Identification of the Promoter for the Biosynthesis Gene of Polyketide Meroterpenoids in Phomopsis tersa FS441

doi:10.13560/j.cnki.biotech.bull.1985.2024-0347

Abstract

Abstract:

【Objective】 Phomeroids are novel skeleton polyketide meroterpenoids isolated from the marine fungus Phomopsis tersa FS441, demonstrating promising anti-tumor activity. To facilitate the transcriptional regulation of the crucial genes for the biosynthesis of phomeroids, including the terpene cyclase gene ctg1509 and the P450 monooxygenase gene ctg1511, the functions for the promoters of these two genes were identified in this study.【Method】 This study cloned the promoter p1509 and p1511 of ctg1509 and ctg1511 genes in marine fungus P. tersa FS441. The transcriptional activities of the p1509 and p1511 promoters were validated using luciferase reporter gene vectors, the plate experiment and the growth curve were employed to identify the function of the two promoters. Moreover, PlantCARE database was used to analyze the regulatory elements of the promoter. 【Result】 The promoter of p1509 has the strongest transcriptional activity, which is stronger than the positive promoter PgpdA. p1509 and p1511 promoters can initiate the transcription of ampicillin resistant gene AmpR in Escherichia coli. p1509 and p1511 promoters’ regions not only contain core components such as CAAT box and TATA box, but also include cis-acting regulatory elements involved in low temperature response and light response. 【Conclusion】 This study confirmed the function of p1509 and p1511 promoters, and excavated a novel strong promoter, p1509, which had stronger transcriptional activity than the positive control and contained abundant regulatory elements.

Key words: promoters for biosynthetic genes, transcriptional activity, Phomopsis tersa, polyketide meroterpenoids, element analysis

LIU Yu-ping, ZHANG Wei-yang, ZHANG Wei-min, YE Wei, LI Dong-li. Identification of the Promoter for the Biosynthesis Gene of Polyketide Meroterpenoids in Phomopsis tersa FS441[J]. Biotechnology Bulletin, 2024, 40(12): 248-255.

Figures/Tables 7

References 30

[1]	Lu SL, Wang JM, Sheng RL, et al. Novel bioactive polyketides isolated from marine actinomycetes: an update review from 2013 to 2019[J]. Chem Biodivers, 2020, 17(12): e2000562.
[2]	时杰, 余玥, 刘冰, 等. 真菌来源杂萜类化合物的研究进展[J]. 中国药学杂志, 2024, 59(6): 476-485. doi: 10.11669/cpj.2024.06.002
	Shi J, Yu Y, Liu B, et al. Research progress of meroterpenoids produced by fungi[J]. Chin Pharm J, 2024, 59(6): 476-485. doi: 10.11669/cpj.2024.06.002
[3]	董佳钰, 李敏, 肖宗华, 等. 米曲霉异源表达天然产物研究进展[J]. 合成生物学, 2022, 3(6): 1126-1149. doi: 10.12211/2096-8280.2022-007
	Dong JY, Li M, Xiao ZH, et al. Recent advances in heterologous production of natural products using Aspergillus oryzae[J]. Synth Biol J, 2022, 3(6): 1126-1149.
[4]	Geris R, Simpson TJ. Meroterpenoids produced by fungi[J]. Nat Prod Rep, 2009, 26(8): 1063-1094. doi: 10.1039/b820413f pmid: 19636450
[5]	李军伟, 段瑞刚, 邹建华, 等. 八角莲内生真菌Penicillium sp.的杂萜和丁内酯类化学成分研究[J]. 药学学报, 2014, 49(6): 913-920.
	Li JW, Duan RG, Zou JH, et al. Meroterpenoids and isoberkedienolactone from endophytic fungus Penicillium sp. associated with Dysosma versipellis[J]. Acta Pharm Sin, 2014, 49(6): 913-920.
[6]	温小安, 张迎霞, 柳军, 等. 杂环并五环三萜化合物的合成及糖原磷酸化酶抑制活性[J]. 中国药科大学学报, 2009, 40(6): 491-496.
	Wen XA, Zhang YX, Liu J, et al. Synthesis and biological activity of heterocycle-fused derivatives of pentacylic triterpenes as glycogen phosphorylase inhibitors[J]. J China Pharm Univ, 2009, 40(6): 491-496.
[7]	Elissawy AM, El-Shazly M, Ebada SS, et al. Bioactive terpenes from marine-derived fungi[J]. Mar Drugs, 2015, 13(4): 1966-1992. doi: 10.3390/md13041966 pmid: 25854644
[8]	Li J, Yang F, Wang Z, et al. Unusual anti-inflammatory meroterpenoids from the marine sponge Dactylospongia sp[J]. Org Biomol Chem, 2018, 16(36): 6773-6782. doi: 10.1039/c8ob01580e pmid: 30191932
[9]	张娜, 傅小雪, 王文婧. 泰国红树内生真菌土曲霉xy03菌株中新杂萜类成分[J]. 菌物学报, 2021, 40(1): 222-231. doi: 10.13346/j.mycosystema.200169
	Zhang N, Fu XX, Wang WJ. A new meroterpenoid from the Thai mangrove endophytic fungus Aspergillus terreus xy03[J]. Mycosystema, 2021, 40(1): 222-231.
[10]	Dai GZ, Sun JP, Peng XP, et al. Astellolides R-W, drimane-type sesquiterpenoids from an Aspergillus parasiticus strain associated with an isopod[J]. J Nat Prod, 2023, 86(7): 1746-1753.
[11]	张浩东, 赵清梅, 薛佳祺, 等. 丝状真菌的转录调控启动子及其功能研究进展[J]. 农业科学研究, 2021, 42(4): 56-64.
	Zhang HD, Zhao QM, Xue JQ, et al. Advances in transcriptional regulatory promoters of filamentous fungi and their functions[J]. J Agric Sci, 2021, 42(4): 56-64.
[12]	李田, 孙景宽, 刘京涛. 植物启动子研究进展[J]. 生物技术通报, 2015, 31(2): 18-25. doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.003
	Li T, Sun JK, Liu JT. Research advances on plant promoter[J]. Biotechnol Bull, 2015, 31(2): 18-25. doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.003
[13]	Peters B, Casey J, Aidley J, et al. A conserved cis-regulatory module determines germline fate through activation of the transcription factor DUO1 promoter[J]. Plant Physiol, 2017, 173(1): 280-293. doi: 10.1104/pp.16.01192 pmid: 27624837
[14]	李广平, 张长青, 章镇, 等. 中国李pgip启动子的克隆及调控元件分析[J]. 园艺学报, 2009, 36(10): 1425-1430.
	Li GP, Zhang CQ, Zhang Z, et al. Cloning of pgip promoter from Prunus salicina and the computational identification of its regulatory element[J]. Acta Hortic Sin, 2009, 36(10): 1425-1430.
[15]	贾小霞, 齐恩芳, 马胜, 等. 马铃薯PYL基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2022, 48(10): 2533-2545. doi: 10.3724/SP.J.1006.2022.14183
	Jia XX, Qi EF, Ma S, et al. Genome-wide identification and expression analysis of potato PYL gene family[J]. Acta Agron Sin, 2022, 48(10): 2533-2545.
[16]	李文静, 张新业, 张智研, 等. 番茄SlNRT2基因家族鉴定、启动子克隆及表达分析[J]. 植物生理学报, 2024, 60(1): 97-107.
	Li WJ, Zhang XY, Zhang ZY, et al. Identification and characterization of the SlNRT2 gene family and cloning and expression analysis of SlNRT2 in Solanum lycopersicum[J]. Plant Physiol J, 2024, 60(1): 97-107.
[17]	Liu DM, Zhu HY, Pujiana D, et al. Cloning and functional characterization of gpd and α-tubulin promoters from Annulohypoxylon stygium, a companion fungus of Tremella fuciformis[J]. Mycoscience, 2020, 61(1): 1-8.
[18]	Song HY, Choi D, Han DM, et al. A novel rapid fungal promoter analysis system using the phosphopantetheinyl transferase gene, npgA, in Aspergillus nidulans[J]. Mycobiology, 2018, 46(4): 429-439. doi: 10.1080/12298093.2018.1548806 pmid: 30637152
[19]	Kofoed VC, Campion C, Rasmussen PU, et al. Exposure to resistant fungi across working environments and time[J]. Sci Total Environ, 2024, 923: 171189.
[20]	彭存智, 常丽丽, 王丹, 等. 尖孢镰刀菌Six1d启动子的克隆及其在真菌分泌表达载体构建中的应用[J]. 热带作物学报, 2023, 44(6): 1203-1213. doi: 10.3969/j.issn.1000-2561.2023.06.014
	Peng CZ, Chang LL, Wang D, et al. Cloning of Six1d promoter of Fusarium oxysporum and its application in the construction of fungal secretory expression vector[J]. Chin J Trop Crops, 2023, 44(6): 1203-1213.
[21]	Chen SC, Liu ZM, Tan HB, et al. Phomeroids A and B: two novel cytotoxic meroterpenoids from the deep-sea-derived fungus Phomopsis tersa FS441[J]. Org Chem Front, 2020, 7(3): 557-562.
[22]	Ashrfai M, Farsi M, Mirshamsi A, et al. Isolation and sequence analysis of GpdII promoter of the white button mushroom(Agaricus bisporus)from strains Holland737 and IM008[J]. J Hortic Sci, 2015, 2: 33-41.
[23]	张莹, 韩晓静, 蔡逸安, 等. 解淀粉芽孢杆菌内源启动子的筛选及表达碱性果胶酶的应用研究[J]. 微生物学报, 2023, 63(4): 1575-1586.
	Zhang Y, Han XJ, Cai YA, et al. Screening of endogenous promoters of Bacillus amyloliquefaciens and application of them in the expression of alkaline pectinase[J]. Acta Microbiol Sin, 2023, 63(4): 1575-1586.
[24]	Keasling JD. Synthetic biology for synthetic chemistry[J]. ACS Chem Biol, 2008, 3(1): 64-76. doi: 10.1021/cb7002434 pmid: 18205292
[25]	杨何宝, 郭世奇, 尹守亮, 等. 利用强启动子对纤维二糖酶基因在里氏木霉中进行异源表达[J]. 纤维素科学与技术, 2022, 30(4): 39-48, 67.
	Yang HB, Guo SQ, Yin SL, et al. Heterologous expression of cellobiase gene in Trichoderma reesei using strong promoters[J]. J Cellul Sci Technol, 2022, 30(4): 39-48, 67.
[26]	黄自磊, 章卫民, 叶伟, 等. 深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定[J]. 生物技术通报, 2018, 34(4): 144-150. doi: 10.13560/j.cnki.biotech.bull.1985.2017-0941
	Huang ZL, Zhang WM, Ye W, et al. Cloning and identification of gene promoter for gliotoxin biosynthesis from deep-sea-derived fungus Dichotomomyces cejpii[J]. Biotechnol Bull, 2018, 34(4): 144-150.
[27]	王振东, 臧晓南, 丛晓梅, 等. 裂殖壶菌pks基因启动子的克隆及活性检测[J]. 海洋湖沼通报, 2022, 44(5): 27-32.
	Wang ZD, Zang XN, Cong XM, et al. Cloning and activity analysis of pks gene promoter of Aurantiochytrium limacinum[J]. Trans Oceanol Limnol, 2022, 44(5): 27-32.
[28]	岑由飞, 朱牧孜, 叶伟, 等. Paramyrothecium roridum中单端孢霉烯毒素生物合成基因启动子的克隆和功能鉴定[J]. 生物技术通报, 2021, 37(8): 85-94. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1386
	Cen YF, Zhu MZ, Ye W, et al. Cloning and functional identification of trichothecene mycotoxin biosynthesis gene promoter from Paramyrothecium roridum[J]. Biotechnol Bull, 2021, 37(8): 85-94.
[29]	陈建, 赵德刚. 植物萜类生物合成相关酶类及其编码基因的研究进展[J]. 分子植物育种, 2004, 2(6): 757-764.
	Chen J, Zhao DG. Research advances on the enzymes and their coding gene involved in plant terpene biosynthesis[J]. Mol Plant Breed, 2004, 2(6): 757-764.
[30]	高齐霖, 杨建, 李蓉, 等. 南沙链霉菌来源细胞色素P450酶CYP154C34的表征及生物转化[J]. 微生物学报, 2024, 64(2): 502-515.
	Gao QL, Yang J, Li R, et al. Characterization and biotransformation of cytochrome P450 CYP154C34 from Streptomyces nanshen-sis[J]. Acta Microbiol Sin, 2024, 64(2): 502-515.

启动子Promoter	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
p1509	CCGCTCGAGCGTTTATAATGAGAATACATAGCG	CCCAAGCTTCCGAACGCGTAGTAAACG
p1511	ACGCGTGCTAGCCCGGGCAACACGTAGGAAGAAGAGGGTC	AACAGTACCGGAATGCCATGACAATTTAAACTOTGTTAAGGTAC

启动子Promoter	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
p1509	CCGCTCGAGCGTTTATAATGAGAATACATAGCG	CCCAAGCTTCCGAACGCGTAGTAAACG
p1511	ACGCGTGCTAGCCCGGGCAACACGTAGGAAGAAGAGGGTC	AACAGTACCGGAATGCCATGACAATTTAAACTOTGTTAAGGTAC

启动子 Promoter	分值 Score	核心序列 Core sequence
p1509	0.97	TTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATG
p1511	0.92	AATATTTTGTTAAAATTCGCGTTTAAATTTTTGTTAAATCAGCTCATTTT

启动子 Promoter	分值 Score	核心序列 Core sequence
p1509	0.97	TTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATG
p1511	0.92	AATATTTTGTTAAAATTCGCGTTTAAATTTTTGTTAAATCAGCTCATTTT

启动子Promoter	元件名称Component name	元件功能Component functions
p1509/p1511	ABRE	参与脱落酸反应性的顺式作用元件Cis-acting elements involved in abscisic acid reactivity
	MBS	参与干旱诱导的MYB结合位点MYB binding sites involved in drought induced stress
	TGACG-motif	参与MeJA反应的顺式调控元件Cis-acting regulatory elements involved in MeJA reaction
	TATA-box	转录开始的核心启动子元件Core promoter elements for transcription initiation
	CAAT-box	启动子和增强子区域中顺式作用元件Cis-acting elements in promoter and enhancer regions
	CGCTA-motif	参与MeJA反应的顺式调控元件Cis-regulating elements involved in MeJA reaction
p1509	G-Box	参与光反应的顺式调节元件Cis-regulating elements involved in light reactions
	GATA-motif	光反应的顺式调节元件Cis-regulating element for light reaction
	GCN4-motif	参与胚乳表达调控元件Elements involved in regulating endosperm expression
	P-Box	赤霉素反应元件Gibberellin reaction element
	TATC-Box	参与赤霉素反应的顺式作用元件Cis-acting elements involved in gibberellin reaction
	TGA-element	激活反应元件Activate reaction elements
p1511	LTR	低温顺式响应元件Low temperature cis-responsive element
	I-Box	光响应元件Photoresponsive element
	ARE	厌氧诱导顺式作用元件Anaerobic induced cis acting element
	TCT-motif	光响应元件Photoresponsive element
	GT1-motif	光响应元件Photoresponsive element