Heterologous production of corosolic acid, a phyto-insulin, in agroinfiltrated Nicotiana benthamiana leaves.

IF 1.4 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jutapat Romsuk, Pisanee Srisawat, Jekson Robertlee, Shuhei Yasumoto, Kenji Miura, Toshiya Muranaka, Hikaru Seki
{"title":"Heterologous production of corosolic acid, a phyto-insulin, in agroinfiltrated <i>Nicotiana benthamiana</i> leaves.","authors":"Jutapat Romsuk, Pisanee Srisawat, Jekson Robertlee, Shuhei Yasumoto, Kenji Miura, Toshiya Muranaka, Hikaru Seki","doi":"10.5511/plantbiotechnology.24.0420a","DOIUrl":null,"url":null,"abstract":"<p><p>Triterpenoids, a group of specialized plant metabolites with substantial structural diversity, are promising for healthcare applications. Ursolic acid, a pentacyclic triterpenoid with therapeutic potential, is also important as a precursor of corosolic acid, which is known as a \"phyto-insulin\" for its insulin-like properties. Ursolic acid is synthesized from a linear 30-carbon precursor 2,3-oxidosqualene via cyclization to produce triterpene scaffold α-amyrin, followed by a series of oxidation steps at the C-28 position mediated by cytochrome P450 monooxygenases (CYPs) in the CYP716A subfamily. The Tsukuba system was developed for the high-level transient expression of foreign proteins in plant cells based on the use of a binary vector equipped with geminiviral replication system and a double terminator. In this study, we used the Tsukuba system to produce ursolic acid in <i>Nicotiana benthamiana</i> leaves via transient pathway reconstruction. We used an oxidosqualene cyclase identified from the medicinal legume <i>Bauhinia forficata</i>, exhibiting a preponderant α-amyrin-producing activity. Wild-type <i>Medicago truncatula</i> CYP716A12 and its mutants were assessed in terms of ursolic acid production. We improved the performance of MtCYP716A12 by co-expressing it with the appropriate cytochrome P450 reductase (CPR) isozyme as an electron-transfer partner and tested different <i>Agrobacterium</i> infiltration ratios to optimize the CPR : CYP ratio to maximize ursolic acid production. We also achieved high yield of corosolic acid by co-expressing <i>Avicennia marina</i> CYP716C53 with ursolic acid biosynthetic enzymes. Moreover, engineering of AmCYP716C53 significantly improved corosolic acid yield, resulting in a yield exceeding the content found in banaba leaves, a well-known rich source of corosolic acid.</p>","PeriodicalId":20411,"journal":{"name":"Plant Biotechnology","volume":"41 3","pages":"277-288"},"PeriodicalIF":1.4000,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11921146/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.5511/plantbiotechnology.24.0420a","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Triterpenoids, a group of specialized plant metabolites with substantial structural diversity, are promising for healthcare applications. Ursolic acid, a pentacyclic triterpenoid with therapeutic potential, is also important as a precursor of corosolic acid, which is known as a "phyto-insulin" for its insulin-like properties. Ursolic acid is synthesized from a linear 30-carbon precursor 2,3-oxidosqualene via cyclization to produce triterpene scaffold α-amyrin, followed by a series of oxidation steps at the C-28 position mediated by cytochrome P450 monooxygenases (CYPs) in the CYP716A subfamily. The Tsukuba system was developed for the high-level transient expression of foreign proteins in plant cells based on the use of a binary vector equipped with geminiviral replication system and a double terminator. In this study, we used the Tsukuba system to produce ursolic acid in Nicotiana benthamiana leaves via transient pathway reconstruction. We used an oxidosqualene cyclase identified from the medicinal legume Bauhinia forficata, exhibiting a preponderant α-amyrin-producing activity. Wild-type Medicago truncatula CYP716A12 and its mutants were assessed in terms of ursolic acid production. We improved the performance of MtCYP716A12 by co-expressing it with the appropriate cytochrome P450 reductase (CPR) isozyme as an electron-transfer partner and tested different Agrobacterium infiltration ratios to optimize the CPR : CYP ratio to maximize ursolic acid production. We also achieved high yield of corosolic acid by co-expressing Avicennia marina CYP716C53 with ursolic acid biosynthetic enzymes. Moreover, engineering of AmCYP716C53 significantly improved corosolic acid yield, resulting in a yield exceeding the content found in banaba leaves, a well-known rich source of corosolic acid.

在农侵的烟叶中异源产生的一种植物胰岛素——科罗索酸。
三萜是一类具有丰富结构多样性的特殊植物代谢物,在医疗保健领域具有广阔的应用前景。熊果酸是一种具有治疗潜力的五环三萜,它也是一种重要的植物果酸前体,因其具有胰岛素样特性而被称为“植物胰岛素”。熊果酸是由线性30碳前体2,3-氧化角鲨烯经环化生成三萜支架α-amyrin合成的,随后由CYP716A亚家族的细胞色素P450单加氧酶(CYPs)介导的C-28位点的一系列氧化步骤。Tsukuba系统是一种基于双病毒复制系统和双终止子的二元载体,用于在植物细胞中高水平瞬时表达外源蛋白的系统。在本研究中,我们利用筑波系统通过瞬时途径重建在烟叶中产生熊果酸。我们使用了一种从药用豆科植物紫荆中鉴定出的氧化角鲨烯环化酶,该酶具有较强的α-淀粉生成活性。对野生型短尾紫花苜蓿CYP716A12及其突变体进行了熊果酸产量评价。我们通过与合适的细胞色素P450还原酶(CPR)同工酶作为电子转移伙伴共表达MtCYP716A12,提高了其性能,并测试了不同的农杆菌浸润比,以优化CPR: CYP的比例,以最大限度地提高熊果酸的产量。我们还通过与熊果酸生物合成酶共表达海葵CYP716C53,获得了高产量的熊果酸。此外,AmCYP716C53基因的工程改造显著提高了花冠果酸的产量,其产量超过了众所周知的花冠果酸丰富来源——香蕉叶片中的含量。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Plant Biotechnology
Plant Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-PLANT SCIENCES
CiteScore
2.90
自引率
18.80%
发文量
45
审稿时长
6-12 weeks
期刊介绍: Plant Biotechnology is an international, open-access, and online journal, published every three months by the Japanese Society for Plant Biotechnology. The journal, first published in 1984 as the predecessor journal, “Plant Tissue Culture Letters” and became its present form in 1997 when the society name was renamed to Japanese Society for Plant Cell and Molecular Biology, publishes findings in the areas from basic- to application research of plant biotechnology. The aim of Plant Biotechnology is to publish original and high-impact papers, in the most rapid turnaround time for reviewing, on the plant biotechnology including tissue culture, production of specialized metabolites, transgenic technology, and genome editing technology, and also on the related research fields including molecular biology, cell biology, genetics, plant breeding, plant physiology and biochemistry, metabolic engineering, synthetic biology, and bioinformatics.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信