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

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

Abstract

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

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.

Figures/Tables 13

Fig. 1 Speculated biosynthetic pathway of cyclopamine

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

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

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

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）

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

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

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

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

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

Fig. 10 Fusion PCR gel electrophoresis

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

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）

References 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