Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids

Kollar, Andreas; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels J.; Punter, M.; Brendolise, Cyril; Cooney, Janine M.; Lateur, M.; Hausman, Jean-François; Larondelle, Yvan; Laing, William A.

doi:10.1111/nph.13996

Cited by 77 publications

(90 citation statements)

References 54 publications

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“…Betula platyphylla), lupeol is oxidized at the C28 position by a cytochrome P450-dependent monooxygenase (CYP) to form BA (Figure 1) (Hamberger and Bak, 2013). Recently, a number of studies have reported enzymes of the CYP716A family to be responsible for this modification (Andre et al, 2016;Carelli et al, 2011;Fukushima et al, 2011;Moses et al, 2014). Based on the literature, we selected the Medicago truncatula CYP716A12 and CPR for expression in P. tricornutum.…”

Section: Introducing a Cyp716a12 Cytochrome P450 And A P450-nadph Redmentioning

confidence: 99%

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

D’Adamo

Visconte

Lowe

et al. 2018

Plant Biotechnology Journal

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Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.

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Section: Introducing a Cyp716a12 Cytochrome P450 And A P450-nadph Redmentioning

confidence: 99%

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

D’Adamo

Visconte

Lowe

et al. 2018

Plant Biotechnology Journal

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“…Therefore, BfOSC2 is the first legume enzyme reported to produce mainly germanicol. Multifunctional OSCs that produce germanicol have been reported in Rhizophora styloza (mangrove; RsM1, produces germanicol : β‐amyrin : lupeol at a 63 : 33 : 4 ratio) (Basyuni et al ., ) and Malus domestica (apple; MdOSC4, produces germanicol : β‐amyrin : lupeol at an 82 : 14 : 4 ratio) (Andre et al ., ). Although BfOSC3 catalyzed the conversion of 2,3‐oxidosqualene to only α‐amyrin, we were puzzled by the inconsistency between the accumulation of α‐amyrin in B. forficata and the low productivity of BfOSC3 in yeast.…”

Section: Discussionmentioning

confidence: 97%

“…Plant/Enzyme name Accession no. et al, 2007) and Malus domestica (apple; MdOSC4, produces germanicol : b-amyrin : lupeol at an 82 : 14 : 4 ratio) (Andre et al, 2016). Although BfOSC3 catalyzed the conversion of 2,3oxidosqualene to only a-amyrin, we were puzzled by the inconsistency between the accumulation of a-amyrin in B. forficata and the low productivity of BfOSC3 in yeast.…”

Section: Enzymementioning

confidence: 94%

“…Codon optimization might enhance the a-amyrin-generating activity of BfOSC3 in yeast or other microbial expression systems. Several OSCs that produce b-amyrin or lupeol as a single product have been reported (Hayashi et al, 2001;Zhang et al, 2003;Kajikawa et al, 2005); however, no OSC that produces a-amyrin as a single product has been found (Andre et al, 2016;Yu et al, 2018), and may not exist in nature (Saimaru et al, 2007). To date, only MdOSC1 from M. domestica, CrAS from Catharanthus roseus, and IaAS1 from Ilax asprella have a product profile comprising ≥ 80% a-amyrin (Brendolise et al, 2011;Huang et al, 2012;Zheng et al, 2015).…”

Section: Enzymementioning

confidence: 99%

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Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α‐amyrin‐producing activity

et al. 2019

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Summary Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography–mass spectrometry. Through transcriptome analyses, homology‐based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α‐amyrin synthase activity. B. forficata highly accumulated α‐amyrin. We discovered an OSC with a preponderant α‐amyrin‐producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β‐amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α‐amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non‐model legume plant.

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“…Furthermore, OSC enzymes can produce precursors for both non-sterol triterpenes, like lupeol, and sterol triterpenes, like cycloartenol [32]. To characterize the enzymatic product, OSC genes are often ectopically expressed in yeast [21, 30, 33–35] or in plants via transient expression in Nicotiana benthamiana [36, 37]. …”

Section: Introductionmentioning

confidence: 99%

Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea

et al. 2016

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The red seaweed Laurencia dendroidea belongs to the Rhodophyta, a phylum of eukaryotic algae that is widely distributed across the oceans and that constitute an important source of bioactive specialized metabolites. Laurencia species have been studied since 1950 and were found to contain a plethora of specialized metabolites, mainly halogenated sesquiterpenes, diterpenes and triterpenes that possess a broad spectrum of pharmacological and ecological activities. The first committed step in the biosynthesis of triterpenes is the cyclization of 2,3-oxidosqualene, an enzymatic reaction carried out by oxidosqualene cyclases (OSCs), giving rise to a broad range of different compounds, such as the sterol precursors cycloartenol and lanosterol, or triterpene precursors such as cucurbitadienol and β-amyrin. Here, we cloned and characterized the first OSC from a red seaweed. The OSC gene was identified through mining of a L. dendroidea transcriptome dataset and subsequently cloned and heterologously expressed in yeast for functional characterization, which indicated that the corresponding enzyme cyclizes 2,3-oxidosqualene to the sterol precursor cycloartenol. Accordingly, the gene was named L. dendroidea cycloartenol synthase (LdCAS). A phylogenetic analysis using OSCs genes from plants, fungi and algae revealed that LdCAS grouped together with OSCs from other red algae, suggesting that cycloartenol could be the common product of the OSC in red seaweeds. Furthermore, profiling of L. dendroidea revealed cholesterol as the major sterol accumulating in this species, implicating red seaweeds contain a ‘hybrid’ sterol synthesis pathway in which the phytosterol precursor cycloartenol is converted into the major animal sterol cholesterol.

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Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids

Cited by 77 publications

References 54 publications

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α‐amyrin‐producing activity

Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea

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