Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

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 β-carotene from the carotenoid gene cluster of Fusarium fujikuroi.
CONCLUSIONS: 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.

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.