藜芦22（R）-羟基胆固醇合成基因功能验证与酵母异源合成

doi:10.13560/j.cnki.biotech.bull.1985.2024-0412

生物技术通报 ›› 2024, Vol. 40 ›› Issue (12): 170-181.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0412

藜芦22（R）-羟基胆固醇合成基因功能验证与酵母异源合成

徐艳姣¹^,²^,³^,⁴(), 洪开云¹^,²^,³, 卢宜旺¹^,²^,³, 汪长清¹^,²^,³, 梁艳丽¹^,²^,³, 和四梅¹^,²^,³()

1.云南农业大学农学与生物技术学院，昆明 650201
2.云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心，昆明 650201
3.云南农业大学云南省药用植物生物学重点实验室，昆明 650201
4.丘北长水实验中学，文山 663200

收稿日期:2024-04-30 出版日期:2024-12-26 发布日期:2025-01-15
通讯作者: 和四梅，女，博士，讲师，研究方向：天然产物生物合成；E-mail: simeiheynau@163.com
作者简介:徐艳姣，女，硕士研究生，研究方向：药用植物生物合成；E-mail: 1191556757@qq.com
基金资助:
云南农业大学科研启动基金项目(KY2022-02);云南省基础研究专项(202201AU070236);云南省科技厅重大科技专项计划(202302AA310030);科技人才与平台计划“中青年学术和技术带头人后备人才项目”(202005AC160040)

Function Verification of Genes Involved in 22 (R)-hydroxycholesterol Biosynthesis in Veratrum nigrum and Their Heterologous Synthesis

XU Yan-jiao¹^,²^,³^,⁴(), HONG Kai-yun¹^,²^,³, LU Yi-wang¹^,²^,³, WANG Chang-qing¹^,²^,³, LIANG Yan-li¹^,²^,³, HE Si-mei¹^,²^,³()

1. College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201
2. National-Local Joint Engineering Research Center on Germplasm Innovation & Utilization of Chinese Medicinal Materials in Southwestern China, Yunnan Agricultural University, Kunming 650201
3. Key Laboratory of Medicinal Plant Biology of Yunnan Province, Yunnan Agricultural University, Kunming 650201
4. Long Spring Experimental Qiubei High School, Wenshan 663200

Received:2024-04-30 Published:2024-12-26 Online:2025-01-15

摘要/Abstract

摘要：

【目的】22（R）-羟基胆固醇是藜芦甾体生物碱合成的重要前体，通过异源表达验证藜芦（Veratrum nigrum）中胆固醇C-22位羟化酶的功能，并构建酵母底盘生产22（R）-羟基胆固醇，为藜芦甾体生物碱的生物合成途径解析奠定基础。【方法】基于藜芦转录组数据，从藜芦根中克隆3条全长的CYP90B亚家族基因序列，构建到酵母Y33表达载体，并转入胆固醇酵母底盘进行功能验证。通过高效液相色谱、液相色谱-质谱联用仪检测酵母摇瓶发酵产物，筛选到具有胆固醇C-22位羟化酶催化功能的酶VnCYP90B27-1，利用多片段组装、同源重组和醋酸锂转化等方法将VnCYP90B27-1整合到酵母染色体，构建22（R）-羟基胆固醇的酵母底盘。【结果】实时荧光定量PCR（RT-qPCR）显示，3条候选基因在根和叶中的表达与转录组表达趋势一致，VnCYP90B27-1在藜芦根中的表达量极显著高于叶。系统发育树结果表明，VnCYP90B27-1与加州藜芦的VcCYP90B27和藜芦的VnCYP90B27有较高的同源性，同属于CYP90B亚家族。此外，酵母异源表达结果表明，VnCYP90B27-1具有胆固醇C-22位羟化酶功能，并成功实现酿酒酵母异源合成22（R）-羟基胆固醇，摇瓶产量达（5.37±0.37）mg/L。【结论】成功克隆并验证了藜芦22（R）-羟基胆固醇合成基因VnCYP90B27-1的功能，构建了22（R）-羟基胆固醇酵母底盘，证明在酿酒酵母中VnCYP90B27-1的催化活性比加州藜芦的VcCYP90B27高，为甾体生物碱异源合成提供基因资源。

关键词: 藜芦, CYP90B27, 功能验证, 22（R）-羟基胆固醇, 酵母底盘

Abstract:

【Objective】 22 (R)-hydroxycholesterol is an important precursor of veratrol alkaloid biosynthesis. The function of cholesterol C-22 hydroxylase in Veratrum nigrum was verified by heteroexpression, and the yeast chassis was constructed to produce 22 (R)-hydroxycholesterol, which lays a foundation for deciphering the biosynthetic pathway of veratrol alkaloid.【Method】 Based on the transcriptome data of V. nigrum, three full-length CYP90B subfamily gene sequences were cloned from the root of V. nigrum, the yeast Y33 expression vector was constructed and transferred into the cholesterol yeast chassis for functional verification. The yeast fermentaed products in shake flask were detected by high performance liquid chromatography and liquid chromatography-mass spectrometry. The enzyme VnCYP90B27-1 with cholesterol C-22 hydroxylase catalytic function was screened. VnCYP90B27-1 was integrated into the yeast chromosome by multi-fragment assembly, homologous recombination and lithium acetate conversion to construct a yeast chassis for 22 (R)-hydroxycholesterol. 【Result】 RT-qPCR showed that the expressions of the three candidate genes in the roots and leaves was consistent with the trend of transcriptome expression. The expression of VnCYP90B27-1 in the roots was significantly higher than that in the leaves. The results of phylogenetic tree showed that VnCYP90B27-1 had high homology with VcCYP90B27 of Veratrum californicum and VnCYP90B27 of V. nigrum, and belonged to CYP90B subfamily. In addition, the results of heterologous expression in the yeast showed that VnCYP90B27-1 had the function of cholesterol C-22 hydroxylase, and successfully achieved the heterologous synthesis of 22 (R)-hydroxycholesterol in S. cerevisiae, with the yield of (5.37±0.37) mg / L in shake flask. 【Conclusion】The function of 22 (R)-hydroxycholesterol synthesis gene VnCYP90B27-1 from V. nigrum is successfully cloned and verified. The 22 (R)-hydroxycholesterol yeast chassis is constructed. It is proved that the catalytic activity of VnCYP90B27-1 in S. cerevisiae is higher than that of VcCYP90B27 from V. californicum, which provides genetic resources for heterologous synthesis of steroid alkaloids.

Key words: Veratrum nigrum, CYP90B27, function verification, 22 (R)-hydroxycholesterol, yeast chassis

徐艳姣, 洪开云, 卢宜旺, 汪长清, 梁艳丽, 和四梅. 藜芦22（R）-羟基胆固醇合成基因功能验证与酵母异源合成[J]. 生物技术通报, 2024, 40(12): 170-181.

XU Yan-jiao, HONG Kai-yun, LU Yi-wang, WANG Chang-qing, LIANG Yan-li, HE Si-mei. Function Verification of Genes Involved in 22 (R)-hydroxycholesterol Biosynthesis in Veratrum nigrum and Their Heterologous Synthesis[J]. Biotechnology Bulletin, 2024, 40(12): 170-181.

图/表 13

图1 环巴胺生物合成途径推测

Fig. 1 Speculated biosynthetic pathway of cyclopamine

表1 RT-qPCR引物和基因克隆引物序列表

Table 1 Sequences of RT-qPCR primers and gene cloning primers

基因 Gene	引物序列 Primer sequence（5'-3'）	用途 Purpose
ACT	CGAGAGCAATGTACGCAAGC GGTGTCAGCCATACTGTCCC	实时荧光定量 PCR RT-qPCR
VnigCYP063	GTCGATCCCCATCAACTTGC ACTCGAGGAAGTAGATGGCG
VnigCYP106	ACGTGGTTAGGTTTGTGCAC TCCACGGATCGAACTGTTGA
VnigCYP119	TCAGGGCGGTACATATGGAC CAACTTGGCGAGCTCAGATC
Y33-VnigCYP063	cagtcgacctcgaatctagaATGGCGATGGAGCTCCTC catgatgcggccctctagaTCAGTCCCCGAGTTTTTCGAG	基因克隆 Gene clone
Y33-VnigCYP106	cagtcgacctcgaatctagaATGTCGACAATAAGAGAGCTACTC acatgatgcggccctctagaTCATGTTACGGCGCGAACCTTG
Y33-VnigCYP119	cagtcgacctcgaatctagaATGGAACCTGTGGCGATTCTAC acatgatgcggccctctagaCTACATCATCTCCATCTCATTTGGC

图2 候选基因与其他物种的22羟化酶的进化树和motif分析

Fig. 2 Phylogenetic tree and motif analysis of candidate genes and 22-hydroxylases of other species

图3 藜芦的CYP90B27序列比对分析

Fig. 3 Multiple sequence alignment analysis of V. nigrum CYP90B27

图4 胆固醇C-22位羟化酶基因表达模式分析 A：转录组表达量TPM值；B：qPCR表达量（**P<0.01，*P<0.05）

Fig. 4 Analysis of gene expression pattern of cholesterol C-22 hydroxylase A : TPM value of transcriptome expression ; B: qPCR expression,（**P<0.01，*P<0.05）

图5 藜芦RNA及基因扩增琼脂糖凝胶电泳图 A：1、2：根；3、4：叶；B：1：VnigCYP063, 2：VnigCYP1063, 3：Vnig-CYP119, 4：VcCYP90B27v1

Fig. 5 Agarose gel electrophoresis of V. nigrum RNA and gene amplification A : 1, 2 : root ; 3, 4 : leaf ; B : 1 : VnigCYP063, 2 : VnigCYP1063, 3 : VnigCYP119, 4 : VcCYP90B27v1. M: Normal Run 250bp-II DNA ladder / DNA marker

图6 Y33载体线性化及菌落PCR凝胶电泳图 A：Y33载体酶切电泳图；B：候选基因重组转菌水PCR

Fig. 6 Y33 vector linearization and colony PCR gel electrophoresis A: Y33 vector enzyme digestion electrophoresis map; B : candidate gene recombinant bacteria water PCR

图7 VnigCYP063和VcCYP90B27在Vg13底盘中的发酵产物

Fig. 7 Fermentated products of VnigCYP063 and VcCYP90B27 in Vg13 chassis

图8 VnigCYP063和VcCYP90B27在 Vg13底盘中的发酵产物LC-MS检测图 A：不同酵母产物TIC（m/z 425）保留时间图；B：22（R）-羟基胆固醇标准品质谱图；C/D：Y33-VnigCYP063和Y33-VcCYP90B27酵母产物22（R）-羟基胆固醇质谱图

Fig. 8 Fermentation products of VnigCYP063 and VcCYP90B27 in Vg13 chassis A: Retention time diagram of TIC（m/z425）of different yeast products; B: 22（R）-hydroxycholesterol standard mass spectrum; C/D: Y33-VnigCYP063 and Y33-VcCYP90B27 yeast products 22（R）-hydroxycholesterol mass spectra

图9 在酿酒酵母中过表达基因模块示意图

Fig. 9 Schematic representation of overexpressing gene module in Saccharomyces cerevisiae

图10 融合PCR 凝胶电泳图

Fig. 10 Fusion PCR gel electrophoresis

图11 22（R）-羟基胆固醇底盘菌产物中的发酵产物（1）22（R）-羟基胆固醇，（2）胆固醇

Fig. 11 22（R）-hydroxy cholesterol chassischia products of fermentated products （1）22（R）-hydroxycholester（1）.（2）Cholesterol

图12 酵母菌发酵产物测定图 A：22（R）-羟基胆固醇标准曲线，B：底盘菌产物定量图，CHOL为胆固醇，22（R）-CHOL为22（R）-羟基胆固醇，小写字母表示不同菌株间产量差异达到（P<0.05）显著水平

Fig. 12 Determination of yeast fermentated products A: Standard curve of 22（R）-hydroxycholesterol. B: Quantitative analysis of chassisobacteria products，CHOL is cholesterol,22（R）-CHOL is 22-（R）-hydroxycholesterol. The lowercase letter indicates that the yield difference between different strains reached a significant level of（P<0.05）

参考文献 29

[1]	成孟华, 饶高雄. 藜芦属植物化学成分和药理作用的研究进展[J]. 中草药, 2021, 52(18): 5758-5774.
	Cheng MH, Rao GX. Research progress on chemical constituents and pharmacological activities of plants from Veratrum[J]. Chin Tradit Herb Drugs, 2021, 52(18): 5758-5774.
[2]	Xiang LM, Wang YH, Yi XM, et al. Steroidal alkaloid glycosides and phenolics from the immature fruits of Solanum nigrum[J]. Fitoterapia, 2019, 137: 104268.
[3]	李文希, 张屏, 李福全, 等. 藜芦甾体生物碱抗肿瘤活性研究进展[J]. 世界科学技术-中医药现代化, 2020, 22(1): 118-125.
	Li WX, Zhang P, Li FQ, et al. Research progress on antitumor activity of steroidal alkaloids in genus Veratrum[J]. Mod Tradit Chin Med Mater Med World Sci Technol, 2020, 22(1): 118-125.
[4]	张蒙珍, 高丽娟, 徐世芳, 等. 藜芦属植物甾体生物碱及其药理活性研究进展[J]. 中国中药杂志, 2020, 45(21): 5129-5142.
	Zhang MZ, Gao LJ, Xu SF, et al. Advances in studies on steroidal alkaloids and their pharmacological activities in genus Veratrum[J]. China J Chin Mater Med, 2020, 45(21): 5129-5142.
[5]	Giannis A, Heretsch P, Sarli V, et al. Synthesis of cyclopamine using a biomimetic and diastereoselective approach[J]. Angew Chem Int Ed Engl, 2009, 48(42): 7911-7914.
[6]	Wang D, Yu ZJ, Guan M, et al. Comparative transcriptome analysis of Veratrum maackii and Veratrum nigrum reveals multiple candidate genes involved in steroidal alkaloid biosynthesis[J]. Sci Rep, 2023, 13(1): 8198.
[7]	Xu LP, Wang D, Chen J, et al. Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production[J]. Metab Eng, 2022, 70: 115-128. doi: 10.1016/j.ymben.2022.01.013 pmid: 35085779
[8]	Lin Y, Wang YN, Zhang GH, et al. Reconstruction of engineered yeast factory for high yield production of ginsenosides Rg3 and Rd[J]. Front Microbiol, 2023, 14: 1191102.
[9]	Bureau JA, Oliva ME, Dong YM, et al. Engineering yeast for the production of plant terpenoids using synthetic biology approaches[J]. Nat Prod Rep, 2023, 40(12): 1822-1848.
[10]	Li MK, Ma MY, Wu ZK, et al. Advances in the biosynthesis and metabolic engineering of rare ginsenosides[J]. Appl Microbiol Biotechnol, 2023, 107(11): 3391-3404. doi: 10.1007/s00253-023-12549-6 pmid: 37126085
[11]	Augustin MM, Ruzicka DR, Shukla AK, et al. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells[J]. Plant J, 2015, 82(6): 991-1003.
[12]	寇呈熹. 藜芦嗪的生物合成研究[D]. 哈尔滨: 东北林业大学, 2023.
	Kou CX. Study on biosynthesis of verazine[D]. Harbin:Northeast Forestry University, 2023.
[13]	Seki H, Tamura K, Muranaka T. P450s and UGTs: key players in the structural diversity of triterpenoid saponins[J]. Plant Cell Physiol, 2015, 56(8): 1463-1471. doi: 10.1093/pcp/pcv062 pmid: 25951908
[14]	Sonawane PD, Pollier J, Panda S, et al. Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism[J]. Nat Plants, 2016, 3: 16205. doi: 10.1038/nplants.2016.205 pmid: 28005066
[15]	Yin Y, Gao L, Zhang X, et al. A cytochrome P450 monooxygenase responsible for the C-22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway[J]. Phytochemistry, 2018, 156: 116-123. doi: S0031-9422(18)30587-9 pmid: 30268044
[16]	Chen Z, Yu HY, Jin GT, et al. 22R-but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis[J]. The Plant Journal, 2022:109,940-951.
[17]	张永辉, 刘正杰, 成钦, 等. 芦笋胆固醇C-22羟化酶基因AoCYP90B27的克隆及表达模式分析[J]. 西北农业学报, 2023, 32(8): 1205-1214.
	Zhang YH, Liu ZJ, Cheng Q, et al. Cloning and expression pattern of cholesterol C-22 hydroxylase gene AoCYP90B27 from Asparagus officinalis[J]. Acta Agric Boreali Occidentalis Sin, 2023, 32(8): 1205-1214.
[18]	Szeliga M, Ciura J, Tyrka M. Representational difference analysis of transcripts involved in jervine biosynthesis[J]. Life, 2020, 10(6): 88.
[19]	郑晓红, 管童伟, 韩旭然, 等. 环巴胺类似物的合成及体外抗肿瘤细胞活性研究[J]. 天然产物研究与开发, 2015, 27(5): 890-895.
	Zheng XH, Guan TW, Han XR, et al. Synthesis and antitumor activity of cyclopamine analogues[J]. Nat Prod Res Dev, 2015, 27(5): 890-895. doi: 10.16333/j.1001-6880.2015.05.027
[20]	Wang Y, Shi Y, Tian WS, et al. Stereoselective synthesis of(-)-verazine and congeners via a cascade ring-switching process of furostan-26-acid[J]. Org Lett, 2020, 22(7): 2761-2765. doi: 10.1021/acs.orglett.0c00747 pmid: 32202118
[21]	温文, 薛兵, 康静静, 等. 响应面法优化毛叶藜芦环巴胺的提取工艺[J]. 食品工业科技, 2014, 35(4): 219-222.
	Wen W, Xue B, Kang JJ, et al. Optimization of cyclopamine extraction from Veratrum grandiflorum Loes by response surface methodology[J]. Sci Technol Food Ind, 2014, 35(4): 219-222.
[22]	Turner MW, Cruz R, Mattos J, et al. Cyclopamine bioactivity by extraction method from Veratrum californicum[J]. Bioorg Med Chem, 2016, 24(16): 3752-3757.
[23]	Turner MW, Rossi M, Campfield V, et al. Steroidal alkaloid variation in Veratrum californicum as determined by modern methods of analytical analysis[J]. Fitoterapia, 2019, 137: 104281.
[24]	Parks LW, Casey WM. Physiological implications of sterol biosynthesis in yeast[J]. Annu Rev Microbiol, 1995, 49: 95-116. pmid: 8561481
[25]	Dai ZB, Liu Y, Zhang XN, et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides[J]. Metab Eng, 2013, 20: 146-156. doi: 10.1016/j.ymben.2013.10.004 pmid: 24126082
[26]	Xu SH, Nes WD. Biosynthesis of cholesterol in the yeast mutant erg6[J]. Biochem Biophys Res Commun, 1988, 155(1): 509-517.
[27]	Mumberg D, Müller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds[J]. Gene, 1995, 156(1): 119-122. doi: 10.1016/0378-1119(95)00037-7 pmid: 7737504
[28]	Zhao FL, Bai P, Liu T, et al. Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae[J]. Biotechnol Bioeng, 2016, 113(8): 1787-1795. doi: 10.1002/bit.25934 pmid: 26757342
[29]	Yang JZ, Liu YG, Zhong DC, et al. Combinatorial optimization and spatial remodeling of CYPs to control product profile[J]. Metab Eng, 2023, 80: 119-129. doi: 10.1016/j.ymben.2023.09.004 pmid: 37703999

编辑推荐 0

Metrics

阅读次数

全文

391

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	3	0	0	388

来源	本网站	其他网站

次数	376	15
比例	96%	4%

摘要

最新录用	在线预览	正式出版

0	0	64

	来源	本网站

	次数	64
	比例	100%

藜芦22（R）-羟基胆固醇合成基因功能验证与酵母异源合成

Function Verification of Genes Involved in 22 (R)-hydroxycholesterol Biosynthesis in Veratrum nigrum and Their Heterologous Synthesis

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 13

参考文献 29

相关文章 15

编辑推荐 0

Metrics

本文评价

[1]	任晓敏, 云岚, 艾芊, 赵乔. 新麦草异戊烯基转移酶PjIPT基因的功能验证[J]. 生物技术通报, 2024, 40(7): 207-215.
[2]	杜泽光, 任少文, 张凤勤, 李梅兰, 李改珍, 齐仙惠. 大白菜BrMLP328的克隆、表达及功能验证[J]. 生物技术通报, 2024, 40(4): 122-129.
[3]	杨伟成, 孙岩, 杨倩, 王壮琳, 马菊花, 薛金爱, 李润植. 陆地棉FAX家族的全基因组鉴定及GhFAX1的功能分析[J]. 生物技术通报, 2024, 40(3): 155-169.
[4]	刘辉, 卢扬, 叶夕苗, 周帅, 李俊, 唐健波, 陈恩发. 外源硫诱导苦荞镉胁迫响应的比较转录组学分析[J]. 生物技术通报, 2023, 39(5): 177-191.
[5]	李峰, 陈雯雯, 邓江丽, 毛清黎, 权文利, 毛雅慧. 白藜芦醇对核桃细菌性黑斑病菌的抑制作用[J]. 生物技术通报, 2021, 37(6): 58-65.
[6]	乔孟欣, 李素贞, 陈景堂. 玉米铁还原酶基因ZmFRO2的功能分析[J]. 生物技术通报, 2020, 36(11): 9-20.
[7]	高原, 王福玲, 徐蓓蕾, 雒江菡, 阎力君, 赵莹莹, 王越. 葡萄籽中白藜芦醇的提取及其对LO2细胞氧化损伤保护及延缓衰老作用[J]. 生物技术通报, 2018, 34(3): 225-229.
[8]	程杏安, 张淑明, 周晓武, 吴波, 林贤伟, 秦湘静 , 黄素青, 刘展眉, 蒋旭红. 两种天然产物对B16F10细胞增殖及黑色素合成抑制机理研究[J]. 生物技术通报, 2017, 33(8): 199-205.
[9]	周敏. 白藜芦醇对IgA大鼠肾组织TH2型炎性因子及血清CIC的影响[J]. 生物技术通报, 2017, 33(6): 128-133.
[10]	冯薇, 胡小妍, 马明娜, 郭萌, 路福平, 李玉. 产β-葡萄糖苷酶细菌的筛选及转化白藜芦醇的研究[J]. 生物技术通报, 2017, 33(11): 130-135.
[11]	姚庆收, 姜吉刚, 武玉永, 梁乘榜. 培养基种类对花生毛状根株系生物量和白藜芦醇含量的影响[J]. 生物技术通报, 2014, 30(5): 174-178.
[12]	柳忠玉, 赵树进. 转PcRS基因拟南芥的抗炭疽病研究[J]. 生物技术通报, 2014, 30(10): 107-112.
[13]	黄秀琴;郭丽琼;李小明;林俊芳;袁致浩;谭铭琛;. 花生白藜芦醇合酶基因的克隆与生物信息学分析[J]. , 2012, 0(03): 69-74.
[14]	张怡;李成伟;. 酵母异源功能互补在植物基因克隆中的应用[J]. , 2010, 0(07): 14-21.
[15]	冯磊;花慧;邱丽颖;金坚;. 白藜芦醇靶点蛋白质的研究[J]. , 2009, 0(08): 128-133.