Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi.

Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

Structures and Biosynthesis of Corvol Ethers--Sesquiterpenes from the Actinomycete Kitasatospora setae.

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one- and two-dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.

An improved technique for the rapid chemical characterisation of bacterial terpene cyclases.

A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2µ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases.

A toolkit for heterologous expression of metabolic pathways in Aspergillus oryzae.

Much has been learned about the activities of the key enzymes involved in eukaryotic natural product synthesis by isolating the relevant genes and expressing them in a suitable foreign host. Aspergillus oryzae has proved to be an amenable host for the functional analysis of megasynthases from other fungi, but secondary metabolites are often the products of suites of enzymes, and understanding their biosynthesis requires simultaneous expression of several genes. This chapter describes the development and use of a molecular toolkit that facilitates the rapid assembly of the genes constituting whole biosynthetic pathways in one or a few multiple gene expression plasmids designed to provide high-level expression in A. oryzae. Conventional DNA manipulation by restriction/ligation is replaced by homologous recombination in yeast and Gateway®-mediated site-specific recombination in vitro. The toolkit comprises an assembly vector used for the simple construction and modification of large genes from overlapping DNA fragments and three multigene expression vectors. Insertion of three tailoring enzyme genes by homologous recombination and one megasynthase gene by Gateway® transfer into each of the expression vectors can be achieved in a little more than 1 week, and alternative selection markers in the expression plasmids permit cotransformation of A. oryzae with up to 12 genes.