CoIN: co-inducible nitrate expression system for secondary metabolites in Aspergillus nidulans

Abstract

BackgroundSequencing of fungal species has demonstrated the existence of thousands of putative secondary metabolite gene clusters, the majority of them harboring a unique set of genes thought to participate in production of distinct small molecules. Despite the ready identification of key enzymes and potential cluster genes by bioinformatics techniques in sequenced genomes, the expression and identification of fungal secondary metabolites in the native host is often hampered as the genes might not be expressed under laboratory conditions and the species might not be amenable to genetic manipulation. To overcome these restrictions, we developed an inducible expression system in the genetic model Aspergillus nidulans.ResultsWe genetically engineered a strain of A. nidulans devoid of producing eight of the most abundant endogenous secondary metabolites to express the sterigmatocystin Zn(II)2Cys6 transcription factor-encoding gene aflR and its cofactor aflS under control of the nitrate inducible niiA/niaD promoter. Furthermore, we identified a subset of promoters from the sterigmatocystin gene cluster that are under nitrate-inducible AflR/S control in our production strain in order to yield coordinated expression without the risks from reusing a single inducible promoter. As proof of concept, we used this system to produce β-carotene from the carotenoid gene cluster of Fusarium fujikuroi.ConclusionUtilizing one-step yeast recombinational cloning, we developed an inducible expression system in the genetic model A. nidulans and show that it can be successfully used to produce commercially valuable metabolites.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0049-2) contains supplementary material, which is available to authorized users.

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB

“…The elements of the selfexcising β-rec/six blaster cassette are shown in Supplementary Figure S1A. The ku70 knockout cassette was constructed using yeast recombination cloning (Wiemann et al, 2018). The three fragments (5 flank Ku70-hygromycin resistance cassette and 3 flank Ku70) were assembled into the pYHC-WA-pyrG plasmid.…”

Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense

“…Wiemann and co-workers 21 used an inducible niiA/niaD promoter sequence to drive the expression of sterigmatocystin Zn(II)2Cys6 transcription factor-encoding gene aflR and its cofactor aflS. These two factors regulate the expression of Aspergillus nidulans endogenous secondary metabolites genes to produce sterigmatocystin.…”

“…This intergenic region behaves as a switch; 'on' state in presence of nitrate and 'off ' in presence of ammonium and glutamine. This nitrate-inducible promoter was used to express the transcription factor AflR and its cofactor AflS that were responsible for activating eight sterigmatocystin (precursor for aflatoxin B1) promoters for sterigmatocystin production in Aspergillus nidulans 21 . Further, this genetic circuit was used to produce β-carotene in Aspergillus nidulans from the carotenoid gene cluster of Fusarium fujikuroi 21 .…”

“…This nitrate-inducible promoter was used to express the transcription factor AflR and its cofactor AflS that were responsible for activating eight sterigmatocystin (precursor for aflatoxin B1) promoters for sterigmatocystin production in Aspergillus nidulans 21 . Further, this genetic circuit was used to produce β-carotene in Aspergillus nidulans from the carotenoid gene cluster of Fusarium fujikuroi 21 . Moreover, these BDPs are regulated by activators, transcription factors and enhancers that facilitate binding of the protein complexes driving expression of the gene pair.…”

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

“…They include promoter replacements in cis, overexpression of cluster-specific or global activators in trans, deletion of global regulators, interference with chromatin-based silencing or trans-expression of whole BGCs in a heterologous host and approaches based on the variation of cultivation conditions (i.e. "OSMAC") (Bode et al, 2002;Brakhage and Schroeckh, 2011;Chiang et al, 2010;Clevenger et al, 2017;Connolly et al, 2013;Gacek-Matthews et al, 2016;Gerke et al, 2012;Gerke and Braus, 2014;Hansen et al, 2015;Keller, 2019;Lyu et al, 2020;Niehaus et al, 2016b;Strauss and Reyes-Dominguez, 2011;Studt et al, 2016;Westphal et al, 2019;Wiemann et al, 2013;Wiemann et al, 2018). Promoter exchanges seem only feasible with small predicted clusters or clusters that harbour a clear candidate of BGC-specific transcription factor (TF) that can be overexpressed and converted to an active state.…”