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.

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-ß-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 ±â€¯0.089 mg/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.

Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes.

Halogenation of natural compounds in planta is rare. Herein, a successful engineering of tryptophan 6-halogenation into the plant context by heterologous expression of the Streptomyces toxytricini Stth gene and localization of its enzymatic product in various tobacco cell compartments is described. When co-expressed with the flavin reductase rebF from Lechevalieria aerocolonigenes, Stth efficiently produced chlorinated tryptophan in the cytosol. Further, supplementation of KBr yielded the brominated metabolite. More strikingly, targeting of the protein to the chloroplasts enabled effective halogenation of tryptophan even in absence of the partner reductase, providing crucial evidence for sufficient, organelle-specific supply of the FADH2 cofactor to drive halogen integration. Incorporation of an alternative enzyme, the 7-halogenase RebH from L. aerocolonigenes, into the metabolic set-up resulted in the formation of 6,7-dichlorotryptophan. Finally, expression of tryptophan decarboxylase (tdc) in concert with stth led to the generation of 6-chlorotryptamine, a new-to-nature precursor of monoterpenoid indole alkaloids. In sum, the report highlights the tremendous application potential of plants as a unique chassis for the engineering of rare and valuable halogenated natural products, with chloroplasts as the cache of reduction equivalents driving metabolic reactions.

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.

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours.

Insight into the structure and inhibition mechanism of O-ß-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-ß-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 Å distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.

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.

Natural products ­ learning chemistry from plants.

Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large-scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant-derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.

Natural products - modifying metabolite pathways in plants.

The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.

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 up. 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.