Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation.

De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions.

Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis.

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts.

Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning.

Simple and reliable DNA editing by uracil excision (a.k.a.

Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form.

Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices.

Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.

Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility.

Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing.

Aspergillus nidulans synthesize insect juvenile hormones upon expression of a heterologous regulatory protein and in response to grazing by Drosophila melanogaster larvae.

Secondary metabolites are known to serve a wide range of specialized functions including communication, developmental control and defense. Genome sequencing of several fungal model species revealed that the majority of predicted secondary metabolite related genes are silent in laboratory strains, indicating that fungal secondary metabolites remain an underexplored resource of bioactive molecules. In this study, we combine heterologous expression of regulatory proteins in Aspergillus nidulans with systematic variation of growth conditions and observe induced synthesis of insect juvenile hormone-III and methyl farnesoate.

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown.

Genetics of Polyketide Metabolism in Aspergillus nidulans.

Secondary metabolites are small molecules that show large structural diversity and a broad range of bioactivities. Some metabolites are attractive as drugs or pigments while others act as harmful mycotoxins. Filamentous fungi have the capacity to produce a wide array of secondary metabolites including polyketides.

Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case.

Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems.

(+)-Geodin from Aspergillus terreus.

The fungal metabolite (+)-geodin [systematic name: (2R)-methyl 5,7-dichloro-4-hydroxy-6'-methoxy-6-methyl-3,4'-dioxospiro[benzofuran-2,1'-cyclohexa-2',5'-diene]-2'-carboxylate], C(17)H(12)Cl(2)O(7), was isolated from Aspergillus terreus. The crystal structure contains two independent molecules in the asymmetric unit. Molecules denoted 1 interact through O-H.

Glucosinolate engineering identifies a gamma-glutamyl peptidase.

Consumption of cruciferous vegetables is associated with reduced risk of developing cancer, a phenomenon attributed to glucosinolates, which are characteristic of these vegetables. We report production of the bioactive benzylglucosinolate in the noncruciferous plant Nicotiana benthamiana through metabolic engineering. The study includes identification of gamma-glutamyl peptidase 1 (GGP1), which substantially increased glucosinolate production by metabolizing an accumulating glutathione conjugate, an activity not previously described for glucosinolate biosynthesis or for proteins containing glutamine amidotransferase domains.

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5' end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3' overhangs designed to specifically complement each other.