Nepeta cataria L. (catnip) can serve as a chassis for the engineering of secondary metabolic pathways.

OBJECTIVE: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences. RESULTS: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 µmol g-1 s-1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 µmol g-1 s-1 and 18 ± 4 µmol g-1 s-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides. CONCLUSION: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

Transcriptome-informed identification and characterization of Planococcus citri cis- and trans-isoprenyl diphosphate synthase genes.

Insect physiology and reproduction depend on several terpenoid compounds, whose biosynthesis is mainly unknown. One enigmatic group of insect monoterpenoids are mealybug sex pheromones, presumably resulting from the irregular coupling activity of unidentified isoprenyl diphosphate synthases (IDSs). Here, we performed a comprehensive search for IDS coding sequences of the pest mealybug Planococcus citri. We queried the available genomic and newly generated short- and long-read P. citri transcriptomic data and identified 18 putative IDS genes, whose phylogenetic analysis indicates several gene family expansion events. In vitro testing confirmed regular short-chain coupling activity with five gene products. With the candidate with highest IDS activity, we also detected low amounts of irregular coupling products, and determined amino acid residues important for chain-length preference and irregular coupling activity. This work therefore provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, including the sex pheromone biosynthesis in P. citri.

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity.

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.

Production of the sheep pox virus structural protein SPPV117 in tobacco chloroplasts.

OBJECTIVE: A chloroplast transgenic approach was assessed in order to produce a structural protein SPPV117 of sheep pox virus in Nicotiana tabacum for the future development of a plant-based subunit vaccine against sheep pox. RESULTS: Two DNA constructs containing SPPV117 coding sequence under the control of chloroplast promoter and terminator of psbA gene or rrn promoter and rbcL terminator were designed and inserted into the chloroplast genome by a biolistic method. The transgenic plants were selected via PCR analysis. Northern and Western blot analysis showed expression of the transgene at transcriptional and translational levels, respectively. The recombinant protein accumulated to about 0.3% and 0.9% of total soluble protein in leaves when expressed from psbA and rrn promoter, respectively. Plant-produced SPPV117 protein was purified using metal affinity chromatography and the protein yield was 19.67  ±  1.25 µg g-1 (FW). The serum of a sheep infected with the virus recognised the chloroplast-produced protein indicating that the protein retains its antigenic properties. CONCLUSIONS: These results demonstrate that chloroplasts are a suitable system for the production of a candidate subunit vaccine against sheep pox.

Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4.

Human drug-metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)-epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.

Correction to: Two novel methylesterases from Olea europaea contribute to the catabolism of oleoside-type secoiridoid esters.

Page 5, paragraph 3, line 14, GenBank Accession Number which should read MK234850 instead of MK23485.

Two novel methylesterases from Olea europaea contribute to the catabolism of oleoside-type secoiridoid esters.

MAIN CONCLUSION: Two newly identified phytohormone cleaving esterases from Olea europaea are responsible for the glucosidase-initiated activation of the specialized metabolites ligstroside and oleuropein. Biosynthetic routes leading to the formation of plant natural products are tightly orchestrated enzymatic sequences usually involving numerous specialized catalysts. After their accumulation in plant cells and tissues, otherwise non-reactive compounds can be enzymatically activated, e.g., in response to environmental threats, like pathogen attack. In olive (Olea europaea), secoiridoid-derived phenolics, such as oleuropein or ligstroside, can be converted by glucosidases and as yet unidentified esterases to oleoside aldehydes. These are not only involved in pathogen defense, but also bear considerable promise as pharmaceuticals or neutraceuticals. Making use of the available olive genomic data, we have identified four novel methylesterases that showed significant homology to the polyneuridine aldehyde esterase (PNAE) from Rauvolfia serpentina, an enzyme acting on a distantly related metabolite group (monoterpenoid indole alkaloids, MIAs) also featuring a secoiridoid structural component. The four olive enzymes belong to the α/ß-hydrolase fold family and showed variable in vitro activity against methyl esters of selected plant hormones, namely jasmonic acid (MeJA), indole acetic acid (MeIAA), as well as salicylic acid (MeSA). None of the identified catalysts were directly active against the olive metabolites oleuropein, ligstroside, or oleoside 11-methyl ester. When employed in a sequential reaction with an appropriate glucosidase, however, two were capable of hydrolyzing these specialized compounds yielding reactive dialdehydes. This suggests that the esterases play a pivotal role in the activation of the olive secoiridoid polyphenols. Finally, we show that several of the investigated methylesterases exhibit a concomitant in vitro transesterification capacity-a novel feature, yielding ethyl esters of jasmonic acid (JA) or indole-3-acetic acid (IAA).

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.

Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds.

Employment of transient expression of foreign genes for bioconversion of pharmaceutically valuable low-molecular-weight compounds, including plant secondary metabolites, is an enticing trend still scantily explored in plant biotechnology. In the present work, an efficient protocol for rapid assessment of synthetic and plant-derived metabolites as potential substrates for human P450s (CYP2D6 and CYP3A4) via Agrobacterium-mediated transient expression in Nicotiana benthamiana is put forth. Animal P450s with broad substrate specificity are promising candidates for transformation of diverse metabolites. The efficiency of P450s in heterologous surroundings is not always satisfactory and depends on the availability of an associated electron-transfer enzyme. Plants represent an attractive assortment of prospective hosts for foreign P450s expression. The optimal composition of genetic blocks providing the highest transient expression efficiency is designed, an effective substrate administration scheme is validated, and biological activity of the investigated P450s against loratadine and several indole alkaloids with different molecular scaffold structures is tested. A novel indole alkaloid, 11-hydroxycorynanthine, is isolated from N. benthamiana plants transiently expressing CYP2D6 and supplemented with corynanthine, and its structure was elucidated. The proposed technique might be of value in realization of combinatorial biosynthesis concept comprising the junction of heterologous enzymes and substrates in different metabolic surroundings.

Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.

OBJECTIVE: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. RESULTS: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g FW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g FW-1 activity towards CBCA production). CONCLUSION: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

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.

Current Perspectives on Biotechnological Cannabinoid Production in Plants.

The plant Cannabis sativa contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of Cannabis for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other Cannabis-derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic Cannabis plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on Cannabis cell culture systems, in vitro propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated.

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.

Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

The case for plant-made veterinary immunotherapeutics.

The excessive use of antibiotics in food animal production has contributed to resistance in pathogenic bacteria, thereby triggering regulations and consumer demands to limit their use. Alternatives for disease control are therefore required that are cost-effective and compatible with intensive production. While vaccines are widely used and effective, they are available against a minority of animal diseases, and development of novel vaccines and other immunotherapeutics is therefore needed. Production of such proteins recombinantly in plants can provide products that are effective and safe, can be orally administered with minimal processing, and are easily scalable with a relatively low capital investment. The present report thus advocates the use of plants for producing vaccines and antibodies to protect farm animals from diseases that have thus far been managed with antibiotics; and highlights recent advances in product efficacy, competitiveness, and regulatory approval.

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.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.