Dataset Information

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

ABSTRACT: Background:Sequencing 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. Results:We 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 ?-carotene from the carotenoid gene cluster of Fusarium fujikuroi. Conclusion:Utilizing 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.

SUBMITTER: Wiemann P

PROVIDER: S-EPMC5851313 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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CoIN: co-inducible nitrate expression system for secondary metabolites in <i>Aspergillus nidulans</i>.

Wiemann Philipp P Soukup Alexandra A AA Folz Jacob S JS Wang Pin-Mei PM Noack Andreas A Keller Nancy P NP

Fungal biology and biotechnology 20180313

<h4>Background</h4>Sequencing 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 gene ...[more]

PMID: 29564145

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Project description:The filamentous fungus Aspergillus nidulans has been a primary workhorse used to understand fungal genetics. Much of this work has focused on elucidating the genetics of biosynthetic gene clusters (BGCs) and the secondary metabolites (SMs) they produce. SMs are both niche defining in fungi and of great economic importance to humans. Despite the focus on A. nidulans, very little is known about the natural diversity in secondary metabolism within this species. We determined the BGC content and looked for evolutionary patterns in BGCs from whole-genome sequences of two clinical isolates and the A4 reference genome of A. nidulans Differences in BGC content were used to explain SM profiles determined using liquid chromatography-high-resolution mass spectrometry. We found that in addition to genetic variation of BGCs contained by all isolates, nine BGCs varied by presence/absence. We discovered the viridicatumtoxin BGC in A. nidulans and suggest that this BGC has undergone a horizontal gene transfer from the Aspergillus section Nigri lineage into Penicillium sometime after the sections Nigri and Nidulantes diverged. We identified the production of viridicatumtoxin and several other compounds previously not known to be produced by A. nidulans One isolate showed a lack of sterigmatocystin production even though it contained an apparently intact sterigmatocystin BGC, raising questions about other genes and processes known to regulate this BGC. Altogether, our work uncovers a large degree of intraspecies diversity in BGC and SM production in this genetic model species and offers new avenues to understand the evolution and regulation of secondary metabolism.IMPORTANCE Much of what we know about the genetics underlying secondary metabolite (SM) production and the function of SMs in the model fungus Aspergillus nidulans comes from a single reference genome. A growing body of research indicates the importance of biosynthetic gene cluster (BGC) and SM diversity within a species. However, there is no information about the natural diversity of secondary metabolism in A. nidulans We discovered six novel clusters that contribute to the considerable variation in both BGC content and SM production within A. nidulans We characterize a diverse set of mutations and emphasize how findings of single nucleotide polymorphisms (SNPs), deletions, and differences in evolutionary history encompass much of the variation observed in nonmodel systems. Our results emphasize that A. nidulans may also be a strong model to use within-species diversity to elucidate regulatory cross talk, fungal ecology, and drug discovery systems.

Project description:1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.

Dataset Information

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

Publications

CoIN: co-inducible nitrate expression system for secondary metabolites in <i>Aspergillus nidulans</i>.

Similar Datasets

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