Publications by authors named "Katarina Cankar"

33 Publications

Physiological Response of to Indole.

Microorganisms 2020 Dec 8;8(12). Epub 2020 Dec 8.

Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, 33615 Bielefeld, Germany.

The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase. that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe to Fe in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of in a deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.
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http://dx.doi.org/10.3390/microorganisms8121945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764795PMC
December 2020

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Arch Biochem Biophys 2020 11 26;695:108647. Epub 2020 Oct 26.

Bioscience, Wageningen Plant Research, Netherlands. Electronic address:

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.
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http://dx.doi.org/10.1016/j.abb.2020.108647DOI Listing
November 2020

Fermentative -Methylanthranilate Production by Engineered .

Microorganisms 2020 Jun 8;8(6). Epub 2020 Jun 8.

Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, 33615 Bielefeld, Germany.

The -functionalized amino acid -methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid -isopropyl -methylanthranilate, the flavor compound -methyl--methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to -alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium are operated industrially at the million tons per annum scale. Fermentative processes using for -alkylated amino acids based on an imine reductase have been developed, while -alkylation of the aromatic amino acid anthranilate with -adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene encoding anthranilate -methyltransferase from resulted in production of -methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene improved -methylanthranilate production. In a test bioreactor culture, the metabolically engineered C1* strain produced NMA to a final titer of 0.5 g·L with a volumetric productivity of 0.01 g·L·h and a yield of 4.8 mg·g glucose.
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http://dx.doi.org/10.3390/microorganisms8060866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356990PMC
June 2020

Silencing of germacrene A synthase genes reduces guaianolide oxalate content in L.

GM Crops Food 2020 31;11(1):54-66. Epub 2019 Oct 31.

Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, Belgrade, Republic of Serbia.

Chicory ( L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by ) and GAS short (encoded by ). AmiRNA silencing was used to obtain plants with reduced gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.
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http://dx.doi.org/10.1080/21645698.2019.1681868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064209PMC
February 2020

Identification of the Bisabolol Synthase in the Endangered Candeia Tree ( (DC) McLeisch).

Front Plant Sci 2018 19;9:1340. Epub 2018 Sep 19.

Wageningen Plant Research, Wageningen, Netherlands.

Candeia ( (DC) McLeisch, Asteraceae) is a Brazilian tree, mainly occurring in the cerrado areas. From ethnobotanical information its essential oil is known to have wound healing and nociceptive properties. These properties are ascribed to result from a sesquiterpene alcohol, (-)-α-bisabolol, which is present at high concentrations in this oil. Bisabolol is highly valued by the cosmetic industry because of its antibacterial, anti-inflammatory, skin-smoothing and wound healing properties. Over the past decades, Candeia timber has been collected at large scale for bisabolol extraction from wild reserves and the species is thereby at risk of extinction. To support the development of breeding and nursing practices that would facilitate sustainable cultivation of Candeia, we identified a terpene synthase gene, EeBOS1, that appears to control biosynthesis (-)-α-bisabolol in the plant. Expression of this gene in showed that EeBOS1 protein is capable of producing (-)-α-bisabolol from farnesyl pyrophosphate . Analysis of gene expression in different tissues from Candeia plants in different life stages showed a high correlation of EeBOS1 expression and accumulation of (-)-α-bisabolol. This work is the first step to unravel the pathway toward (-)-α-bisabolol in Candeia, and in the further study of the control of (-)-α-bisabolol production.
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http://dx.doi.org/10.3389/fpls.2018.01340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158398PMC
September 2018

The effect of isabelin, a sesquiterpene lactone from Ambrosia artemisiifolia on soil microorganisms and human pathogens.

FEMS Microbiol Lett 2018 02;365(4)

Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, PO BOX 50, 6700 AB Wageningen, The Netherlands.

Ambrosia artemisiifolia L. (common ragweed) is an invasive weed, which is well known for the strong allergenic effect of its pollen as well as for its invasiveness and impact in crop fields (e.g. causing yield losses). This species produces a broad range of sesquiterpenoids. In recent years, new bioactive molecules have been discovered in this plant, e.g. isabelin, a sesquiterpene dilactone. The bioactivity of isabelin has been already demonstrated on allergy-related receptors and its inhibitory effect on seeds of various plant species. Isabelin was tested for potential antimicrobial effects by using a selection of soil-borne bacteria and fungi and three human pathogens as model organisms. For the majority of microorganisms tested, no antimicrobial activity of isabelin was observed. However, isabelin revealed strong antimicrobial activity against the Gram-positive soil bacterium Paenibacillus sp. and against the Gram-positive, multidrug-resistant Staphylococcus aureus. The observed inhibitory activity of isabelin can enlighten the importance to study similar compounds for their effect on human pathogens and on soil and rhizosphere microorganisms.
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http://dx.doi.org/10.1093/femsle/fny001DOI Listing
February 2018

Identification of major loci and genomic regions controlling acid and volatile content in tomato fruit: implications for flavor improvement.

New Phytol 2017 Jul 6;215(2):624-641. Epub 2017 Jun 6.

INRA, UR1052, GAFL, 67 Allée des Chênes Domaine Saint Maurice - CS60094, Montfavet Cedex, 84143, France.

Plant metabolites are important to world food security due to their roles in crop yield and nutritional quality. Here we report the metabolic profile of 300 tomato accessions (Solanum lycopersicum and related wild species) by quantifying 60 primary and secondary metabolites, including volatile organic compounds, over a period of 2 yr. Metabolite content and genetic inheritance of metabolites varied broadly, both within and between different genetic groups. Using genotype information gained from 10 000 single nucleotide polymorphism markers, we performed a metabolite genome-wide association mapping (GWAS) study. We identified 79 associations influencing 13 primary and 19 secondary metabolites with large effects at high resolution. Four genome regions were detected, highlighting clusters of associations controlling the variation of several metabolites. Local linkage disequilibrium analysis and allele mining identified possible candidate genes which may modulate the content of metabolites that are of significant importance for human diet and fruit consumption. We precisely characterized two associations involved in fruit acidity and phenylpropanoid volatile production. Taken together, this study reveals complex and distinct metabolite regulation in tomato subspecies and demonstrates that GWAS is a powerful tool for gene-metabolite annotation and identification, pathways elucidation, and further crop improvement.
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http://dx.doi.org/10.1111/nph.14615DOI Listing
July 2017

Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.

Sci Rep 2017 04 13;7(1):862. Epub 2017 Apr 13.

Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, 6700 AB, Wageningen, The Netherlands.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.
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http://dx.doi.org/10.1038/s41598-017-00893-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429845PMC
April 2017

Genome-Wide Identification of BAHD Acyltransferases and Characterization of HQT-like Enzymes Involved in Caffeoylquinic Acid Synthesis in Globe Artichoke.

Front Plant Sci 2016 23;7:1424. Epub 2016 Sep 23.

Department of Agricultural, Forest and Food Sciences, University of Torino Grugliasco, Italy.

Globe artichoke ( L. var. ) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2, and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues. For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing approach with the goal of assessing the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in leaves we also established the sub-cellular localization of these three enzymes.
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http://dx.doi.org/10.3389/fpls.2016.01424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033976PMC
September 2016

Biotechnological production of limonene in microorganisms.

Appl Microbiol Biotechnol 2016 Apr 26;100(7):2927-38. Epub 2016 Feb 26.

Plant Research International, PO Box 16, 6700, AA, Wageningen, The Netherlands.

This mini review describes novel, biotechnology-based, ways of producing the monoterpene limonene. Limonene is applied in relatively highly priced products, such as fragrances, and also has applications with lower value but large production volume, such as biomaterials. Limonene is currently produced as a side product from the citrus juice industry, but the availability and quality are fluctuating and may be insufficient for novel bulk applications. Therefore, complementary microbial production of limonene would be interesting. Since limonene can be derivatized to high-value compounds, microbial platforms also have a great potential beyond just producing limonene. In this review, we discuss the ins and outs of microbial limonene production in comparison with plant-based and chemical production. Achievements and specific challenges for microbial production of limonene are discussed, especially in the light of bulk applications such as biomaterials.
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http://dx.doi.org/10.1007/s00253-016-7337-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786606PMC
April 2016

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas.

BMC Genomics 2015 Dec 24;16:1103. Epub 2015 Dec 24.

Netherlands Institute of Ecology, Department of Microbial Ecology, Droevendaalsesteeg 10, Wageningen, 6708 PB, The Netherlands.

Background: Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities.

Results: In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7% of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities.

Conclusions: Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.
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http://dx.doi.org/10.1186/s12864-015-2289-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690342PMC
December 2015

Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae.

Yeast 2015 Jan 17;32(1):159-71. Epub 2014 Sep 17.

Laboratory of Plant Physiology, Wageningen University, The Netherlands.

Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028 mg/l (+)-limonene and 0.060 mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12 mg/l (+)-limonene and 0.49 mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems.
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http://dx.doi.org/10.1002/yea.3038DOI Listing
January 2015

(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways.

Biotechnol J 2015 Jan 18;10(1):180-9. Epub 2014 Sep 18.

Laboratory of Plant Physiology, Wageningen University and Research Centre, Wageningen, The Netherlands; Plant Research International, Business Unit Bioscience, Wageningen University and Research Centre, Wageningen, The Netherlands.

Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants.
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http://dx.doi.org/10.1002/biot.201400288DOI Listing
January 2015

Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway.

Plant Sci 2014 Jun 13;223:59-68. Epub 2014 Mar 13.

Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands. Electronic address:

Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivated taxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrations of sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recently attracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costunolide is considered the common precursor of the STLs and three enzymes are involved in its biosynthetic pathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolide synthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), in a set of ∼19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta. The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 and CYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates in Nicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes. The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast with the corresponding terpene synthases.
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http://dx.doi.org/10.1016/j.plantsci.2014.03.007DOI Listing
June 2014

Production of (+)-valencene in the mushroom-forming fungus S. commune.

Appl Microbiol Biotechnol 2014 Jun 16;98(11):5059-68. Epub 2014 Feb 16.

Department of Microbiology, Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands,

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.
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http://dx.doi.org/10.1007/s00253-014-5581-2DOI Listing
June 2014

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis).

FEBS Lett 2014 Mar 11;588(6):1001-7. Epub 2014 Feb 11.

Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Electronic address:

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.
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http://dx.doi.org/10.1016/j.febslet.2014.01.061DOI Listing
March 2014

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development.

Plant Biotechnol J 2014 May 16;12(4):492-502. Epub 2014 Jan 16.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, the Netherlands.

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure-function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.
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http://dx.doi.org/10.1111/pbi.12156DOI Listing
May 2014

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

Plant Biotechnol J 2014 Feb 25;12(2):174-82. Epub 2013 Sep 25.

Plant Research International, Wageningen, the Netherlands; Platform Green Synthetic Biology, Delft University of Technology, Delft, the Netherlands.

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential.
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http://dx.doi.org/10.1111/pbi.12124DOI Listing
February 2014

Genetic mapping and characterization of the globe artichoke (+)-germacrene A synthase gene, encoding the first dedicated enzyme for biosynthesis of the bitter sesquiterpene lactone cynaropicrin.

Plant Sci 2012 Jul 23;190:1-8. Epub 2012 Mar 23.

DIVAPRA, Plant Genetics and Breeding, University of Torino, via L. da Vinci 44, 10095 Grugliasco (TO), Italy.

Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative germacrene A synthase (GAS) gene was identified in a set of ~19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway.
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http://dx.doi.org/10.1016/j.plantsci.2012.03.006DOI Listing
July 2012

The identification and interpretation of differences in the transcriptomes of organically and conventionally grown potato tubers.

J Agric Food Chem 2012 Mar 22;60(9):2090-101. Epub 2012 Feb 22.

RIKILT-Institute of Food Safety (Wageningen UR), Wageningen, The Netherlands.

In the European integrated research project SAFEFOODS, one of the aims was to further establish the potential of transcriptomics for the assessment of differences between plant varieties grown under different environmental conditions. Making use of the knowledge of cellular processes and interactions is one of the ways to obtain a better understanding of the differences found with transcriptomics. For the present study the potato genotype Santé was grown under both organic and conventional fertilizer, and each combined with either organic or conventional crop protection, giving four different treatments. Samples were derived from the European project QualityLowInputFood (QLIF). Microarray data were analyzed using different statistical tools (multivariate, principal components analysis (PCA); univariate, analysis of variance (ANOVA)) and with pathway analysis (hypergeometric distribution (HGD) and gene set enrichment analysis (GSEA)). Several biological processes were implicated as a result of the different treatments of the plants. Most obvious were the lipoxygenase pathway, with higher expression in organic fertilizer and lower expression in organic crop protection; the starch synthase pathway, with higher expression in both organic crop protection and fertilizer; and the biotic stress pathway, with higher expression in organic fertilizer. This study confirmed that gene expression profiling in combination with pathway analysis can identify and characterize differences between plants grown under different environmental conditions.
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http://dx.doi.org/10.1021/jf204696wDOI Listing
March 2012

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

PLoS One 2011 15;6(8):e23255. Epub 2011 Aug 15.

Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023255PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156125PMC
February 2012

Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.).

Phytochemistry 2011 Oct 26;72(14-15):1739-50. Epub 2011 May 26.

Plant Breeding and Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran.

Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-β-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur.
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http://dx.doi.org/10.1016/j.phytochem.2011.04.021DOI Listing
October 2011

Nicotiana benthamiana as a production platform for artemisinin precursors.

PLoS One 2010 Dec 3;5(12):e14222. Epub 2010 Dec 3.

Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.

Background: Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment.

Methodology/principal Findings: Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg x kg(-1) fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana.

Conclusion/significance: This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014222PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2997059PMC
December 2010

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene.

FEBS Lett 2011 Jan 26;585(1):178-82. Epub 2010 Nov 26.

Laboratory of Plant Physiology, Wageningen University and Research Centre, Wageningen, The Netherlands.

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.
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http://dx.doi.org/10.1016/j.febslet.2010.11.040DOI Listing
January 2011

PVY(NTN) elicits a diverse gene expression response in different potato genotypes in the first 12 h after inoculation.

Mol Plant Pathol 2009 Mar;10(2):263-75

Department of Plant Physiology and Biotechnology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia.

Host gene expression changes in the early response to potato virus Y(NTN) interaction were compared in two differently sensitive potato cultivars: the resistant cultivar Santé and the sensitive cultivar Igor. Hybridization of potato TIGR cDNA microarrays allowed us to monitor the expression of approximately 10,000 genes simultaneously at 0.5 and 12 h post-inoculation (hpi). Microarray data, analysed by statistics and data mining, were complemented by subtraction library construction and sequence analysis to validate the findings. The expression profiles of the two cultivars were similar and faint at 0.5 hpi, but they differed substantially at 12 hpi. Although, at 0.5 hpi, cv. Santé responded by the differential expression of a greater number of genes, at 12 hpi the number was higher in cv. Igor. The majority of genes in this cultivar were down-regulated at 12 hpi, indicating a host gene shut-off. Suites of genes that exhibited altered transcript abundance in response to the virus were identified, and included genes involved in the processes of photosynthesis, perception, signalling and defence responses. The expression of the considerable number of genes associated with photosynthesis was surprisingly up-regulated as early as 0.5 hpi and down-regulated at 12 hpi in both cultivars. The expression of genes involved in perception and signalling was increased in the sensitive cultivar at 12 hpi. By contrast, a simultaneous strong defence response at the transcriptional level was evident in the resistant cultivar, as shown by the up-regulation of genes involved in brassinosteroid, polyamine and secondary metabolite biosynthesis, and of genes coding for pathogenesis-related proteins.
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http://dx.doi.org/10.1111/j.1364-3703.2008.00530.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640473PMC
March 2009

Transcriptome analysis of potato tubers--effects of different agricultural practices.

J Agric Food Chem 2009 Feb;57(4):1612-23

RIKILT Institute of Food Safety, Bornsesteeg 45, P.O. Box 230, 6700 AE Wageningen, The Netherlands.

The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments.
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http://dx.doi.org/10.1021/jf802815dDOI Listing
February 2009

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize.

Anal Biochem 2008 May 21;376(2):189-99. Epub 2008 Feb 21.

Department of Plant Physiology and Biotechnology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia.

Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.
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http://dx.doi.org/10.1016/j.ab.2008.02.013DOI Listing
May 2008

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms.

BMC Biotechnol 2008 Mar 6;8:26. Epub 2008 Mar 6.

Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia.

Background: The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan and SYBR Green real-time PCR chemistries. In our study four alternative chemistries: Lux, Plexor, Cycling Probe Technology and LNA were extensively evaluated and compared using TaqMan chemistry as a reference system.

Results: Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.

Conclusion: Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA technology is an alternative to TaqMan when designing assays for quantitative analysis. Because LNA probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan probe is difficult or even impossible due to sequence characteristics. Plexor on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.
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http://dx.doi.org/10.1186/1472-6750-8-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970PMC
March 2008

Calculation of measurement uncertainty in quantitative analysis of genetically modified organisms using intermediate precision--a practical approach.

J AOAC Int 2007 Mar-Apr;90(2):582-6

National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia.

Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.
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July 2007

Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

BMC Biotechnol 2006 Aug 14;6:37. Epub 2006 Aug 14.

Department of Plant Physiology and Biotechnology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia.

Background: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available.

Results: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated.

Conclusion: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.
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http://dx.doi.org/10.1186/1472-6750-6-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569826PMC
August 2006