A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM.

A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags.

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.

Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform.

Epidemiological studies have shown that consumption of cruciferous vegetables, such as, broccoli and cabbages, is associated with a reduced risk of developing cancer. This phenomenon has been attributed to specific glucosinolates among the ~30 glucosinolates that are typically present as natural products characteristic of cruciferous plants. Accordingly, there has been a strong interest to produce these compounds in microbial cell factories as it will allow production of selected beneficial glucosinolates.

Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid.

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P.

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi.

Background: Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides.

Adaptive evolution of drug targets in producer and non-producer organisms.

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance.

A genome-wide polyketide synthase deletion library uncovers novel genetic links to polyketides and meroterpenoids in Aspergillus nidulans.

Fungi possess an advanced secondary metabolism that is regulated and coordinated in a complex manner depending on environmental challenges. To understand this complexity, a holistic approach is necessary. We initiated such an analysis in the important model fungus Aspergillus nidulans by systematically deleting all 32 individual genes encoding polyketide synthases.

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications.

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.

Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum.

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum.

The complex genetic architecture of the metabolome.

Discovering links between the genotype of an organism and its metabolite levels can increase our understanding of metabolism, its controls, and the indirect effects of metabolism on other quantitative traits. Recent technological advances in both DNA sequencing and metabolite profiling allow the use of broad-spectrum, untargeted metabolite profiling to generate phenotypic data for genome-wide association studies that investigate quantitative genetic control of metabolism within species. We conducted a genome-wide association study of natural variation in plant metabolism using the results of untargeted metabolite analyses performed on a collection of wild Arabidopsis thaliana accessions.

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis.

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance.

Controlled indole-3-acetaldoxime production through ethanol-induced expression of CYP79B2.

Indole-3-acetaldoxime (IAOx) is a key branching point between primary and secondary metabolism. IAOx serves as an intermediate in the biosynthesis of indole glucosinolates (I-GLSs), camalexin and the plant hormone indole-3-acetic acid (IAA). The cytochrome P450s CYP79B2 and CYP79B3 catalyze the conversion of tryptophan to IAOx.

A novel 2-oxoacid-dependent dioxygenase involved in the formation of the goiterogenic 2-hydroxybut-3-enyl glucosinolate and generalist insect resistance in Arabidopsis,

Glucosinolates are secondary metabolites found almost exclusively in the order Brassicales. They are synthesized from a variety of amino acids and can have numerous side chain modifications that control biological function. We investigated the biosynthesis of 2-hydroxybut-3-enyl glucosinolate, which has biological activities including toxicity to Caenorhabditis elegans, inhibition of seed germination, induction of goiter disease in mammals, and production of bitter flavors in Brassica vegetable crops.

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain.

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain.

Subclade of flavin-monooxygenases involved in aliphatic glucosinolate biosynthesis.

Glucosinolates (GSLs) are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. We previously identified the flavin-monooxygenase FMO(GS-OX1) as an enzyme in the biosynthesis of aliphatic GSLs in Arabidopsis (Arabidopsis thaliana) that catalyzes the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Here, we report the fine mapping of a quantitative trait locus for the S-oxygenating activity in Arabidopsis.

Biochemical networks and epistasis shape the Arabidopsis thaliana metabolome.

Genomic approaches have accelerated the study of the quantitative genetics that underlie phenotypic variation. These approaches associate genome-scale analyses such as transcript profiling with targeted phenotypes such as measurements of specific metabolites. Additionally, these approaches can help identify uncharacterized networks or pathways.

Identifying the molecular basis of QTLs: eQTLs add a new dimension.

Natural genetic variation within plant species is at the core of plant science ranging from agriculture to evolution. Whereas much progress has been made in mapping quantitative trait loci (QTLs) controlling this natural variation, the elucidation of the underlying molecular mechanisms has remained a bottleneck. Recent systems biology tools have significantly shortened the time required to proceed from a mapped locus to testing of candidate genes.

A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates.

Background: Glucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools.

Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways.

Phenotypic variation between individuals of a species is often under quantitative genetic control. Genomic analysis of gene expression polymorphisms between individuals is rapidly gaining popularity as a way to query the underlying mechanistic causes of variation between individuals. However, there is little direct evidence of a linkage between global gene expression polymorphisms and phenotypic consequences.

Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis.

The cancer-preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4-methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer-preventive properties, whereas the isothiocyanate of 4-methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin-monooxygenase (FMO) enzyme, FMO(GS-OX1), which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs.

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments.

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy.

New insight into the biosynthesis and regulation of indole compounds in Arabidopsis thaliana.

In spite of their silent and sessile life, plants are dynamic organisms that have developed advanced defence strategies in their adaptation to the pressure of herbivores and pathogens. Natural plant products play an important role as chemical weapons in this warfare. Characteristic of cruciferous plants is the synthesis of nitrogen- and sulphur-rich compounds, such as glucosinolates (Mikkelsen et al.