Comparison of expression profiles for Nicotiana benthamiana agroinfiltrated with aryophyllene synthase and SNARE-RNAi [Nicotiana benthamiana]
Krol, Sander van der; Ting, Jimmy - \ 2020
Wageningen University & Research
GSE60061 - PRJNA257401 - Nicotiana benthamiana
Here we show for the first time that vesicle fusion is one of multiple pathways in plant cells involved in terpene emissions. We demonstrate a positive feedback on terpene production when vesicle fusion is inhibited in cells producing high levels of terpenes, which relates to an induced proteasome malfunction
Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes
Jongedijk, Esmer ; Müller, Sebastian ; Dijk, Aalt D.J. Van; Schijlen, Elio ; Champagne, Antoine ; Boutry, Marc ; Levisson, Mark ; Krol, Sander Van Der; Bouwmeester, Harro ; Beekwilder, Jules ; Takahashi, Hideki - \ 2020
Journal of Experimental Botany 71 (2020)10. - ISSN 0022-0957 - p. 3052 - 3065.
Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals
Histone Deacetylase 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion
Woude, Lennard C. van der; Perrella, Giorgio ; Snoek, Basten L. ; Hoogdalem, M.D. van; Novák, O. ; Verk, Marcel C. van; Kooten, Heleen N. van; Zorn, Lennert E. ; Tonckens, Rolf ; Dongus, Joram ; Praat, Myrthe ; Stouten, Evelien ; Proveniers, Marcel C.G. ; Vellutini, Elisa ; Patitaki, Eirini ; Shapulatov, Umidjon ; Kohlen, W. ; Balasubramanian, Sureshkumar ; Ljung, Karin ; Krol, A.R. van der; Smeekens, S. ; Kaiserli, Eirini ; Zanten, Martijn van - \ 2019
Proceedings of the National Academy of Sciences of the United States of America 116 (2019)50. - ISSN 0027-8424 - p. 25343 - 25354.
Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis.
Insights into heterologous biosynthesis of Arteannuin B and artemisinin in physcomitrella patens
Ikram, Nur Kusaira Khairul ; Kashkooli, Arman Beyraghdar ; Peramuna, Anantha ; Krol, Alexander R. Van Der; Bouwmeester, Harro ; Simonsen, Henrik Toft - \ 2019
Molecules 24 (2019)21. - ISSN 1420-3049
Artemisinin - Biotechnology - Malaria - Physcomitrella patens - Sesquiterpenoids
Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADSCYP71AV1- ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.
Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production
Beyraghdar Kashkooli, Arman ; Krol, Alexander R. van der; Rabe, Patrick ; Dickschat, Jeroen S. ; Bouwmeester, Harro - \ 2019
Metabolic Engineering 54 (2019). - ISSN 1096-7176 - p. 12 - 23.
Combinatorial metabolic engineering - Dihydroparthenolide - Double bond reductase - Feverfew - Sesquiterpene lactone - Sweet wormwood
The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3β-hydroxy-dihydrocostunolide and 3β-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.
Functional intron-derived miRNAs and host-gene expression in plants
Shapulatov, Umidjon ; Hoogdalem, M.D. van; Schreuder, M.E.L. ; Bouwmeester, H.J. ; Abdurakhmonov, Ibrokhim Y. ; Krol, A.R. van der - \ 2018
Plant Methods 14 (2018). - ISSN 1746-4811
Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.
Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens
Khairul Ikram, Nur Kusaira Binti ; Beyraghdar Kashkooli, A. ; Peramuna, Anantha V. ; Krol, A.R. van der; Bouwmeester, H. ; Simonsen, Henrik T. - \ 2017
Frontiers in Bioengineering and Biotechnology 5 (2017). - ISSN 2296-4185 - 8 p.
Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.
Low-phosphate induction of plastidal stromules is dependent on strigolactones but not on the canonical strigolactone signaling component MAX2
Vismans, Gilles ; Meer, Tom van der; Langevoort, Olivier ; Schreuder, Marielle ; Bouwmeester, Harro ; Peisker, Helga ; Dörman, Peter ; Ketelaar, Tijs ; Krol, Sander van der - \ 2016
Plant Physiology 172 (2016)4. - ISSN 0032-0889 - p. 2235 - 2244.
Stromules are highly dynamic protrusions of the plastids in plants. Several factors, such as drought and light conditions, influence the stromule frequency (SF) in a positive or negative way. A relatively recently discovered class of plant hormones are the strigolactones; strigolactones inhibit branching of the shoots and promote beneficial interactions between roots and arbuscular mycorrhizal fungi. Here, we investigate the link between the formation of stromules and strigolactones. This research shows a strong link between strigolactones and the formation of stromules: SF correlates with strigolactone levels in the wild type and strigolactone mutants (max2-1 max3-9), and SF is stimulated by strigolactone GR24 and reduced by strigolactone inhibitor D2.
Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua
Wang, Bo ; Kashkooli, Arman Beyraghdar ; Sallets, Adrienne ; Ting, Jimmy ; Ruijter, Norbert C.A. de; Olofsson, Linda ; Brodelius, Peter ; Olofsson, Linda ; Boutry, Marc ; Bouwmeester, Harro ; Krol, Sander van der - \ 2016
Metabolic Engineering 38 (2016). - ISSN 1096-7176 - p. 159 - 169.
ABC transporters - Artemisia annua - Artemisinin - Lipid transfer proteins - Nicotiana benthamiana - Pleiotropic Drug Resistance protein
Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.
Secrets of the world's most popular bedding plant unlocked
Krol, Sander van der; Immink, Richard G.H. - \ 2016
Nature Plants 2 (2016). - ISSN 2055-026X
Boosted by next-generation sequencing technology, there is now an ever-growing list of fully sequenced plant genomes. Recent additions to this list are two presumed ancestors of Petunia hybrida, the most popular bedding plant worldwide. These genome sequences provide new information on a species at a key position in plant phylogeny, and support the use of petunia as a research model plant species.
RNA interference for functional genomics and improvement of cotton (Gossypium sp.)
Abdurakhmonov, Ibrokhim Y. ; Ayubov, Mirzakamol S. ; Ubaydullaeva, Khurshida A. ; Buriev, Zabardast T. ; Shermatov, Shukhrat E. ; Ruziboev, Haydarali S. ; Shapulatov, Umidjon ; Saha, Sukumar ; Ulloa, Mauricio ; Yu, John Z. ; Percy, Richard G. ; Devor, Eric J. ; Sharma, Govind C. ; Sripathi, Venkateswara R. ; Kumpatla, Siva P. ; Krol, Sander van der; Kater, Hake D. ; Khamidov, Khakimdjan ; Salikhov, Shavkat I. ; Jenkins, Johnie N. ; Abdukarimov, Abdusattor ; Pepper, Alan E. - \ 2016
Frontiers in Plant Science 7 (2016)FEB2016. - ISSN 1664-462X
Antisense - Cotton pest control - Disease resistance - Fiber quality - Gene silencing - Gossypium
RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.
Monoterpene biosynthesis potential of plant subcellular compartments
Dong, L. ; Jongedijk, E.J. ; Bouwmeester, H.J. ; Krol, A.R. van der - \ 2016
New Phytologist 209 (2016)2. - ISSN 0028-646X - p. 679 - 690.
Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.
SNARE-RNAi results in higher terpene emission from ectopically expressed caryophyllene synthase in nicotiana benthamiana
Ting, Jimmy ; Delatte, Thierry L. ; Kolkman, P. ; Misas-Villamil, Johana C. ; Hoorn, Renier A.L. Van Der; Bouwmeester, Harro J. ; Krol, Sander van der - \ 2015
Molecular Plant 8 (2015)3. - ISSN 1674-2052 - p. 454 - 466.
caryophyllene synthase - linalool synthase - Nicotiana benthamiana - proteasome - terpene transport - vesicle-associated membrane proteins (VAMP72)
Plants produce numerous terpenes and much effort has been dedicated to the identification and characterization of the terpene biosynthetic genes. However, little is known about how terpenes are transported within the cell and from the cell into the apoplast. To investigate a putative role of vesicle fusion in this process, we used Agrobacterium tumefaciens-mediated transient coexpression in Nicotiana benthamiana of an MtVAMP721e-RNAi construct (Vi) with either a caryophyllene synthase or a linalool synthase, respectively. Headspace analysis of the leaves showed that caryophyllene or linalool emission increased about five-fold when N. benthamiana VAMP72 function was blocked. RNA sequencing and protein ubiquitination analysis of the agroinfiltrated N. benthamiana leaf extracts suggested that increased terpene emissions may be attributed to proteasome malfunction based on three observations: leaves with TPS+Vi showed (1) a higher level of a DsRed marker protein, (2) a higher level of ubiquitinated proteins, and (3) coordinated induced expression of multiple proteasome genes, presumably caused by the lack of proteasome-mediated feedback regulation. However, caryophyllene or linalool did not inhibit proteasome-related protease activity in the in vitro assays. While the results are not conclusive for a role of vesicle fusion in terpene transport, they do show a strong interaction between inhibition of vesicle fusion and ectopic expression of certain terpenes. The results have potential applications in metabolic engineering.
Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2015
Yeast 32 (2015)1. - ISSN 0749-503X - p. 159 - 171.
monoterpene biosynthesis - escherichia-coli - synthase - precursor
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. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production
Masakapalli, S.K. ; Ritala, A. ; Dong, L.M. ; Krol, A.R. van der; Oksman-Caldentey, K.M. ; Ratcliffe, R.G. ; Sweetlove, L.J. - \ 2014
Phytochemistry 99 (2014). - ISSN 0031-9422 - p. 73 - 85.
heterotrophic arabidopsis cells - central carbon metabolism - alkaloid biosynthesis - mevalonate pathway - mass-spectrometry - synthase - networks - plants - quantification - expression
The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-C-13]-, [2-C-13]- or [C-13(6)]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-C-14]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 +/- 24 ng/mg dry weight) and the de nova synthesis of geraniol in pGES was confirmed by C-13 labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest.demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far. (C) 2013 Elsevier Ltd. All rights reserved.
Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis
Zhang, Y. ; Dijk, A.D.J. van; Scaffidi, A. ; Flematti, G.R. ; Hofmann, M. ; Charnikhova, T. ; Verstappen, F.W.A. ; Hepworth, J. ; Krol, A.R. van der; Leyser, O. - \ 2014
Nature Chemical Biology 10 (2014). - ISSN 1552-4450 - p. 1028 - 1033.
arbuscular mycorrhizal fungi - structural requirements - germination stimulants - biological-activities - arabidopsis-thaliana - crystal-structures - plant hormones - protein - inhibition - expression
Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-¿-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis MORE AXILLARY GROWTH 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.
Assessment of pleiotropic transcriptome perturbations in Arabidopsis engineered for indirect insect defence
Houshyani Hassanzadeh, B. ; Krol, A.R. van der; Bino, R.J. ; Bouwmeester, H.J. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229
global gene-expression - metabolomics - thaliana - gm - biosynthesis - metabolism - emission - synthase - crops - wheat
Background: Molecular characterization is an essential step of risk/safety assessment of genetically modified (GM) crops. Holistic approaches for molecular characterization using omics platforms can be used to confirm the intended impact of the genetic engineering, but can also reveal the unintended changes at the omics level as a first assessment of potential risks. The potential of omics platforms for risk assessment of GM crops has rarely been used for this purpose because of the lack of a consensus reference and statistical methods to judge the significance or importance of the pleiotropic changes in GM plants. Here we propose a meta data analysis approach to the analysis of GM plants, by measuring the transcriptome distance to untransformed wild-types. Results: In the statistical analysis of the transcriptome distance between GM and wild-type plants, values are compared with naturally occurring transcriptome distances in non-GM counterparts obtained from a database. Using this approach we show that the pleiotropic effect of genes involved in indirect insect defence traits is substantially equivalent to the variation in gene expression occurring naturally in Arabidopsis. Conclusion: Transcriptome distance is a useful screening method to obtain insight in the pleiotropic effects of genetic modification.
Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway
Ritala, A. ; Dong, L. ; Imseng, N. ; Seppanen-Laakso, T. ; Vasilev, N. ; Krol, A.R. van der; Rischer, H. ; Maaheimo, H. ; Virkki, A. ; Brandli, J. ; Schillberg, S. ; Eibl, R. ; Bouwmeester, H.J. ; Oksman-Caldentey, K.M. - \ 2014
Journal of Biotechnology 176 (2014). - ISSN 0168-1656 - p. 20 - 28.
catharanthus-roseus - isoprenoid biosynthesis - plastidial pathways - essential oils - key enzyme - monoterpene - cultures - synthase - cells - bioreactors
The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastidtargeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC–MS screening tool was developed for the rapid evaluation of production clones. The GC–MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 g/g dry weight (DW) and a maximum of 31.3 g/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 g/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.
Natural products – learning chemistry from plants
Staniek, A. ; Bouwmeester, H.J. ; Fraser, P.D. ; Kayser, O. ; Martens, S. ; Tissier, A. ; Krol, A.R. van der; Wessjohann, L. ; Warzecha, H. - \ 2014
Biotechnology Journal 9 (2014)3. - ISSN 1860-6768 - p. 326 - 336.
escherichia-coli - benzylisoquinoline alkaloids - saccharomyces-cerevisiae - vanillin production - synthetic biology - organic-synthesis - biosynthesis - biocatalysis - artemisinin - enzymes
Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large-scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant-derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.
Comparison of plant-based expression platforms for the heterologous production of geraniol
Vasilev, N. ; Schmitz, C. ; Dong, L. ; Ritala, A. ; Imseng, N. ; Hakkinen, S.T. ; Krol, A.R. van der; Eibl, R. ; Oksman-Caldentey, K.M. ; Bouwmeester, H.J. ; Fischer, R. ; Schillberg, S. - \ 2014
Plant Cell, Tissue and Organ Culture: an international journal on in vitro culture of higher plants 117 (2014)3. - ISSN 0167-6857 - p. 373 - 380.
mevalonate kinase-deficiency - pelargonium-graveolens - chemical-composition - catharanthus-roseus - response factor - mentha-spicata - essential oils - sweet basil - mouse model - biosynthesis
We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 µg/g fresh weight, fw), followed by the transient expression system (27 µg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 µg/g fw), and finally hairy root cultures (9 µg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 µg/g fresh biomass per day, whereas transient expression produced 5.9 µg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 µg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol