(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways
Cankar, K. ; Jongedijk, E.J. ; Klompmaker, M. ; Majdic, T. ; Mumm, R. ; Bouwmeester, H.J. ; Bosch, H.J. ; Beekwilder, M.J. - \ 2015
Biotechnology Journal 10 (2015)1. - ISSN 1860-6768 - p. 180 - 189.
plant transformation - biosynthetic-pathway - terpenoid metabolism - squalene synthase - tobacco - expression - artemisinin - arabidopsis - reductase - precursors
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. Keywords: 5-Epi-aristolochene synthase · Metabolic engineering · RNAi · Squalene synthase
Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study
Mulders, K.J.M. ; Weesepoel, Y.J.A. ; Bodenes, C. ; Lamers, P.P. ; Vincken, J.P. ; Martens, D.E. ; Gruppen, H. ; Wijffels, R.H. - \ 2015
Journal of Applied Phycology 27 (2015)1. - ISSN 0921-8971 - p. 125 - 140.
alga haematococcus-pluvialis - green-alga - triacylglycerol accumulation - biosynthetic-pathway - dunaliella-salina - light - chlorophyceae - inhibition - microalgae - complex
Natural carotenoids such as astaxanthin, ß,ß-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultrahigh performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and ß,ß-carotene. The DPA treatment decreased the overall production of ß,ß-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of ß,ß-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. ofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.
Metabolomic engineering for the microbial production of cartenoids and related products with a focus on the rare C50 carotenoids
Heider, S.A.E. ; Peters-Wendisch, P. ; Wendisch, V.F. ; Beekwilder, M.J. - \ 2014
Applied Microbiology and Biotechnology 98 (2014)10. - ISSN 0175-7598 - p. 4355 - 4368.
alga haematococcus-pluvialis - blue-light photoreception - escherichia-coli - bacterial carotenoids - biosynthetic-pathway - bacillus-subtilis - astaxanthin biosynthesis - functional-analysis - beta-carotene - corynebacterium-glutamicum
Carotenoids, a subfamily of terpenoids, are yellowtored-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived fromisopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.
Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae
Onkokesung, N. ; Reichelt, M. ; Doorn, A. van; Schuurink, R.C. ; Loon, J.J.A. van; Dicke, M. - \ 2014
Journal of Experimental Botany 65 (2014)8. - ISSN 0022-0957 - p. 2203 - 2217.
plant-responses - anthocyanin accumulation - lepidopteran herbivores - coexpression analysis - biosynthetic-pathway - indole-glucosinolate - functional genomics - signaling pathways - defense responses - complex
Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin and flavonol levels which enhances plant resistance to generalist caterpillars. However, how these metabolites affect specialist herbivores has remained unknown. Performance of a specialist aphid (Brevicoryne brassicae) was unaffected after feeding on oxMYB75 plants, whereas a specialist caterpillar (Pieris brassicae) gained significantly higher body mass when feeding on this plant. An increase in anthocyanin and total flavonol glycoside levels correlated negatively with the body mass of caterpillars fed on oxMYB75 plants. However, a significant reduction of kaempferol-3,7-dirhamnoside (KRR) corresponded to an increased susceptibility of oxMYB75 plants to caterpillar feeding. Pieris brassicae caterpillars also grew less on an artificial diet containing KRR or on oxMYB75 plants that were exogenously treated with KRR, supporting KRR's function in direct defence against this specialist caterpillar. The results show that enhancing the activity of the anthocyanin pathway in oxMYB75 plants results in re-channelling of quercetin/kaempferol metabolites which has a negative effect on the accumulation of KRR, a novel defensive metabolite against a specialist caterpillar.
Fragmentation of an aflatoxin-like gene cluster in a forest pathogen
Bradshaw, R.E. ; Slot, J.C. ; Moore, G.G. ; Chettri, P. ; Wit, P.J.G.M. de; Ehrlich, K.C. ; Ganley, A.R.D. ; Olson, M.A. ; Rokas, A. ; Carbone, I. ; Cox, M.P. - \ 2013
New Phytologist 198 (2013)2. - ISSN 0028-646X - p. 525 - 535.
aspergillus-parasiticus - dothistroma-septosporum - phylogenetic analyses - biosynthetic-pathway - recombination events - secondary metabolism - functional-analysis - horizontal transfer - filamentous fungi - evolution
Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D.septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D.septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.
Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.
Ryden, A.M. ; Ruyter-Spira, C.P. ; Litjens, R. ; Takahashi, S. ; Quax, W.J. ; Osada, H. ; Bouwmeester, H.J. ; Kayser, O. - \ 2010
Plant and Cell Physiology 51 (2010)7. - ISSN 0032-0781 - p. 1219 - 1228.
chain dehydrogenases/reductases sdrs - amorpha-4,11-diene synthase - functional assignments - biosynthetic-pathway - essential oil - key enzyme - expression - reductase - peppermint - acid
From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, and in particular functions as a menthone:neomenthol oxidoreductase. The kinetic parameter kcat/Km was determined to be 939 fold more efficient for the reduction of (-)-menthone to (+)-neomenthol, than results previously reported for the menthone:neomenthol reductase from Mentha x piperita. Based on its kinetic properties, the possible use of Red1 in biological crop protection is discussed.
High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous
Verwaal, R. ; Wang, J. ; Meijnen, J.P. ; Visser, H. ; Sandmann, G. ; Berg, J.A. van den; Ooyen, A.J.J. van - \ 2007
Applied and Environmental Microbiology 73 (2007)13. - ISSN 0099-2240 - p. 4342 - 4350.
yeast candida-utilis - coenzyme-a reductase - phaffia-rhodozyma - escherichia-coli - biosynthetic-pathway - erwinia-uredovora - hxt5 expression - astaxanthin - lycopene - synthase
To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially ß-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product ß-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of ß-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of ß-carotene by S. cerevisiae.
Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase
Ojima, K. ; Breitenbach, J. ; Visser, J.H. ; Setoguchi, Y. ; Tabata, K. ; Hoshino, T. ; Berg, J.A. van den; Sandmann, G. - \ 2006
Molecular Genetics and Genomics 275 (2006)2. - ISSN 1617-4615 - p. 148 - 158.
escherichia-coli - functional-characterization - haematococcus-pluvialis - biosynthetic-pathway - yeast - family - host - canthaxanthin - hydroxylase - expression
A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a ß-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the ß-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this ß-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from ß-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of ß-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both ß-ionone ends
Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit
Schijlen, E.G.W.M. ; Vos, C.H. de; Jonker, H.H. ; Broeck, H.C. van den; Molthoff, J.W. ; Tunen, A.J. van; Martens, S. ; Bovy, A.G. - \ 2006
Plant Biotechnology Journal 4 (2006)4. - ISSN 1467-7644 - p. 433 - 444.
signal-transduction pathways - stilbene synthase gene - carcinoma cell-lines - heart-disease - biosynthetic-pathway - antioxidant activity - dietary flavonoids - petunia-hybrida - french paradox - male-sterility
Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.
Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs
Romano, A. ; Plas, L.H.W. van der; Witholt, B. ; Eggink, G. ; Mooibroek, A. - \ 2005
Planta 220 (2005)3. - ISSN 0032-0935 - p. 455 - 464.
nonrelated carbon-sources - unsaturated fatty-acids - beta-oxidation - pseudomonas-aeruginosa - polyhydroxyalkanoic acid - fluorescent pseudomonads - functional expression - biosynthetic-pathway - particle bombardment - plants
Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes [B. Rehm et al. (1998) J. Biol Chem 273:24044–24051] as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.
Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon
Lücker, J. ; Schwab, W. ; Hautum, B. van; Blaas, J. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
Plant Physiology 134 (2004)1. - ISSN 0032-0889 - p. 510 - 519.
s-linalool synthase - biosynthetic-pathway - volatile compounds - nicotiana-tabacum - gene-expression - floral scents - flowers - transformation - emission - clarkia
Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting -pinene, limonene, and -terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes