Molecular Characterization and Event-Specific Real-Time PCR Detection of Two Dissimilar Groups of Genetically Modified Petunia (Petunia x hybrida) Sold on the Market.

Petunia plants with unusual orange flowers were noticed on the European market and confirmed to be genetically modified (GM) by the Finnish authorities in spring 2017. Later in 2017, inspections and controls performed by several official laboratories of national competent authorities in the European Union detected several GM petunia varieties with orange flowers, but also another group of unusually colored flowers. In the latter group, a so far undetected gene coding for a flavonoid 3'5' hydroxylase (F3'5'H) responsible for the purple color was identified by German and Dutch authorities, suggesting that the petunias found on the markets contain different genetic constructs. Here, a strategy is described for the identification of GM petunia varieties. It is based on an initial GMO screening for known elements using (real-time) PCR and subsequent identification of the insertion sites by a gene walking-like approach called ALF (amplification of linearly-enriched fragments) in combination with Sanger and MinION sequencing. The results indicate that the positively identified GM petunias can be traced back to two dissimilar GM events used for breeding of the different varieties. The test results also confirm that the transgenic petunia event RL01-17 used in the first German field trial in 1991 is not the origin of the GM petunias sold on the market. On basis of the obtained sequence data, event-specific real-time PCR confirmatory methods were developed and validated. These methods are applicable for the rapid detection and identification of GM petunias in routine analysis. In addition, a decision support system was developed for revealing the most likely origin of the GM petunia.

Dirigent proteins: molecular characteristics and potential biotechnological applications.

Dirigent proteins (DIRs) are thought to play important roles in plant secondary metabolism. They lack catalytic activity but direct the outcome of bimolecular coupling reactions toward regio- and stereospecific product formation. Functionally described DIRs confer specificity to the oxidative coupling of coniferyl alcohol resulting in the preferred production of either (+)- or (-)-pinoresinol, which are the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIRs are extracellular glycoproteins with high ß-strand content and have been found in all land plants investigated so far. Their ability to capture and orientate radicals represents a unique naturally evolved concept for the control of radical dimerization reactions. Although oxidative coupling is commonly used in biological systems, its wider application in chemical synthesis is often limited by insufficient selectivity. This minireview gives an overview of functionally described DIRs and their molecular characteristics and wants to inspire further research for their use in biotechnological applications.

Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function.

Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (-)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg L⁻¹ of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the ß-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.

Identification and characterization of a kunzeaol synthase from Thapsia garganica: implications for the biosynthesis of the pharmaceutical thapsigargin.

Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6ß-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 µM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.

Enzymatic synthesis of valerena-4,7(11)-diene by a unique sesquiterpene synthase from the valerian plant (Valeriana officinalis).

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 µm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.

A model of dirigent proteins derived from structural and functional similarities with allene oxide cyclase and lipocalins.

Dirigent proteins impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)- or (-)-pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all-beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N-linked glycans at the solvent-exposed protein surface. The model shows AtDIR6 as an eight-stranded antiparallel ß-barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand-binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product.

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities.

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.