Diterpene-producing unicellular organism

The present invention is directed to a unicellular organism system, such as a yeast, for producing geranylgeranyl pyrophosphate and a diterpene in vivo. The yeast cell preferably comprises an inducible nucleic acid sequence encoding geranylgeranyl pyrophosphate synthase, an inducible nucleic acid sequence encoding a soluble form of HMG-CoA reductase, a nucleic acid sequence of an allele that confers an increase in sterol metabolic flux and, in the diterpene-producing cell, a diterpene synthase

Nerolidol- terpene- and terpene deriviative synthesis

According to one embodiment, the description relates to a method of nerolidol production. The method includes culturing a yeast strain lacking functional squalene synthase and overproducing HMG CoA reductase in synthetic medium lacking uracil and producing nerolidol. The pH of the medium may be adjusted to an acidic level to further increase nerolidol production. Other chemicals may also be produced by this method. The nerolidol or other chemicals may be removed from the yeast or medium or both. The medium may additionally contain a polyaromatic resin, which may adsorb nerolidol or other chemicals

Astertarone A Synthase from Chinese Cabbage Does Not Produce the C4-Epimer: Mechanistic Insights

The Brassica rapa oxidosqualene cyclase Bra032185 makes 60% astertarone A (<b>1</b>) and 20 minor triterpenes (0.1–11%). Mechanistic analysis indicates the absence of an enol intermediate to generate the 4<i>S</i> epimer of <b>1</b>, unless workup involves saponification. Bra032185 and its closest Arabidopsis thaliana homologue diverged markedly in product profiles over a short evolutionary distance, while developing opposite C18 configurations. Active-site residue comparison of Bra032185 with friedelin and shionone synthases suggests convergent evolution to 3-ketotriterpenes

Are Isoursenol and γ-Amyrin Rare Triterpenes in Nature or Simply Overlooked by Usual Analytical Methods?

Among pentacyclic triterpenes commonly found in plants, γ-amyrin and isoursenol are seldom reported and considered rare in nature. It was hypothesized that these triterpenes are instead routinely overlooked due to inadequate spectral characterization. γ-Amyrin was prepared by HCOOH isomerization of α-amyrin, and isoursenol was isolated from products of a heterologously expressed oxidosqualene cyclase. With precise NMR and GC-MS data, a metabolomics strategy was used to identify isoursenol and γ-amyrin in a wide range of plants

New insights regarding the cyclization pathway for sterol biosynthesis from (S)-2,3-oxidosqualene

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Cholesterol Import by Aspergillus fumigatus and Its Influence on Antifungal Potency of Sterol Biosynthesis Inhibitors

High mortality rates from invasive aspergillosis in immunocompromised patients are prompting research toward improved antifungal therapy and better understanding of fungal physiology. Herein we show that Aspergillus fumigatus, the major pathogen in aspergillosis, imports exogenous cholesterol under aerobic conditions and thus compromises the antifungal potency of sterol biosynthesis inhibitors. Adding serum to RPMI medium led to enhanced growth of A. fumigatus and extensive import of cholesterol, most of which was stored as ester. Growth enhancement and sterol import also occurred when the medium was supplemented with purified cholesterol instead of serum. Cells cultured in RPMI medium with the sterol biosynthesis inhibitors itraconazole or voriconazole showed retarded growth, a dose-dependent decrease in ergosterol levels, and accumulation of aberrant sterol intermediates. Adding serum or cholesterol to the medium partially rescued the cells from the drug-induced growth inhibition. We conclude that cholesterol import attenuates the potency of sterol biosynthesis inhibitors, perhaps in part by providing a substitute for membrane ergosterol. Our findings establish significant differences in sterol homeostasis between filamentous fungi and yeast. These differences indicate the potential value of screening aspergillosis antifungal agents in serum or other cholesterol-containing medium. Our results also suggest an explanation for the antagonism between itraconazole and amphotericin B, the potential use of Aspergillus as a model for sterol trafficking, and new insights for antifungal drug development