Project description:Terpenes synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single active site. We have determined that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD+ yet does not utilise it chemically for a non-oxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.
Project description:Wild yeast and many clinical strains form complex, biofilm colonies, providing protection from desiccation, drugs and other stresses. Few transcriptomic studies have focused on sub-surface invasive “roots” due to the challenges of cell isolation from agar and subpopulation cross-contamination. Here we present, a first transcriptomic analysis via RNA sequencing of root and aerial cells, separated by a novel method. We identified 719 coding genes with upregulated root expression, mainly involved in ribosome structure/biogenesis, biosynthesis and translation and 529 loci/genes with upregulated aerial expression, mainly involved in meiosis/sporulation, stress defense and protein degradation; all indicating that aerial cells are resting and root cells are metabolically active cells. On the basis of 172 root-upregulated and 233 aerial-upregulated non-coding loci detected, anti-regulated gene/lncRNA pairs have been identified that may contribute to negative regulation of gene expression. Importantly, cells showing typical markers of “roots” correspond with cells embedded in extracellular matrix and cells showing typical markers of “aerial” parts with ECM-free cells.
Project description:Vasculogenic mimicry has been generally accepted as a new form of tumor vascularization and regarded as an unfavorable prognostic factor in multiple aggressive malignancies. We previously reported the presence of vasculogenic mimicry in osteosarcoma patients. The mechanistic basis for osteosarcoma VM remains unclear. We used microarrays to detail the global programme of gene expression between 143B cells and HOS cells exposed to Matrigel which showed greatly different vasculogenic mimicry formation potential and identified distinct classes of vasculogenic mimicry-realted genes during this process.
Project description:Phytoplasmas are obligate plant pathogens that include species causing severe developmental abnormalities in diverse plant families. Phytoplasma infections may result in dwarfism, witches’ broom, virescence and phyllody, the conversion of reproductive organs into leaf-like structures. Phyllody is due to the activity of phytoplasma effector proteins, called phyllogens, that interact with the host MADS transcription factor (MTF) network. This interaction was believed to be specific for members of the AP1/SEP/AGL6 superclade of tetrameric MTFs, key regulators of floral meristem identity and organogenesis. Here, we determined the molecular and atomic basis for the interaction between the phyllogen from Onions Yellow phytoplasma (PHYL1OY) and the floral MTFs, APETALA1, SEPALLATA and AGAMOUS using biochemical, structural and genetic experiments. The DNA-binding patterns of homo and heteromeric MTFs with PHYL1OY are shown using electrophoretic mobility shift assays, revealing broader interaction patterns between PHYL1OY and MTFs that depend on highly conserved hydrophobic residues. Point mutations of these residues allowed gain or loss of interaction between MTFs and PHYL1OY. Expressing PHYL1OY under the control of the SEP3 and AP1 promoter in Arabidopsis further demonstrated whorl specific phenotypes, recapitulating the in vitro interaction and DNA-binding patterns. These findings reveal how PHYL1OY targets specific MTFs through structural mimicry, resulting in impaired MTF tetramer formation and homeotic conversion of floral organs due to loss of active MTF tetramers.
Project description:Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product's synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives.
Project description:The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. Strains in which the rep1 gene is inactivated (∆rep1 strain) are affected in aerial hyphae formation. This makes these strains instrumental to assess changes in global gene expression as a consequence of aerial growth. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of >= 2. Twenty-two of these genes are up-regulated and half of them encode small secreted proteins (SSP’s) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode secreted cysteine-rich proteins (SCRP’s). Interestingly, most of the SCRP’s are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that only minor changes occur in the expression profile when U. maydis forms aerial structures. Key words: aerial hypha, repellent, hydrophobin-like protein, Ustilago maydis, SSP, SCRP, fungal pathogenicity.
Project description:Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa (“kratom”), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ~290 µg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.