Endophytes as alternative paclitaxel sources : chemistry and genetics of Taxomyces andreanae and the endophytic flora of Wollemia nobilis

Whether suffering a pathogenic attack, basking in symbiotic comfort, or seemingly symptomless, plants constantly participate in molecular interplay with various classes of microbial organisms. One of the means of interorganismal communication in this dynamic continuum are secondary metabolites. The chemical diversity bearing pharmaceutical potential thus implied reaches beyond the plant kingdom and offers an expended view promising to transform glimpses of reductionist research of the past years to snapshots of an exuberant world of systems biology. Endophytes seem to fit perfectly into this natural ‘warehouse’, only a small part of which we have been able to tap into so far. The introductory section of the hereby presented thesis (chapter 2) provides an elaborate overview on the current state of knowledge about endophytic organisms – microbes colonizing internal tissues of all plant species, creating a huge biodiversity with yet unknown novel natural products presumed to push forward the frontiers of drug discovery (Staniek et al., 2008). Paclitaxel, the world’s first billion dollar anticancer blockbuster, was primarily obtained from Taxus brevifolia. While the search for alternative sources of the powerful antineoplastic agent brought an array of reports on paclitaxel producing endophytes, causing quite a controversy over the past two decades, the world’s market still relies on yew-derived supply of the valuable diterpene.

Endophytes:Exploiting biodiversity for the improvement of natural product-based drug discovery

Endophytes, microorganisms that colonize internal tissues of all plant species, create a huge biodiversity with yet unknown novel natural products, presumed to push forward the frontiers of drug discovery. Next to the clinically acknowledged antineoplastic agent, paclitaxel, endophyte research has yielded potential drug lead compounds with antibacterial, antiviral, antioxidant, insulin mimetic, anti-neurodegenerative and immunosuppressant properties. Furthermore, while being implicated in livestock neurotoxicosis, some endophyte-produced alkaloids have been shown to display insecticidal activity. The endophyte-host relationship is postulated to be a 'balanced antagonism'. Moreover, the plausibility of horizontal gene transfer (HGT) hypothesis is taken into account. Knowledge of the genetic background of endophytic natural product biosynthesis is discussed on the basis of loline alkaloids, ergopeptines, lolitrems and maytansinoids. The current dynamic progress in genomics will contribute to a better understanding of endophytic microbes and to further exploiting them as a source of pharmaceutically relevant compounds

Taxomyces andreanae: A Presumed Paclitaxel Producer Demystified?

The 1990s brought an abundance of reports on paclitaxel-producing endophytes, initially heralded as a discovery having tremendous implications for cancer therapy. As the vision of large-scale fermentation tanks producing vast quantities of relatively inexpensive paclitaxel and novel taxanes has faded and has been replaced by controversial silence, we carried out an in-depth investigation of Taxomyces andreanae - the very first Presumed endophytic synthesizer of the diterpenoid. On one hand, metabolic profiling by means of chromatographic, spectroscopic and immunoenzymatic techniques predominant in literature was taken LIP. On the other, the experimental procedure was brought to an alternative, previously unattempted level aiming at revealing the genetic background of paclitaxel biosynthesis in the endophyte. The profound PCR-based screening for taxadiene synthase (txs) - a gene unique to the formation of the primary taxane-skeleton, as well as phenylpropanoyl transferase (bapt) encoding for the catalyst of the final acylation of the core structure rendering the ultimate efficacy of the drug, confirmed the molecular blueprint for paclitaxel biosynthesis to be an inherent genetic trait of the endophyte. However, as the thorough metabolic analysis of Taxomyces andreanae commercial isolate brought no confirmation of endophytic paclitaxel production even after considerable up-scaling endeavors, we postulate that proclaiming the strain "a fungus factory for Taxol" might have been premature

Essential oil constituents derived from different organs of a relictual conifer Wollemia nobilis

The chemical composition of the essential oil of leaves (0.9%, w/v) and twigs (0.33%, w/v) of Wollemia nobilis (Araucariaceae) - a remnant species thought to have been extinct for 65 million years - was investigated by GC/MS. The main constituents of both leaf- and twig-derived oil samples were 16-kaurene (61.8% and 38.2%, respectively) and germacrene D (9.9% and 22%). The principal difference was a considerably more pronounced sesquiterpene presence in the twig-oil, amounting to 33.5%, than in its folial counterpart (23.4%). On the contrary, while remaining the dominant group in both oil samples under investigation, diterpenoids were relatively more abundant in leaf-derived oil constituting 65.3%, versus 41.7% detected in twigs. To our knowledge, this is the first report dealing with the essential oil composition of Wollemi pine twigs, as opposed to the leaf-derived volatiles. (C) 2009 Elsevier Ltd. All rights reserved

Combinatorial biosynthesis of small molecules in plants: Engineering strategies and tools.

Biosynthetic capacity of plants, rooted in a near inexhaustible supply of photosynthetic energy and founded upon an intricate matrix of metabolic networks, makes them versatile chemists producing myriad specialized compounds. Along with tremendous success in elucidation of several plant biosynthetic routes, their reestablishment in heterologous hosts has been a hallmark of recent bioengineering endeavors. However, current efforts in the field are, in the main, aimed at grafting the pathways to fermentable recipient organisms, like bacteria or yeast. Conversely, while harboring orthologous metabolic trails, select plant species now emerge as viable vehicles for mobilization and engineering of complex biosynthetic pathways. Their distinctive features, like intricate cell compartmentalization and formation of specialized production and storage structures on tissue and organ level, make plants an especially promising chassis for the manufacture of considerable amounts of high-value natural small molecules. Inspired by the fundamental tenets of synthetic biology, capitalizing on the versatility of the transient plant transformation system, and drawing on the unique compartmentation of plant cells, we explore combinatorial approaches affording production of natural and new-to-nature, bespoke chemicals of potential importance. Here, we focus on the transient engineering of P450 monooxygenases, alone or in concert with other orthogonal catalysts, like tryptophan halogenases

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia.

MAIN CONCLUSION Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites

A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors.

Plastid transformation is a powerful tool for basic research, but also for the generation of stable genetically engineered plants producing recombinant proteins at high levels or for metabolic engineering purposes. However, due to the genetic makeup of plastids and the distinct features of the transformation process, vector design, and the use of specific genetic elements, a large set of basic transformation vectors is required, making cloning a tedious and time-consuming effort. Here, we describe the adoption of standardized modular cloning (GoldenBraid) to the design and assembly of the full spectrum of plastid transformation vectors. The modular design of genetic elements allows straightforward and time-efficient build-up of transcriptional units as well as construction of vectors targeting any homologous recombination site of choice. In a three-level assembly process, we established a vector fostering gene expression and formation of griffithsin, a potential viral entry inhibitor and HIV prophylactic, in the plastids of tobacco. Successful transformation as well as transcript and protein production could be shown. In concert with the aforesaid endeavor, a set of modules facilitating plastid transformation was generated, thus augmenting the GoldenBraid toolbox. In short, the work presented in this study enables efficient application of synthetic biology methods to plastid transformation in plants

Engineering of new-to-nature halogenated indigo precursors in plants

Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products. Indican (indolyl-β-d-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 ±0.089mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle. Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants

Schematic representation of the modular build-up and the overall GB-based cloning strategy of expression vectors.

<p><b>A)</b> Generic structure of a plastid transformation vector. Magnified details show the modular build-up of a transcriptional unit and how it can be assembled from a set of standardized parts. Numbers within the boxes represent part identity and compatibility. Prom, promoter; Term, terminator; LTR, left targeting region; RTR, right targeting region; UTR, untranslated region; SM, selection marker; GOI, gene of interest; CDS, coding sequence. <b>B)</b> Schematic representation of the cloning strategy yielding the expression vector used in this study. In the pool of standardized parts, circles represent pUPD vectors harboring genetic elements (parts). Elliptical structures represent the different α- and Ω-vectors. Two intertwined ellipses on an arrow represent a one-pot restriction/ligation reaction (GB reaction) combining all the relevant parts. Boxes represent the parts and their assembly.</p