Functional Characterization of CYP716 Family P450 Enzymes in Triterpenoid Biosynthesis in Tomato

Yasumoto, Shuhei; Seki, Hikaru; Shimizu, Yuko; Fukushima, Ery Odette; Muranaka, Toshiya

doi:10.3389/fpls.2017.00021

Cited by 42 publications

(36 citation statements)

References 42 publications

(63 reference statements)

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“…This study contributes to an increased understanding of the role of CYP716A enzymes in the diversification of the triterpenoid backbone (Figure 4; Miettinen et al 2017;Misra et al 2017;Tamura et al 2017aTamura et al , 2017bYasumoto et al 2016Yasumoto et al , 2017. We also identified previously reported putative compounds, oleanolic, ursolic, and betulinic aldehydes (3, 7, 11), by synthesizing authentic standards and comparing their Rts and mass spectra with those of chromatographic peaks obtained from heterologous yeast extracts.…”

Section: Discussionmentioning

confidence: 72%

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

Suzuki

Fukushima

Umemoto

et al. 2018

Plant Biotechnology

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Several enzymes of the CYP716A subfamily have been reported to be involved in triterpenoid biosynthesis. Members of this subfamily oxidize various positions along the triterpenoid backbone and the majority of them catalyze a three-step oxidation at the C-28 position. Interestingly, C-28 oxidation is a common feature in oleanolic acid, ursolic acid, and betulinic acid, which are widely distributed in plants and exhibit important biological activities. In this work, three additional CYP716A enzymes isolated from olive, sugar beet, and coffee, were characterized as multifunctional C-28 oxidases. Semi-quantitative comparisons of in vivo catalytic activity were made against the previously characterized enzymes CYP716A12, CYP716A15, and CYP716A52v2. When heterologously expressed in yeast, the isolated enzymes differed in both catalytic activity and substrate specificity. This study indicates that the screening of enzymes from different plants could be a useful means of identifying enzymes with enhanced catalytic activity and desired substrate specificity. Furthermore, we show that "naturally-evolved" enzymes can be useful in the heterologous production of pharmacologically and industrially important triterpenoids.

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Section: Discussionmentioning

confidence: 72%

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

Suzuki

Fukushima

Umemoto

et al. 2018

Plant Biotechnology

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“…The CYP716A and CYP72A subfamilies are the primary CYP450 gene families involved in pentacyclic triterpenoid saponin diversification, with the CYP716A family being the largest multifunctional C28oxidase family involved in such oleanane-type triterpenoid saponins biosynthesis [16,27,28] (Fig. 8).…”

Section: Identification Of Candidate Cyp450s Involved In Triterpenoidmentioning

confidence: 99%

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponin biosynthesis

Cheng

Liu

Tong

et al. 2020

Preprint

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Background: Members of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. Results: In the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding >300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum.Conclusions: This study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.

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“…To further explore the functional roles and evolutionary relationships for these A. elata CYP450s, we next constructed a phylogenetic tree incorporating these 111 CYP540s along with 59 CYP450s from other plants, including A. thaliana (37), P. ginseng (20), Daucus carota (2) and Polygala tenuifolia (1).…”

Section: Cyp450 Family Member Phylogenetic and Conserved Motif Analysesmentioning

confidence: 99%

Identification and analysis of CYP450 supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponins biosynthesis

Cheng

Liu

Tong

et al. 2020

Preprint

View full text Add to dashboard Cite

BackgroundMembers of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating secondary metabolites biosynthesis. However, the systematic identification and bioinformatics analysis of CYP450s have not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. ResultsIn the present study we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following the annotation and classification of these unigenes, we were able to identify two pathways and 19 putative genes associated with the synthesis of triterpenoid saponins in these plants, with qRT-PCR subsequently being used to validate these gene expression patterns. Scanning with the CYP450 model from Pfam resulted in the identification of 111 full-length and 143 partial-length CYP450s, with the full-length CYP450s being further clustered into 7 clans and 36 families. Through phylogenetic and conserved motif analyses, we were further able to group these CYP450 proteins into two primary branches: A-type (53%) and non-A type (47%). We further conducted representative protein sequence alignment for these CYP450 family members, with secondary elements being assigned in light of the recently published Arabidopsis CYP90B1 structure. Using the available sequence information, we further identified predicted substrate recognition sites (SRSs) and substrate binding sites within these putative proteins.We further assessed the expression patterns of these 111 CYP450 genes across A. elata tissues, with 12 members of this gene family being selected at random for qRT-PCR validation. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 was identified as being the most likely to play a role in hederagenin biosynthesis. Finally, we assessed the subcellular localization of these CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum.ConclusionsThis study presents a systematic analysis of the CYP450 gene family in A. elata and provided a foundation for further functional characterization of CYP450 genes.

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Functional Characterization of CYP716 Family P450 Enzymes in Triterpenoid Biosynthesis in Tomato

Cited by 42 publications

References 42 publications

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponin biosynthesis

Identification and analysis of CYP450 supergene family members from the transcriptome of Aralia elata (Miq.) Seem reveal candidate genes for triterpenoid saponins biosynthesis

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