens Laurids

doi:10.1016/j.fgb.2019.103248

Cited by 21 publications

(14 citation statements)

References 89 publications

(122 reference statements)

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“…Specifically, were the two integration border sites amplified from a non-coding region between the genes NECHADRAFT_103550 (Conserved, hypothetical Fusarium protein) and NECHADRAFT_91300 (Putative ATP-binding ABC transporter), found just upstream from NECHADRAFT_66759 (β-Tubulin). Finally, a plasmid backbone was PCR amplified from in-house vector pSHUT3 [31] with primers C094 + C095. This vector is based on U-GOAL [32] and comprise the bacterial elements kan R , IncP, trfA to which the yeast auxotrophic selection marker URA3 from pYES2 (Invitrogen) and replication origin CEN6/ARSH4 from pRS315 (ATCC ® 77144) [33] were added.…”

Section: Methodsmentioning

confidence: 99%

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

Nielsen

Holzwarth

Brew

et al. 2019

Fungal Biol Biotechnol

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BackgroundBesides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.ResultsTo remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.ConclusionBy creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094.Graphic abstract

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Section: Methodsmentioning

confidence: 99%

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

Nielsen

Holzwarth

Brew

et al. 2019

Fungal Biol Biotechnol

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“…Laboratory trials for DKPs-microbial induction have faced some constraints. Although producing DKPs from microorganisms via an expression system is feasible; the optimization is long and not a straightforward process [58]. The biosynthesis of DKPs relies mainly on two enzymes, non-ribosomal peptide synthetases (NRPs) and tRNA-dependent cyclodipeptide synthases (CDPs) [59].…”

Section: Occurence and Biosynthesis Of Dkpsmentioning

confidence: 99%

Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold

et al. 2021

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Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.

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“…In contrast, genetically tractable filamentous fungi have compatible transcription, translation, post-translational modification, and secretion machineries for the expression of foreign fungal genes and therefore are better suited for the heterologous expression of full-length BGCs. Intact BGCs of penicillin, citrinin, fusatins, W493, bikaverin, and flavoglaucin have been transferred and successfully expressed in fungal hosts. Moreover, Bok and Clevenger et al developed fungal artificial chromosomes to introduce entire BGCs from three Aspergillus species into A. nidulans , and about 27% of the transferred BGCs produced detectable products. , …”

Section: Introductionmentioning

confidence: 99%

Total Heterologous Biosynthesis of Fungal Natural Products in Aspergillus nidulans

2022

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Fungal natural products comprise a wide range of bioactive compounds including important drugs and agrochemicals. Intriguingly, bioinformatic analyses of fungal genomes have revealed that fungi have the potential to produce significantly more natural products than what have been discovered so far. It has thus become widely accepted that most biosynthesis pathways of fungal natural products are silent or expressed at very low levels under laboratory cultivation conditions. To tap into this vast chemical reservoir, the reconstitution of entire biosynthetic pathways in genetically tractable fungal hosts (total heterologous biosynthesis) has become increasingly employed in recent years. This review summarizes total heterologous biosynthesis of fungal natural products accomplished before 2020 using Aspergillus nidulans as heterologous hosts. We review here Aspergillus transformation, A. nidulans hosts, shuttle vectors for episomal expression, and chromosomal integration expression. These tools, collectively, not only facilitate the discovery of cryptic natural products but can also be used to generate high-yield strains with clean metabolite backgrounds. In comparison with total synthesis, total heterologous biosynthesis offers a simplified strategy to construct complex molecules and holds potential for commercial application.

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Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum

Cited by 21 publications

References 89 publications

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold

Total Heterologous Biosynthesis of Fungal Natural Products in Aspergillus nidulans

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