Functional analysis of orthologous artemisinic aldehyde &amp;#916;11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing &lt;i&gt;Artemisia absinthium&lt;/i&gt;

Muangphrom, Paskorn; Suzuki, Munenori; Seki, Hikaru; Fukushima, Ery Odette; Muranaka, Toshiya

doi:10.5511/plantbiotechnology.14.0807a

Cited by 9 publications

(8 citation statements)

References 34 publications

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“…Primers for PCR are listed in Table S1. These PCR products were then cloned directly into a pENTR/D‐TOPO entry vector (Invitrogen, Carlsbad, CA, USA), as previously described (Muangphrom et al ., ). Sequence determination of these genes within the entry clones was then performed.…”

Section: Methodsmentioning

confidence: 97%

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Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

et al. 2019

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SummaryGlycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins.

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Section: Methodsmentioning

confidence: 97%

“…Site‐directed mutagenesis of UGT73P12 was performed in the entry clone by inverse PCR with the gene‐specific primers, 19–26. Expression vectors were produced by an LR clonase reaction between the entry clones and a modified pColdTF vector (Novagen, Madison, WI, USA) harboring a gateway cassette (Muangphrom et al ., ).…”

Section: Methodsmentioning

confidence: 97%

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

et al. 2019

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“…In the artemisinin biosynthesis, artemisinic aldehyde Δ11(13) reductase (DBR) can convert artemisinic aldehyde into dihydroartemisinic aldehyde [30]. DBR1 has been cloned by Zhang [33], which can perform catalytic hydrogenation of 2,3 unsaturated aldehyde.…”

Section: Discussionmentioning

confidence: 99%

“…The enzymatic activity of the recombinant DBR1 enzyme was assayed according to the method by Muangphrom et al [30] and modified. The activity was determined GC using β-ionone as substrate.…”

Section: Methodsmentioning

confidence: 99%

Cloning and characterization of enoate reductase with high β-ionone to dihydro-β-ionone bioconversion productivity

et al. 2018

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BackgroundDihydro-β-ionone is a principal aroma compound and has received considerable attention by flavor and fragrance industry. The traditional method of preparing dihydro-β-ionone has many drawbacks, which has restricted its industrial application. Therefore, it is necessary to find a biotechnological method to produce dihydro-β-ionone.ResultsIn this study, the enoate reductase with high conversion efficiency of β-ionone to dihydro-β-ionone, DBR1, was obtained by screening four genetically engineered bacteria. The product, dihydro-β-ionone, was analyzed by GC and GC-MS. The highest dihydro-β-ionone production with 308.3 mg/L was detected in the recombinant strain expressing DBR1 which was later on expressed and purified. Its optimal temperature and pH were 45 °C and 6.5, respectively. The greatest activity of the purified enzyme was 356.39 U/mg using β-ionone as substrate. In the enzymatic conversion system, 1 mM of β-ionone was transformed into 91.08 mg/L of dihydro-β-ionone with 93.80% of molar conversion.ConclusionDBR1 had high selectivity to hydrogenated the 10,11-unsaturated double bond of β-ionone as well as high catalytic efficiency for the conversion of β-ionone to dihydro-β-ionone. It is the first report on the bioconversion of β-ionone to dihydro-β-ionone by using enoate reductase.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0438-x) contains supplementary material, which is available to authorized users.

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“…Column chromatography was performed using silica gel 60N (spherical, neutral), with particle sizes 63-210 µm (Kanto Chemical, Tokyo, Japan) or 10% AgNO 3 impregnated silica gel (Wako, Osaka, Japan). The amorpha-4,11-diene and koidzumiol standards were prepared as described previously (Muangphrom et al 2014(Muangphrom et al , 2016. α-Bisabolol was purchased from Tokyo Chemical Industry (Tokyo, Japan).…”

Section: General Experimental Proceduresmentioning

confidence: 99%

Identification and characterization of a novel sesquiterpene synthase, 4-amorphen-11-ol synthase, from <i>Artemisia maritima</i>

Muangphrom

Seki

Matsumoto

et al. 2018

Plant Biotechnology

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Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

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Functional analysis of orthologous artemisinic aldehyde Δ11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing <i>Artemisia absinthium</i>

Cited by 9 publications

References 34 publications

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

Cloning and characterization of enoate reductase with high β-ionone to dihydro-β-ionone bioconversion productivity

Identification and characterization of a novel sesquiterpene synthase, 4-amorphen-11-ol synthase, from <i>Artemisia maritima</i>

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