Metabolic engineering of plant monoterpenes, sesquiterpenes and diterpenes—current status and future opportunities

All enzymes of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway are localized to plastids.

“In contrast, all enzymes of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway are localized to plastids”

• cites Hsieh et al., 2008Not checked yet

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• cites Suire et al., 2000Not checked yet

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Alpha-copaene was not a major contributor to potato aroma.

“The authors thus concluded that a-copaene was not a major contributor to potato aroma.”

• cites Morris et al., 2011Not checked yet

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When the FaNES1 gene product was targeted to mitochondria of transgenic Arabidopsis plants, a metabolic conversion of (3S,6E)-nerolidol to 4,8-dimethyl-1,3(E),7-nonatriene was reported.

“When the FaNES1 gene product was targeted to mitochondria of transgenic Arabidopsis plants, a metabolic conversion of (3S,6E)-nerolidol to 4,8-dimethyl-1,3(E),7-nonatriene was reported”

• cites Kappers et al., 2005Not checked yet

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Constitutive expression of the S-linalool synthase gene from Clarkia breweri in carnation resulted in the formation of S-linalool (up to 6% of total volatiles) and cis- and trans-S-linalool oxides (up to 4.5% of total volatiles) in flowers.

“resulted in the formation of S-linalool (up to 6% of total volatiles) but also cis-and trans-S-linalool oxides (up to 4.5% of total volatiles) in flowers”

• cites Lavy et al., 2002Not checked yet

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Tomato transformed with the Clarkia breweri linalool synthase gene under the fruit-specific E8 promoter showed increased production of various volatiles, including S-linalool and its oxidation product 8-hydroxylinalool, in ripening fruit.

“The authors reported an increased production of various volatiles, including S-linalool and its oxidation product, 8-hydroxylinalool, in ripening fruit”

• cites Lewinsohn et al., 2001Not checked yet

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Many mono-, sesqui-, and diterpenes accumulate in distinct anatomical structures and cell types.

“While some terpenoids are synthesized in all plant cells, many mono-, sesqui-and diterpenes accumulate in distinct anatomical structures and cell types.”

Flowers of Petunia hybrida emit a blend of volatiles dominated by phenylpropanoids and benzenoids, with small amounts of fatty acid derivatives and sesquiterpenes.

“Flowers of Petunia 9 hybrida (Solanaceae) emit a blend of volatiles that is dominated by phenylpropanoids and benzenoids, with small amounts of fatty acid derivatives and sesquiterpenes”

• cites Verdonk et al., 2003Not checked yet

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Volatiles emitted from transgenic tobacco plants with plastid-targeted PTS and FPS significantly deterred tobacco hornworms from feeding on leaves.

“Interestingly, the authors also demonstrated that the volatiles emitted from these transgenic plants significantly deterred tobacco hornworms from feeding on leaves”

• cites Wu et al., 2006Not checked yet

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A novel terpenoid introduced by metabolic engineering was again metabolized by endogenous enzymatic activities, generating metabolites that neither occur naturally in the source organism of the transgene nor in the host plant used in the transformation.

“A novel terpenoid, introduced by metabolic engineering, was again metabolized by endogenous enzymatic activities, thus generating metabolites that neither occur naturally in the source organism of the transgene nor in the host plant used in the transformation.”

• cites Lücker et al., 2004bNot checked yet

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Tomato transformed with a geraniol synthase gene from sweet basil under a fruit ripening-specific polygalacturonase promoter released a range of terpenoid volatiles derived from geraniol, including oxidation products, reduced metabolites, isomerization products, and acetylated conjugates.

“Transgenic plants released a whole range of terpenoid volatiles metabolically derived from geraniol, including oxidation products (e.g.geranial and geranic acid), C-C double bond-reduced metabolites (citronellol and its oxidation products), C-C double bond cis/trans isomerization products (neral and its oxidized and reduced forms) and acetylated conjugates (geranyl and citronellyl acetates).”

• cites Davidovich-Rikanati et al., 2007Not checked yet

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A cytosolic pool of GPP for further conversion into monoterpenes by ZIS has to be present in ripening tomato fruit.

“This means that a cytosolic pool of GPP for further conversion into monoterpenes by ZIS has to be present in ripening tomato fruit.”

Tobacco can be used as an experimental model system to test metabolic engineering strategies for monoterpenoid volatile production.

“These studies established that tobacco, which is readily transformable and has a short regeneration time, can be used as an experimental model system to test metabolic”

• cites Lücker et al., 2004aNot checked yet

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• cites El Tamer et al., 2003Not checked yet

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The ability of ZIS to convert GPP partially explains why transgenic tomato plants expressing the ZIS gene accumulated both mono- and sesquiterpenes.

“which partially explains the fact that transgenic plants expressing the ZIS gene accumulated both mono-and sesquiterpenes”

• cites Iijima et al., 2004Not checked yet

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Transgenic maize expressing a geraniol synthase gene from Lippia dulcis accumulated geranyl acetate and several hydroxylated and glycosylated derivatives of geranic acid.

“Transgenic plants accumulated geranyl acetate and several hydroxylated and glycosylated derivatives of geranic acid, but bioassays indicated that there was no improved resistance to fungi, possibly because of the further metabolization of geranic acid into inactive derivatives.”

• cites Yang et al., 2011Not checked yet

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The lack of improved fungal resistance in geraniol synthase-expressing maize may be because geranic acid is further metabolized into inactive derivatives.

“Transgenic plants accumulated geranyl acetate and several hydroxylated and glycosylated derivatives of geranic acid, but bioassays indicated that there was no improved resistance to fungi, possibly because of the further metabolization of geranic acid into inactive derivatives.”

Constitutive expression of a truncated Arabidopsis HMGR gene in spike lavender resulted in up to ~2-fold higher essential oil yields in leaves and flowers compared to controls.

“Interestingly, the constitutive expression of a truncated gene encoding only the catalytic domain of the Arabidopsis 3-hydroxy-3-methylglutaryl-CoA reductase, an enzyme of the MVA pathway (Friesen and Rodwell, 2004), in spike lavender also resulted in significant increases in oil yields (up to ~2-fold higher than in leaves and flowers of control plants) (Mun ˜oz-Bertomeu et al., 2007) (Table 3).”

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Progress in engineering terpenoid essential oil yield and composition has been confined to mint, lavender and Eucalyptus.

“To date, progress in engineering terpenoid essential oil yield and composition has been confined to mint, lavender and Eucalyptus.”

Transgenic plants emitting enhanced levels of certain terpenoids due to expression of heterologous terpene synthases deter the feeding of hornworms.

“In summary, transgenic plants that emitted enhanced levels of certain terpenoids due to the expression of heterologous terpene synthases were found to have a reduced aphid colonization (Aharoni et al., 2003;Beale et al., 2006;Yu et al., 2012a), deter the feeding of hornworms (Wu et al., 2006) or attract predatory mites as 'bodyguards' against arthropod infestation (Kappers et al., 2005).”

• cites Wu et al., 2006Not checked yet

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In transgenic plants emitting the endogenous sesquiterpene b-caryophyllene, aphid colonization was significantly diminished compared to untransformed controls.

“Aphid colonization was significantly diminished in transgenic plants when compared to untransformed controls, while at the same time aphid predators were attracted by the volatile emissions (Beale et al., 2006).”

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When challenged with a fungus or a bacterium, transgenic orange fruit showed increased resistance compared to fruit peel of wild-type controls.

“When challenged with a fungus or a bacterium, transgenic fruit showed increased resistance (compared to fruit peel of wildtype controls).”

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Transgenic orange trees with antisense reduction of limonene synthase had dramatically reduced limonene concentrations in fruit peel.

“The peel of fruit form transgenic orange trees had dramatically reduced limonene concentrations (no absolute quantitation data provided).”

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The p-menthane biosynthetic pathway is completely defined.

“The developmentally controlled distribution of glandular trichomes has been studied in detail (Turner et al., 2000a,b), and the p-menthane biosynthetic pathway is completely defined (Croteau et al., 2005) (Fig. 3).”

• cites Croteau et al., 2005Not checked yet

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Monoterpenes in peppermint are derived exclusively from the MEP pathway.

“Feeding studies with isotope-labelled precursors established that monoterpenes in peppermint (Mentha 9 piperita L. cv.Black Mitcham; Lamiaceae) are derived exclusively from the MEP pathway (Eisenreich et al., 1997), whereas the MVA pathway is inactive in gland cells (McCaskill and Croteau, 1995).”

• cites Eisenreich et al., 1997Not checked yet

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Antisense approaches are useful in metabolic engineering of disease-related traits.

“Exudate harvested from antisense-expressing plants had increased activity (compared to wild-type controls) against aphids, which demonstrates the utility of antisense approaches in the metabolic engineering of diseaserelated traits.”

Transgenic plants emitting enhanced levels of certain terpenoids due to expression of heterologous terpene synthases have reduced aphid colonization.

“In summary, transgenic plants that emitted enhanced levels of certain terpenoids due to the expression of heterologous terpene synthases were found to have a reduced aphid colonization (Aharoni et al., 2003;Beale et al., 2006;Yu et al., 2012a), deter the feeding of hornworms (Wu et al., 2006) or attract predatory mites as 'bodyguards' against arthropod infestation (Kappers et al., 2005).”

• cites Aharoni et al., 2003Not checked yet

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• cites Beale et al., 2006Not checked yet

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• cites Yu et al., 2012aNot checked yet

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Expression of an Ebf gene from Artemisia annua in tobacco resulted in increased attraction of aphid predators.

“Diminished aphid colonization and increased attraction of aphid predators were also observed when an Ebf gene from sweet wormwood (Artemisia annua L. strain 025) was expressed in tobacco (N.tabacum L. cv.W38) (Yu et al., 2012a) (Table 2).”

• cites Yu et al., 2012aNot checked yet

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Expression of an Ebf gene from Artemisia annua in tobacco resulted in diminished aphid colonization.

“Diminished aphid colonization and increased attraction of aphid predators were also observed when an Ebf gene from sweet wormwood (Artemisia annua L. strain 025) was expressed in tobacco (N.tabacum L. cv.W38) (Yu et al., 2012a) (Table 2).”

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Fruit flies were less attracted to orange fruit accumulating less limonene compared to those with wild-type limonene levels.

“In addition, fruit flies were less attracted to fruit accumulating less limonene compared to those with wild-type limonene levels.”

• cites Rodrı ´guez et al., 2011Not checked yet

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Exudate harvested from antisense-expressing tobacco plants had increased activity against aphids compared to wild-type controls.

“Exudate harvested from antisense-expressing plants had increased activity (compared to wild-type controls) against aphids, which demonstrates the utility of antisense approaches in the metabolic engineering of diseaserelated traits.”

• cites Wang et al., 2001Not checked yet

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Constitutive overexpression of an Arabidopsis DXS cDNA in spike lavender led to up to 359% increased essential oil yields in leaves and up to 74% in flowers, with essentially no negative effects on oil composition.

“The constitutive overexpression of an Arabidopsis DXS cDNA in spike lavender led to the most highly increased essential oil yields reported thus far (up to 359% in leaves and up to 74% in flowers), with essentially no negative effects on oil composition (Mun ˜oz-Bertomeu et al., 2006) (Table 3), which might be interpreted as an indication for the importance of the MEP pathway for precursor supply.”

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The high yield increases from DXS overexpression in spike lavender might indicate the importance of the MEP pathway for precursor supply.

“The constitutive overexpression of an Arabidopsis DXS cDNA in spike lavender led to the most highly increased essential oil yields reported thus far (up to 359% in leaves and up to 74% in flowers), with essentially no negative effects on oil composition (Mun ˜oz-Bertomeu et al., 2006) (Table 3), which might be interpreted as an indication for the importance of the MEP pathway for precursor supply.”

Downregulation of expression levels of genes involved in terpenoid biosynthesis has been used successfully to increase disease resistance against aphids or fruit flies.

“Interestingly, the downregulation of the expression levels of genes involved in terpenoid biosynthesis has also been used successfully to increase disease resistance against aphids or fruit flies (Rodrı ´guez et al., 2011;Wang et al., 2001).”

• cites Rodrı ´guez et al., 2011Not checked yet

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• cites Wang et al., 2001Not checked yet

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Field testing over several growth seasons indicated that transgenic peppermint plants overexpressing DXR had up to 18% higher oil yields than wild-type controls after establishing a strong root system.

“Field testing over several growth seasons indicated that, after a period that was required to establish a strong root system, transgenic peppermint plants overexpressing DXR had up to 18% higher oil yields than wild-type controls (Lange et al., 2011).”

• cites Lange et al., 2011Not checked yet

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The MVA pathway is inactive in peppermint gland cells.

“Feeding studies with isotope-labelled precursors established that monoterpenes in peppermint (Mentha 9 piperita L. cv.Black Mitcham; Lamiaceae) are derived exclusively from the MEP pathway (Eisenreich et al., 1997), whereas the MVA pathway is inactive in gland cells (McCaskill and Croteau, 1995).”

• cites McCaskill and Croteau, 1995Not checked yet

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Constitutive overexpression of DXS did not result in significant essential oil yield increases in transgenic peppermint plants.

“The constitutive overexpression of the gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS; note that DXPS is also used as an acronym in the literature), which catalyses the entry step into the MEP pathway (note that this is not the committed first step as the thiamin and pyridoxol biosynthetic pathways also involve DXS), did not result in significant yield increases in transgenic peppermin”

• cites Lange et al., 2011Not checked yet

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In transgenic plants emitting b-caryophyllene, aphid predators were attracted by the volatile emissions.

“Aphid colonization was significantly diminished in transgenic plants when compared to untransformed controls, while at the same time aphid predators were attracted by the volatile emissions (Beale et al., 2006).”

• cites Beale et al., 2006Not checked yet

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Transgenic tobacco with antisense knockdown of a putative cytochrome P450 monooxygenase accumulated ~19-fold higher concentrations of cembratrienol (up to 4.3% of dry leaf biomass) in glandular trichomes compared to wild-type controls.

“Transgenics accumulated ~19-fold higher concentrations (compared to wild-type controls) of cembratrienol (up to 4.3% of dry leaf biomass) in glandular trichomes.”

• cites Wang et al., 2001Not checked yet

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Transgenic plants emitting enhanced levels of certain terpenoids due to expression of heterologous terpene synthases attract predatory mites as bodyguards against arthropod infestation.

“In summary, transgenic plants that emitted enhanced levels of certain terpenoids due to the expression of heterologous terpene synthases were found to have a reduced aphid colonization (Aharoni et al., 2003;Beale et al., 2006;Yu et al., 2012a), deter the feeding of hornworms (Wu et al., 2006) or attract predatory mites as 'bodyguards' against arthropod infestation (Kappers et al., 2005).”

• cites Kappers et al., 2005Not checked yet

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Bioassays indicated that transgenic maize expressing geraniol synthase had no improved resistance to fungi.

“Transgenic plants accumulated geranyl acetate and several hydroxylated and glycosylated derivatives of geranic acid, but bioassays indicated that there was no improved resistance to fungi, possibly because of the further metabolization of geranic acid into inactive derivatives.”

• cites Yang et al., 2011Not checked yet

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The antisense approach could potentially be used to improve pest resistance in the field.

“As no other phenotypic differences between transgenic plants and controls were observed, the antisense approach could potentially be used to improve pest resistance in the field.”

Overexpression of endogenous DXR in transgenic peppermint plants resulted in up to 44% increased oil yields compared to wild-type controls.

“a second set of lines, in which DXR was truly overexpressed, had up to 44% increased oil yields when compared with wild-type controls (Mahmoud and Croteau, 2001) (Table 3).”

• cites Mahmoud and Croteau, 2001Not checked yet

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(+)-Menthofuran was selectively retained in specialized cells of peppermint glandular trichomes.

“It was shown that (+)menthofuran was selectively retained in these specialized cells of glandular trichomes and that, when peppermint plants were exposed to abiotic stresses, the concentration of (+)-menthofuran rose up to the millimolar range, thus resulting in a significant (+)pulegone reductase inhibition (Rios-Estepa et al., 2008).”

• cites Rios-Estepa et al., 2008Not checked yet

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Cytosolic accumulation of LS in Eucalyptus camaldulensis resulted in higher amounts of extractable limonene than expression of LS in plastids.

“A cytosolic accumulation of LS resulted in higher amounts of extractable limonene than the expression of LS in plastids (Ohara et al., 2010) (Table 3), although the availability of GPP, the substrate for LS, is generally considered to be much lower in the cytosol compared to plastids.”

• cites Ohara et al., 2010Not checked yet

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In corn mint, expressing spearmint LS resulted in oil yields similar to or lower than wild-type controls.

“In a separate set of transformations, in which spearmint LS was expressed in corn mint (Mentha arvensis L.), the oil yields were similar to or lower than in wild-type controls (Diemer et al., 2001) (Table 3).”

• cites Diemer et al., 2001Not checked yet

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Antisense expression of (+)-menthofuran synthase cDNA in transgenic peppermint plants decreased (+)-menthofuran levels.

“To decrease (+)-menthofuran levels, an antisense construct of the (+)-menthofuran synthase (MFS) cDNA was expressed constitutively in transgenic peppermint plants (Mahmoud and Croteau, 2001) (Table 3).”

• cites Mahmoud and Croteau, 2001Not checked yet

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Overexpression of endogenous L3H gene in transgenic peppermint plants was unsuccessful, as none of the transgenics contained elevated L3H activity or increased oil yield.

“The attempt to overexpress the endogenous L3H gene in transgenic peppermint plants was unsuccessful, as none of the transgenics contained elevated levels of L3H activity or increased oil yield (Mahmoud et al., 2004) (Table 3).”

• cites Mahmoud et al., 2004Not checked yet

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A developmental time course study indicated that differences between transgenic and control spike lavender plants were most pronounced in young leaves.

“A developmental time course study indicated that the differences between transgenic and control plants were most pronounced in young leaves.”

• cites Mun ˜oz-Bertomeu et al., 2008Not checked yet

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In LS-expressing Eucalyptus transgenics, limonene accumulation and LS transcript levels did not correlate.

“Furthermore, it was also reported that limonene accumulation and LS transcript levels did not correlate.”

• cites Ohara et al., 2010Not checked yet

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In transgenic peppermint plants constitutively overexpressing the spearmint LS gene, oil yield and monoterpene composition were the same as in wild-type controls despite high transcript abundance, protein concentration and enzyme activity.

“Mahmoud et al. (2004) reported the constitutive overexpression of the spearmint LS gene, as evidenced by high transcript abundance, protein concentration and enzyme activity, in glandular trichomes of transgenic peppermint plants (Table 3).However, the oil yield and monoterpene composition were the same as in wild-type controls.”

• cites Mahmoud et al., 2004Not checked yet

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MFS antisense peppermint plants accumulated significantly more essential oil (35% yield increase) compared to wild-type controls.

“As another surprise, MFS antisense plants accumulated significantly more essential oil (35% yield increase) compared to wild-type controls (Mahmoud and Croteau, 2001).”

• cites Mahmoud and Croteau, 2001Not checked yet

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Cosuppressed transgenic peppermint lines with reduction of endogenous L3H transcript levels accumulated oil highly enriched in (-)-limonene (up to 80% of total monoterpenes) but depleted of downstream metabolites.

“However, the authors analysed several cosuppressed transgenic lines (reduction of endogenous L3H transcript levels), which accumulated oil that was highly enriched in (À)-limonene (up to 80% of the total monoterpenes) but depleted of the downstream metabolites in the pathway (Mahmoud et al., 2004) (Table 3).”

• cites Mahmoud et al., 2004Not checked yet

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(+)-Menthofuran is a weak competitive inhibitor of (+)-pulegone reductase, the enzyme that converts (+)-pulegone to (-)-menthone and (+)-isomenthone in peppermint glandular trichomes.

“It was demonstrated that (+)-menthofuran is a weak competitive inhibitor of (+)-pulegone reductase, the enzyme that converts (+)-pulegone to (À)-menthone and (+)isomenthone in peppermint glandular trichomes (Rios-Estepa et al., 2008).”

• cites Rios-Estepa et al., 2008Not checked yet

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The relative abundance of (+)-pulegone and (+)-menthofuran can increase to 15-20% under stress conditions like high night temperatures, low day light intensities, and nutrient and water deficiency.

“The relative abundance of these intermediates can increase to 15-20% under stress conditions like high night temperatures, low day light intensities, and nutrient and water deficiency (Burbott and Loomis, 1967;Clark and Menary, 1980;Court et al., 1993).”

• cites Burbott and Loomis, 1967Not checked yet

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• cites Clark and Menary, 1980Not checked yet

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• cites Court et al., 1993Not checked yet

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The MVA pathway in spike lavender glandular trichomes, in contrast to peppermint, is operational for essential oil biosynthesis.

“These results indicate that the MVA pathway in spike lavender glandular trichomes, in contrast to peppermint (McCaskill and Croteau, 1995), is operational for essential oil biosynthesis.”

The authors reported a good correlation between LS transcript levels and (-)-limonene accumulation in spike lavender.

“In general, the authors reported a good correlation between LS transcript levels and (À)-limonene accumulation (Mun ˜oz- Bertomeu et al., 2008).”

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Some MFS antisense transgenic peppermint lines also had decreased relative amounts of (+)-pulegone compared to wild-type controls.

“Surprisingly, some of the transgenic lines did not only have desirably low (+)menthofuran contents, but the relative amounts of (+)-pulegone were also decreased when compared to wild-type controls.”

• cites Mahmoud and Croteau, 2001Not checked yet

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Progress has been made in identifying gland cell-specific promoters.

“An excellent recent review (Tissier, 2012) highlights progress in identifying such promoters, and we will discuss successful metabolic engineering efforts with tobacco glandular trichomes below (Table 6).”

• cites Tissier, 2012Not checked yet

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Overexpressing both HMGR and ADS under two constitutive promoters in A. annua produced a transgenic line with 7.7-fold higher artemisinin level in a very low-yielding genetic background (wild-type artemisinin content 0.02% of dry weight biomass).

“By overexpressing the genes encoding both HMGR (enzyme of MVA precursor supply pathway) and ADS (first committed enzyme of the artemisinin biosynthetic pathway), under the control of two different constitutive promoters, led to the production of a transgenic line that had a 7.7-fold higher artemisinin level in a very low-yielding genetic background (wildtype artemisinin content 0.02% of dry weight”

• cites Alam and Abdin, 2011Not checked yet

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Antisense suppression of b-caryophyllene synthase transcript led to a 55% increase in artemisinin content compared with wild-type controls.

“This approach led to a 55% increase in artemisinin content compared with wild-type controls.”

• cites Chen et al., 2011aNot checked yet

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Overexpression of an endogenous FPS gene in a high-yielding A. annua strain (artemisinin content approximately 0.65% of dry weight biomass) led to a roughly 34% increase in artemisinin compared to wild-type controls.

“When an endogenous FPS gene was overexpressed in a highyielding A. annua strain (artemisinin content approximately 0.65% of dry weight biomass), a roughly 34% increase (compared to wild-type controls) was reported for transgenic lines (Han et al., 2006) (Table 4).”

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Expression of an HMGR gene from Catharanthus roseus in transgenic A. annua under a constitutive promoter led to a 22.5% increase in artemisinin levels in shoots compared to control plants.

“These discrepancies notwithstanding, the expression of an HMGR gene from Madagascar periwinkle (Catharanthus roseus (L.) G. Don.) in transgenic A. annua, under control of a constitutive promoter, led to modest increases ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal,”

• cites Aquil et al., 2009Not checked yet

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When ADS expression levels were decreased using an antisense approach, the amounts of sesquiterpenes (E)-b-farnesene and germacrene D increased.

“Conversely, when ADS expression levels were decreased using an antisense approach, the amounts of the sesquiterpenes (E)-b-farnesene and germacrene D increased (Table 4) (Ma et al., 2009a).”

• cites Ma et al., 2009aNot checked yet

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Despite notable decreases in sterol levels, the transgenic A. annua plants apparently had a growth performance similar to wild-type controls.

“Despite notable decreases in sterol levels, the transgenic plants apparently had a growth performance similar to wild-type controls.”

• cites Zhang et al., 2009Not checked yet

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Metabolic engineering was used successfully to modulate two commercially relevant traits in peppermint.

“Several of the resulting transgenic lines had significantly increased oil yields (up to 78% higher than wild-type controls in field trials with BD7A-4 line) and a favourable composition (low (+)-menthofuran (1-5%) and (+)-pulegone ( 0.5%), and high (À)-menthone and (À)-menthol levels) (Lange et al., 2011), indicating that metabolic engineering was used successfully to modulate two commercially”

Production of artemisinin has also been achieved in transgenic plants (A. annua and tobacco).

“Alternatively, the production of artemisinin has also been achieved in transgenic plants (A.annua and tobacco).”

All efforts to modulate peppermint essential oil composition and yield have relied on constructs that result in constitutive expression of transgenes.

“Thus far, all efforts to modulate peppermint essential oil composition and yield have relied on constructs that result in a constitutive expression of transgenes.”

Artemisinin yields from the natural producer are fairly low, ranging from 0.01% to 2% of dry weight biomass.

“However, the artemisinin yields from the natural producer are fairly low (ranging from 0.01% to 2% of dry weight biomass), which might be partially explained by the fact that the biosynthesis of this metabolite is restricted to glandular trichomes on the surfaces of leaves and floral bracts (Duke and Paul, 1993;Duke et al., 1994;Olsson et al., 2009;Pu et al., 2012).”

• cites Duke and Paul, 1993Not checked yet

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• cites Duke et al., 1994Not checked yet

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• cites Olsson et al., 2009Not checked yet

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Transgenic A. annua lines with squalene synthase suppression evaluated in field trials showed a 2-fold increase of artemisinin yield from 0.45% to 0.9% of dry weight biomass.

“Transgenic lines were also evaluated in field trials, for which a 2-fold increase of artemisinin yield (from 0.45% to 0.9% of dry weight biomass) was reported (Zhang et al., 2009).”

• cites Zhang et al., 2009Not checked yet

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Suppression of squalene synthase gene by hairpin-RNA-mediated RNAi led to >3-fold increases in artemisinin concentration (up to 3.1% of dry weight biomass) in greenhouse-grown transgenic A. annua compared to wild-type controls.

“The suppression of the expression of the squalene synthase gene, which codes for an early enzyme of the sterol biosynthetic pathway, by means of hairpin-RNA-mediated RNA interference (RNAi), led to significant increases (> 3-fold compared to wild-type controls) of the artemisinin concentration (up to 3.1% of dry weight biomass) in greenhouse-grown transgenic A. annua plants (Zhang et al., 2009) (”

• cites Zhang et al., 2009Not checked yet

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Cytokinin addition increased artemisinin yields in tissue cultures.

“It was known that cytokinin addition increased artemisinin yields in tissue cultures (Whipkey et al., 1992), and the rationale was to increase endogenous cytokinin levels.”

• cites Whipkey et al., 1992Not checked yet

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Increased demand for artemisinin-based combination therapies has led to supply shortages.

“Increased demand for artemisinin-based combination therapies has led to supply shortages (Covello, 2008).”

• cites Covello, 2008Not checked yet

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The same experiment was later repeated by Nafis et al. (2011) with essentially identical results.

“The same experiment was later repeated by Nafis et al. (2011) with essentially identical results.”

• cites Nafis et al., 2011Not checked yet

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Artemisinin biosynthesis is restricted to glandular trichomes on the surfaces of leaves and floral bracts.

“However, the artemisinin yields from the natural producer are fairly low (ranging from 0.01% to 2% of dry weight biomass), which might be partially explained by the fact that the biosynthesis of this metabolite is restricted to glandular trichomes on the surfaces of leaves and floral bracts (Duke and Paul, 1993;Duke et al., 1994;Olsson et al., 2009;Pu et al., 2012).”

• cites Duke and Paul, 1993Not checked yet

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• cites Duke et al., 1994Not checked yet

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• cites Olsson et al., 2009Not checked yet

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Overexpression of FPS in A. annua achieved a 2.5-fold increase in artemisinin yield from 0.5% to 1.3% of dry weight biomass.

“Banyai et al. (2010) achieved a 2.5-fold increase in artemisinin yield (from 0.5% to 1.3% of dry weight biomass) using the same approach (Table 4).”

• cites Banyai et al., 2010Not checked yet

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Follow-up studies by Schramek et al. (2010) indicated a predominant synthesis of artemisinin via the MEP pathway.

“While follow-up studies by Schramek et al. (2010) indicated a predominant synthesis of artemisinin via the MEP pathway, Ram et al. (2010) concluded that the MVA pathway, in particular the activity of 3-hydroxy-3methylglutaryl-CoA reductase (HMGR), was a limiting factor for artemisinin accumulation.”

• cites Schramek et al., 2010Not checked yet

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Total synthesis of artemisinin is not a commercially viable option.

“As the total synthesis of artemisinin is not a commercially viable option (Schmid and Hofheinz, 1983), research efforts aimed at developing alternative production processes, using advanced breeding (Graham et al., 2010) and metabolic engineering (Covello, 2008), were initiated in the early 2000s.”

• cites Schmid and Hofheinz, 1983Not checked yet

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Both MVA and MEP pathways contribute to artemisinin formation in A. annua.

“Based on experiments with specific inhibitors of the MVA and MEP pathways, Towler and Weathers (2007) demonstrated that both pathways contribute to artemisinin formation in A. annua.”

• cites Towler and Weathers, 2007Not checked yet

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Constitutive co-expression of ADS, FPS and CYP71AV1 in tobacco resulted in production of amorpha-1,4-diene and artemisinic alcohol up to 0.0006% of fresh weight biomass.

“co-expression of the ADS gene from A. annua (gene product targeted to plastids), an FPS gene from chicken (gene product targeted to plastids) and the CYP71AV1 gene from A. annua (codes for cytochrome P450-dependent monooxygenase responsible for oxidation of amorpha-1,4-diene) in tobacco resulted in the production of amorpha-1,4-diene and artemisinic alcohol (up to 0.0006% of fresh weight biomass)”

• cites Zhang et al., 2011Not checked yet

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Taken together, the studies discussed above indicate that dramatic (> 3-fold) increases in artemisinin yield were achieved mostly with low-yielding A. annua cultivars.

“Taken together, the studies discussed above indicate that dramatic (> 3-fold) increases in artemisinin yield were achieved mostly with low-yielding A. annua cultivars.”

Expression of cryptochrome photoreceptor gene from Arabidopsis in A. annua resulted in transgenic plants with various phenotypic irregularities, particularly when grown under blue light.

“The expression of the cryptochrome photoreceptor gene from Arabidopsis in A. annua resulted in the regeneration of transgenic plants with various phenotypic irregularities, particularly when grown under blue light (Hong et al., 2009).”

• cites Hong et al., 2009Not checked yet

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The accumulation levels of artemisinin in tobacco are currently too low to be competitive with other production alternatives.

“the accumulation levels of the antimalarial metabolite in tobacco are currently too low to be competitive with other production alternatives.”

Co-expression of plastid-targeted ADS and FPS in transgenic tobacco achieved an approximately 5,000-fold yield increase of amorpha-4,11-diene up to 0.0025% of fresh weight biomass compared to transgenics accumulating ADS in the cytosol.

“Wu et al. (2006) co-expressed genes coding for plastid-targeted ADS (from A. annua) and FPS (from chicken) in transgenic tobacco plants and achieved an approximately 5,000-fold yield increase of amorpha-4,11-diene (up to 0.0025% of fresh weight biomass) compared to transgenics accumulating ADS in the cytosol (Table 5).”

• cites Wu et al., 2006Not checked yet

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Transgenic isopentenyl transferase-overexpressing A. annua plants accumulated elevated levels of cytokinins, chlorophylls and artemisinin (up to 1.6% of dry weight biomass).

“Interestingly, transgenic isopentenyl transferase-overexpressing A. annua plants accumulated elevated levels of cytokinins, chlorophylls and artemisinin (up to 1.6% of the dry weight biomass).”

• cites Sa et al., 2001Not checked yet

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Currently, the artemisinin yield limit appears to be approximately 2% in the field.

“and approximately 2% in the field (Graham et al., 2010)”

• cites Graham et al., 2010Not checked yet

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Currently, the artemisinin yield limit appears to be around 3% of the dry weight under ideal growth conditions.

“Currently, the artemisinin yield limit appears to be around 3% of the dry weight under ideal growth conditions (Zhang et al., 2009)”

• cites Zhang et al., 2009Not checked yet

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Constitutive expression of a flowering promoting factor gene or CONSTANS gene from Arabidopsis in A. annua did not have notable effects on artemisinin yields.

“However, the constitutive expressions of a flowering promoting factor gene or the CONSTANS gene (both from Arabidopsis) in A. annua did not have notable effects on artemisinin yields (Wang et al., 2004(Wang et al., , 2007)).”

• cites Wang et al., 2004Not checked yet

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• cites Wang et al., 2007Not checked yet

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The main product of CYP71AV1 when accumulated recombinantly in yeast was artemisinic acid.

“Interestingly, the main product of CYP71AV1, when accumulated recombinantly in yeast, was artemisinic acid (Ro et al., 2006;Teoh et al., 2006)”

• cites Ro et al., 2006Not checked yet

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• cites Teoh et al., 2006Not checked yet

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Artemisinic acid was not detected in transgenic tobacco lines co-expressing ADS, FPS and CYP71AV1.

“which was not detected in the transgenic tobacco lines mentioned above.”

• cites Zhang et al., 2011Not checked yet

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An unidentified metabolite accumulated at high levels up to 0.02% of fresh weight biomass in transgenic plants expressing the triple-gene construct.

“A potentially yieldlimiting factor was the accumulation of an as yet unidentified metabolite at high levels (up to 0.02% of fresh weight biomass) in transgenic plants.”

• cites van Herpen et al., 2010Not checked yet

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Tobacco transformed with the ADS gene from A. annua accumulated amorpha-4,11-diene in trace amounts below 0.0000002% of fresh weight biomass.

“tobacco (Nicotiana tabacum L. cv.Xanthi) was transformed with the ADS gene from A. annua, which resulted in the accumulation of amorpha-4,11-diene, the product of ADS catalysis, in trace amounts (below 0.0000002% of fresh weight biomass) (Wallaart et al., 2001)”

• cites Wallaart et al., 2001Not checked yet

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Overexpression of a heterologous sesquiterpene synthase gene alone was not sufficient for production of high levels of sesquiterpenes in tobacco.

“As the cytosolic FPP supply appeared to be highly regulated, the overexpression of a heterologous sesquiterpene synthase gene alone was not sufficient for the production of high levels of sesquiterpenes in tobacco (Hohn and Ohlrogge, 1991;Wallaart et al., 2001;Wu et al., 2006).”

• cites Hohn and Ohlrogge, 1991Not checked yet

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• cites Wallaart et al., 2001Not checked yet

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• cites Wu et al., 2006Not checked yet

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Constitutive expression of mitochondrial-targeted A. annua ADS in infiltrated transgenic tobacco accumulated amorpha-1,4-diene to 0.0009% of fresh weight biomass.

“van Herpen et al. (2010) first tested a construct that constitutively expressed an A. annua ADS, targeted to mitochondria, in infiltrated transgenic tobacco (Nicotiana benthamiana Domin.).Amorpha-1,4-diene was accumulated to 0.0009% of fresh weight biomass.”

• cites van Herpen et al., 2010Not checked yet

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The artemisinin biosynthetic pathway can be expressed in a heterologous host plant.

“While the groundbreaking work by Farhi et al. (2011) demonstrated conclusively that the artemisinin biosynthetic pathway can be expressed in a heterologous host plant”

• cites Farhi et al., 2011Not checked yet

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The utility of AaWRKY1 for increasing artemisinin levels has not yet been demonstrated.

“Although the utility of such a transcription factor for increasing artemisinin levels has not yet been demonstrated, it is conceivable that the concept of a global pathway regulator is a viable strategy, as previously for several pathways involved in the formation of specialized plant metabolites (Kliebenstein and Osbourn, 2012).”

A triple-gene construct containing ADS, truncated HMGR and FPS led to a 7-fold yield increase of extractable amorpha-1,4-diene up to 0.006% of fresh weight biomass.

“The use of a triple- gene construct containing ADS (from A. annua; mitochondrial targeting), a truncated HMGR (from Arabidopsis; cytosolic targeting) and an FPS (from Arabidopsis; mitochondrial targeting) led to significant (7-fold) yield increases of extractable amorpha-1,4-diene (up to 0.006% of fresh weight biomass) (van Herpen et al., 2010).”

• cites van Herpen et al., 2010Not checked yet

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The corresponding amorphane acids were not detected in transgenic tobacco lines expressing DBR2.

“Once again, the corresponding amorphane acids were not detected.”

• cites Zhang et al., 2011Not checked yet

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AaWRKY1 transcript was represented at high abundance in a glandular trichome-specific cDNA library from A. annua.

“Ma et al. (2009b) noticed that the transcript of a transcription factor containing a WRKY motif (designated AaWRKY1) was represented at high abundance in a glandular trichome-specific cDNA library from A. annua.”

• cites Ma et al., 2009bNot checked yet

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Mitochondrial localization of a heterologous sesquiterpene synthase in Arabidopsis led to higher sesquiterpenoid accumulation levels compared to cytosolic expression.

“Previous work with Arabidopsis had demonstrated that a mitochondrial localization of a heterologous sesquiterpene synthase led to higher sesquiterpenoid accumulation levels compared to transgenics in which the gene product was expressed cytosolically, probably due to a higher plasticity of the FPP pool in mitochondria (Kappers et al., 2005).”

• cites Kappers et al., 2005Not checked yet

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A balance between various factors, including glandular trichome density, plant height and stature, leaf area, artemisinin concentration and agronomic performance, needed to be maintained to achieve high yields in the field.

“Not surprisingly, a balance between various factors, including glandular trichome density, plant height and stature, leaf area, artemisinin concentration and agronomic performance, needed to be maintained to achieve high yields in the field.”

• cites Graham et al., 2010Not checked yet

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Transcription factors to up-regulate the entire terpenoid pathway or large parts of it are currently unavailable.

“Transcription factors to up-regulate the entire terpenoid pathway (or large parts of it) are currently unavailable.”

The utility of regulators affecting initiation and density of glandular trichomes for increasing numbers of glandular trichomes has not yet been demonstrated conclusively for terpenoid producers.

“The utility of such regulators for increasing the numbers of glandular trichomes has not yet been demonstrated conclusively for terpenoid producers.”

Tobacco accumulates cembrane diterpenoids in leaf glandular trichomes.

“Tobacco accumulates cembrane diterpenoids in leaf glandular trichomes (Keene and Wagner, 1985).”

• cites Keene and Wagner, 1985Not checked yet

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Glandular trichome-specific promoters were used successfully to downregulate expression levels of CBTSs and a cembratrienol hydroxylase in tobacco.

“Glandular trichome-specific promoters were used successfully to downregulate the expression levels of cembratrienol synthases (CBTSs) and a cembratrienol hydroxylase in tobacco (Wang and Wagner, 2003;Ennajdaoui et al., 2010).”

• cites Wang and Wagner, 2003Not checked yet

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• cites Ennajdaoui et al., 2010Not checked yet

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Anthocyanin biosynthesis has been a paradigm for coordinate control of gene expression by bHLH transcription factors that physically interact with R2R3 MYB proteins to activate essentially all genes of the pathway.

“Following seminal discoveries in the 1990s, anthocyanin biosynthesis has been a paradigm for the coordinate control of gene expression by bHLH transcription factors that physically interact with R2R3 MYB proteins to activate essentially all genes of the pathway (reviewed in Grotewold, 2006).”

• cites Grotewold, 2006Not checked yet

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Without much optimization, transgenic tomato fruit produced taxa-4(5),11(12)-diene in quantities similar to those reported for taxol in Pacific yew bark.

“These data indicate that, without much optimization, transgenic tomato fruit produced taxa-4(5),11(12)diene in quantities similar to those reported for taxol in Pacific yew bark.”

• cites Kovacs et al., 2007Not checked yet

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There are very few examples in the literature where such promoters have been employed to drive expression of genes involved in terpenoid biosynthesis.

“There are very few examples in the literature where such promoters have been employed to drive the expression of genes involved in terpenoid biosynthesis.”

Several studies aimed at producing target terpenoids in experimental model plants have resulted in accumulation of breakdown products, apparently generated by nonspecific enzyme activities present in the host plant.

“Several studies aimed at producing certain target terpenoids in experimental model plants have resulted in the accumulation of breakdown products, apparently generated by nonspecific enzyme activities present in the host plant (Fig. 2).”

• cites Aharoni et al., 2003Not checked yet

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• cites Lewinsohn et al., 2001Not checked yet

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• cites Kappers et al., 2005Not checked yet

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Expression of TXS in Nicotiana sylvestris using the trichome-specific CBTS promoter yielded taxa-4(5),11(12)-diene at 0.002% of leaf fresh weight biomass.

“When the TXS gene was expressed in Nicotiana sylvestris (Speg.& Comes) using the trichome-specific CBTS promoter, a taxa-4(5),11(12)diene yield of 0.002% of leaf fresh weight biomass was reported (Tissier et al., 2008).”

• cites Tissier et al., 2008Not checked yet

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Substantial strain improvements will be necessary to enable cost-competitive synthesis of taxol in E. coli.

“indicating that substantial strain improvements will be necessary to enable a cost-competitive synthesis of the complex, highly functionalized, natural product in E. coli.”

• cites Ajikumar et al., 2010Not checked yet

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The types of metabolic conversions appear to depend on the host organism and the specific organ targeted for accumulation of an engineered terpenoid.

“The types of metabolic conversions appear to depend on the host organism and the specific organ targeted for the accumulation of an engineered terpenoid.”

Metabolism of target metabolites may also depend on their chemical structure.

“In addition, metabolism of target metabolites may also depend on their chemical structure.”

Inducible expression of TXS in Arabidopsis led to a 24-fold increase in taxa-4(5),11(12)-diene production up to 0.00006% compared with constitutive expression.

“When the TXS gene was expressed in Arabidopsis under control of a two-component glucocorticoid-inducible promoter, spraying of mature leaves with the inducer dexamethasone resulted in a more efficient recruitment of GGPP and consequently led to significant increases of taxa-4(5),11(12)-diene production up to 0.00006% (24-fold increase compared with production yields for lines constitutively expre”

• cites Besumbes et al., 2004Not checked yet

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Heterologous high-level production of taxa-4(5),11(12)-diene was achieved in E. coli, with elite strains producing about 1 g/L.

“Heterologous high-level production (elite strains produced about 1 g/L) of taxa-4(5),11(12)-diene, the first pathway-specific intermediate in taxol biosynthesis (Fig. 5), was recently achieved in E. coli by expressing a taxadiene synthase (TXS) gene (after codon optimization) from Taxus brevifolia (Ajikumar et al., 2010).”

• cites Ajikumar et al., 2010Not checked yet

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The yields of engineered terpenoids in experimental model plants have been extremely low, with the highest reported accumulation levels being around 0.01% of fresh weight biomass.

“Unfortunately, the yields of engineered terpenoids in experimental model plants have been extremely low, with the highest reported accumulation levels being around 0.01% of fresh weight biomass.”

Inducible promoter technology has potential for improving terpenoid yields.

“indicating the potential for improving yields by using inducible promoter technology (Besumbes et al., 2004) (Table 6).”

• cites Besumbes et al., 2004Not checked yet

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The flux from GGPP towards taxa-4(5),11(12)-diene was negligible compared to that required for synthesis of carotenoids and chlorophylls.

“The authors correctly noted that the flux from the common intermediate GGPP towards taxa-4(5),11(12)-diene was negligible compared to that required for the synthesis of carotenoids and chlorophylls”

• cites Besumbes et al., 2004Not checked yet

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Accumulation of both FPS and ADS in plastids resulted in a roughly 5,000-fold increase in amorpha-4,11-diene concentration in transgenic tobacco compared to transgenic plants expressing unaltered ADS gene encoding a cytosolic product.

“The most impressive example from their work is the accumulation of both FPS and ADS in plastids, which resulted in a roughly 5,000-fold increase in the concentration of amorpha-4,11-diene in transgenic tobacco plants when compared to transgenic plants expressing the unaltered ADS gene (encoding a cytosolic gene product).”

• cites Wu et al., 2006Not checked yet

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A new product, later identified as 5(12)-oxa-3(11)-cyclotaxane, accumulated in transgenic tobacco lines harbouring TXS, T5H and a cembratrienol synthase RNAi construct.

“Instead, a new product, which was later found to be 5(12)-oxa-3(11)-cyclotaxane (Fig. 5), was identified as being accumulated in transgenic tobacco lines (Table 6).”

• cites Rontein et al., 2008Not checked yet

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Localization of terpene synthases and, if necessary, appropriate prenyl diphosphate synthases in plastids or mitochondria is generally preferable over cytosolic localization because of tight regulation of cytosolic prenyl diphosphate pools.

“A localization of terpene synthases and, if necessary, the appropriate prenyl diphosphate synthases, in plastids or mitochondria is generally preferable over a cytosolic localization, because of the tight regulation of cytosolic prenyl diphosphate pools.”

Expression of a plastid-targeted TXS gene from T. baccata in Arabidopsis resulted in taxa-4(5),11(12)-diene accumulation of about 0.0000025% of leaf dry weight biomass.

“A TXS gene isolated from T. baccata (L.) (targeted to plastids) was constitutively expressed in Arabidopsis (Col-3 ecotype), which resulted in taxa-4(5),11(12)-diene accumulation of about 0.0000025% of leaf dry weight biomass (Besumbes et al., 2004) (Table 6).”

• cites Besumbes et al., 2004Not checked yet

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After introduction of T5H and a cytochrome P450 reductase from yew, the yield of taxa-4(5),11(12)-diene-5a-ol was under 60 mg/L.

“After introduction of the next following gene of the taxol pathway, which codes for taxadien-5a-ol hydroxylase (T5H), and a gene encoding an appropriate cytochrome P450 reductase from yew, a much lower yield of the expected taxane end product, taxa-4 (5),11(12)-diene-5a-ol, was reported (under 60 mg/L) (Ajikumar et al., 2010)”

• cites Ajikumar et al., 2010Not checked yet

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A similar undesirable phenotype was reported from transgenic Arabidopsis plants expressing the TXS gene from European yew.

“A similar, undesirable phenotype was also reported from transgenic Arabidopsis plants expressing the TXS gene from European yew.”

• cites Besumbes et al., 2004Not checked yet

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The moss might be developed into a production host for diterpenoids.

“indicating that the moss might be developed into a production host for diterpenoids.”

• cites Anterola et al., 2009Not checked yet

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In transgenic tobacco harbouring TXS, T5H and a cembratrienol synthase RNAi construct, the expected 5a-hydroxy-taxa-4(20),11(12)-diene could not be detected.

“In transgenic plants harbouring all three constructs, the expected 5a-hydroxy-taxa-4(20),11(12)-diene could not be detected (Rontein et al., 2008).”

• cites Rontein et al., 2008Not checked yet

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Tissue- or cell-type-specific promoters can provide advantages over constitutive promoters if the target terpenoid can be accumulated in specialized anatomical structures such as glandular trichomes or secretory cavities.

“Tissue-or celltype-specific promoters can provide advantages over constitutive promoters, if the target terpenoid can be accumulated in specialized anatomical structures such as glandular trichomes or secretory cavities.”

Several promoters that drive expression of transgenes in a glandular trichome-specific fashion have now been characterized.

“Several promoters that drive the expression of transgenes in a glandular trichomespecific fashion have now been characterized.”

Terpenoid volatiles emitted from transgenic plants can have positive effects on disease resistance.

“terpenoid volatiles emitted from transgenic plants can have positive effects on disease resistance (Aharoni et al., 2003;Beale et al., 2006;Kappers et al., 2005;Wu et al., 2006;Yu et al., 2012a).”

• cites Aharoni et al., 2003Not checked yet

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• cites Beale et al., 2006Not checked yet

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• cites Kappers et al., 2005Not checked yet

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Even in tobacco, where diterpene production in glandular trichomes is comparatively high, expression of heterologous genes has not yet resulted in high yields of novel diterpenoids.

“However, even in tobacco, where diterpene production in glandular trichomes is comparatively high, the expression of heterologous genes has not yet resulted in high yields of novel diterpenoids.”

If a reference line contains extremely low levels of a target metabolite, one might determine a several thousand fold increase in concentration while not achieving relevant production levels.

“If the reference line contains extremely low levels of a target metabolite, one might determine a several thousand fold increase in the concentration of that metabolite, while not achieving relevant production levels.”

In Artemisia annua engineered with HMGR, artemisinin increased.

“Artemisinin ↑”

• Also stated in: § Conclusions and future opportunities · plain

  “Artemisinin ↑”

• cites Aquil et al. (2009)Not checked yet

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• cites Nafis et al. (2011)Not checked yet

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In Artemisia annua engineered with CONSTANS zinc finger transcription factor (CO), no artemisinin increase was observed.

“No artemisinin increase”

• cites Wang et al. (2007)Not checked yet

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In Petunia hybrida W115 engineered with linalool synthase (LIS), no change in terpenoid profile was observed.

“No change”

• cites Wei et al. (2004)Not checked yet

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