Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary Metabolites in the Post-Genomic Era

Rendsvig, Jakob Kræmmer Haar; Futyma, Malgorzata E.; Jarczynska, Zofia Dorota; Mortensen, Uffe Hasbro

doi:10.1007/978-3-030-49924-2_10

Cited by 3 publications

(5 citation statements)

References 266 publications

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“…During protoplast transformation, it is common to obtain copy number variants 41 , however such high copy insertions, especially tandem insertions on this scale, are rare. The integration of multi-copies of a transgene are often the result of several copies entering a single nucleus and recombination prior to genomic integration into a single locus, frequently leading to higher levels of transgene expression 42 . However, in fungi and plants, integration of multiple transgene copies can also suppress transgene expression with varying degrees of silencing, a phenomenon that has been well studied and termed ‘quelling’ in Neurospora where it occurs at the post-transcriptional level 43 .…”

Section: Discussionmentioning

confidence: 99%

“…To enhance expression of AvrSr50 in a reproducible manner, further optimisation of the MoT system is required to consistently elevate the number of transgene copies integrated or enhance expression of the transgene by targeting transgenes to locations of high transcriptional activity and/or through synthetic design. For instance, variation of the terminator sequence, development of synthetic promoters and transcriptional regulators have all been utilised to control and enhance production of gene expression cassettes 42 . If these limitations can be overcome, this would present MoT as an ideal accessible fungal surrogate system for studying the virulence/avirulence function of candidate effectors in future, particularly from intractable obligate biotrophic pathogens that are recalcitrance to genetic transformation.…”

Section: Discussionmentioning

confidence: 99%

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Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat

Jensen

Saunders

2023

Sci Rep

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Plant pathogens deliver effector proteins to reprogramme a host plants circuitry, supporting their own growth and development, whilst thwarting defence responses. A subset of these effectors are termed avirulence factors (Avr) and can be recognised by corresponding host resistance (R) proteins, creating a strong evolutionary pressure on pathogen Avr effectors that favours their modification/deletion to evade the immune response. Hence, identifying Avr effectors and tracking their allele frequencies in a population is critical for understanding the loss of host recognition. However, the current systems available to confirm Avr effector function, particularly for obligate biotrophic fungi, remain limited and challenging. Here, we explored the utility of the genetically tractable wheat blast pathogen Magnaporthe oryzae pathotype Triticum (MoT) as a suitable heterologous expression system in wheat. Using the recently confirmed wheat stem rust pathogen (Puccina graminis f. sp. tritici) avirulence effector AvrSr50 as a proof-of-concept, we found that delivery of AvrSr50 via MoT could elicit a visible Sr50-dependant cell death phenotype. However, activation of Sr50-mediated cell death correlated with a high transgene copy number and transcript abundance in MoT transformants. This illustrates that MoT can act as an effective heterologous delivery system for fungal effectors from distantly related fungal species, but only when enough transgene copies and/or transcript abundance is achieved.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat

Jensen

Saunders

2023

Sci Rep

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“…The majority of the tools require the assembly of DNA constructs using Escherichia coli based cloning strategies or in vitro assembly by fusion-PCR reactions. 8 For high-throughput experiments, these DNA assembly processes are often bottlenecks in strain construction due to the time requirements, costs, low efficiency of multipart assembly, and need of optimization.…”

Section: Introductionmentioning

confidence: 99%

“…Over the years, the fungal genetic-engineering toolbox has been ever expanding and includes, e.g., episomal AMA1 based plasmids and circular mini chromosomes, collections of synthetic biology based building blocks including markers, promoters, and terminators, etc., and efficient gene-targeting and gene-editing technologies based on strains that are deficient in nonhomologous end-joining (NHEJ) and/or by CRISPR technologies. The majority of the tools require the assembly of DNA constructs using Escherichia coli based cloning strategies or in vitro assembly by fusion-PCR reactions . For high-throughput experiments, these DNA assembly processes are often bottlenecks in strain construction due to the time requirements, costs, low efficiency of multipart assembly, and need of optimization.…”

Section: Introductionmentioning

confidence: 99%

A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering

et al. 2022

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Efficient homologous recombination in baker’s yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering.

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“…Moreover, there are a number of examples where heterologous expression of a specific pathway in A. oryzae has exceeded the titer of the natural product reported in the native producer [18,19]. Advantages of fungi over other heterologous hosts are: the use of monocistronic operons, meaning separate genes can be individually controlled; numerous cellular compartments, where selective reactions may be contained to overcome toxicity; and tailoring enzymes that catalyze diverse and unique chemical modifications not accessible to other organisms [20]. However, A. oryzae has not yet been explored as a host for the production of bacterial polyketides.…”

Section: Introductionmentioning

confidence: 99%

Engineering Aspergillus oryzae for the Heterologous Expression of a Bacterial Modular Polyketide Synthase

Feng

Hauser

Cox

et al. 2021

JoF

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Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products.

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Filamentous Fungi as Hosts for Heterologous Production of Proteins and Secondary Metabolites in the Post-Genomic Era

Cited by 3 publications

References 266 publications

Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat

Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat

A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering

Engineering Aspergillus oryzae for the Heterologous Expression of a Bacterial Modular Polyketide Synthase

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