Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots

Tsukagoshi, Yuki; Ohyama, Kiyoshi; Seki, Hikaru; Akashi, Tomoyoshi; Muranaka, Toshiya; Suzuki, Hideyuki; Fujimoto, Yoshinori

doi:10.1016/j.phytochem.2016.03.010

Cited by 22 publications

(14 citation statements)

References 37 publications

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“…The hydroxylation steps in plants are presumably mediated by CYP450s as they are in insects [ 12 ]. Tsukagoshi et al [ 13 ] identified a CYP450 enzyme CYP71D443, which catalyzes the C-22 hydroxylation of 20E in Ajuga hairy roots. There was only one reported CYP450 enzyme to be involved in the 20E biosynthesis of plants so far [ 13 ].…”

Section: Resultsmentioning

confidence: 99%

“…Tsukagoshi et al [ 13 ] identified a CYP450 enzyme CYP71D443, which catalyzes the C-22 hydroxylation of 20E in Ajuga hairy roots. There was only one reported CYP450 enzyme to be involved in the 20E biosynthesis of plants so far [ 13 ]. In order to identify the possible involvement of CYP450s in 20E biosynthesis, up-regulated CYP450s unigenes in roots were selected from DEGs.…”

Section: Resultsmentioning

confidence: 99%

“…In insects, cytochrome P-450 monooxygenases (CYP450s), which catalyze the bioreaction from cholesterol to 20E, have been cloned and identified. However, only one CYP450 enzyme in plants has been identified to be involved in 20E biosynthesis in the hairy roots of Ajuga reptans [ 13 ]. To date, the biosynthesis of 20E, especially the origin of the precursor IPP and its downstream pathway, remains largely unknown.…”

Section: Introductionmentioning

confidence: 99%

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Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea

Lei

Xia

Wang

et al. 2018

IJMS

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Cyanotis arachnoidea contains a rich array of phytoecdysteroids, including 20-hydroxyecdysone (20E), which displays important agrochemical, medicinal, and pharmacological effects. To date, the biosynthetic pathway of 20E, especially the downstream pathway, remains largely unknown. To identify candidate genes involved in 20E biosynthesis, the comparative transcriptome of C. arachnoidea leaf and root was constructed. In total, 86.5 million clean reads were obtained and assembled into 79,835 unigenes, of which 39,425 unigenes were successfully annotated. The expression levels of 2427 unigenes were up-regualted in roots with a higher accumulation of 20E. Further assignments with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified 49 unigenes referring to the phytoecdysteroid backbone biosynthesis (including 15 mevalonate pathway genes, 15 non-mevalonate pathway genes, and 19 genes for the biosynthesis from farnesyl pyrophosphate to cholesterol). Moreover, higher expression levels of mevalonate pathway genes in roots of C. arachniodea were confirmed by real-time quantitative PCR. Twenty unigenes encoding CYP450s were identified to be new candidate genes for the bioreaction from cholesterol to 20E. In addition, 90 transcription factors highly expressed in the roots and 15,315 unigenes containing 19,158 simple sequence repeats (SSRs) were identified. The transcriptome data of our study provides a valuable resource for the understanding of 20E biosynthesis in C. arachnoidea.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea

Lei

Xia

Wang

et al. 2018

IJMS

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“…cDNA Cloning of ArDWF1-The details of the preparation of an expression sequence tag library from the Ajuga hairy roots will be reported elsewhere (26). Keyword searches of the annotated expression sequence tags identified only one DWF1 contig.…”

Section: Methodsmentioning

confidence: 99%

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Tsukagoshi

Suzuki

Seki

et al. 2016

Journal of Biological Chemistry

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Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C 28 -and C 29 -⌬ 24(28) -olefinic sterols to 24-methyl-and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the ⌬ 24(25) intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-2 H 3 ,28-13 C)-24-methylenecholesterol, followed by 13 C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a ⌬ 24(25) intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.In vertebrates, cholesterol is the major sterol, whereas, in higher plants, commonly occurring sterols are C 29 sterols, sitosterol (1) and stigmasterol, and a C 28 sterol, campesterol (2). Campesterol is frequently accompanied by dihydrobrassicasterol (3) (1, 2). Latter steps in phytosterol biosynthesis have received considerable attention because of their intriguing chemistry, biochemistry, and biology. Recent studies through genetic mutations have uncovered an essential role of these phytosterols at the cellular level in hormone signaling, organized divisions, and embryo patterning (3).It is generally regarded that these typical plant sterols are biosynthesized from precursor ⌬ 24(28) -olefinic sterols, i.e. isofucosterol (4) and 24-methylenecholesterol (5), in two steps: a double-bond isomerization from ⌬ 24(28) to ⌬ 24(25) (leading to 24-ethyldesmosterol (6) and 24-methyldesmosterol (7)) and a reduction of the ⌬ 24(25) intermediates to the saturated C 29 and C 28 sterols (1 and 2/3). Interestingly, the reduction of 24-ethyldesmosterol affords only sitosterol, whereas the reduction of 24-methyldesmosterol yields a C-24 epimeric mixture of campesterol and dihydrobrassicasterol (1, 2). The two-step mechanism has been proposed mainly on the basis of the m...

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“…CYP5150AN1 was identified to form a 2β-hydroxyl on RSS and produced 2β-OH-RSS as the sole product [53]. In addition to the fungal enzymes, CYP71D443 from Ajuga hairy roots was characterized in yeast as a C-22 hydroxylase that functions on 3β-hydroxy-5β-cholestan-6-one (5β-ketone) and 3β-hydroxy-5β-cholest-7-en-6-one (5β-ketol) [54].…”

Section: Steroid Bioproduction In Yeast Through Biotransformationmentioning

confidence: 99%

Yeast as a promising heterologous host for steroid bioproduction

2020

Journal of Industrial Microbiology and Biotechnology

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With the rapid development of synthetic biology and metabolic engineering technologies, yeast has been generally considered as promising hosts for the bioproduction of secondary metabolites. Sterols are essential components of cell membrane, and are the precursors for the biosynthesis of steroid hormones, signaling molecules, and defense molecules in the higher eukaryotes, which are of pharmaceutical and agricultural significance. In this mini-review, we summarize the recent engineering efforts of using yeast to synthesize various steroids, and discuss the structural diversity that the current steroidproducing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes.

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Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots

Cited by 22 publications

References 37 publications

Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea

Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Yeast as a promising heterologous host for steroid bioproduction

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