基于油质蛋白oleosin鉴定短序列脂滴定位信号<sup>*</sup>

doi:10.13523/j.cb.2302003

中国生物工程杂志

2023, Vol. 43

Issue (7): 36-43 DOI: 10.13523/j.cb.2302003

研究报告

基于油质蛋白oleosin鉴定短序列脂滴定位信号^*

李佳文^1,²,范雨萱²,李福利^2,³,张朝辉^1,^**(

),王士安^2,^3,^**(

)

1 中国海洋大学食品科学与工程学院青岛 266003
2 中国科学院青岛生物能源与过程研究所青岛 266101
3 山东能源研究院青岛 266101

Ide.pngication of Short Peptides from Oleosin for Lipid Droplet Localization in Xanthophyllomyces dendrorhous

Jia-wen LI^1,²,Yu-xuan FAN²,Fu-li LI^2,³,Zhao-hui ZHANG^1,^**(

),Shi-an WANG^2,^3,^**(

)

1 College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
2 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
3 Shandong Energy Institute, Qingdao 266101, China

全文: PDF(1715 KB) HTML

摘要：

目的:人工区室化是代谢工程及合成生物学的重要技术手段,针对脂滴区室化技术未得到有效评价的现状,以产油微生物红法夫酵母为检验体系,评价筛选脂滴定位信号。方法:基于芝麻油质蛋白oleosin的三维结构及其跨膜结构域分析,设计短序列脂滴定位信号,构建定位信号与eGFP荧光蛋白基因融合的表达载体,转化红法夫酵母,采用荧光显微观察评价定位信号的定位功能。结果:完整的oleosin蛋白Ols1_(1-145)和肽段Ols2_(1-63)、Ols4_(32-63)、Ols5_(44-63)均能引导eGFP定位于红法夫酵母的脂滴内部,肽段Ols3_(64-145)不能有效引导eGFP定位于脂滴,仅在细胞质中游离表达。结论:筛选得到了一个短序列脂滴定位信号Ols5_(44-63),其仅含19个氨基酸,能够有效引导eGFP定位于脂滴内部。

关键词： 亚细胞定位; 脂滴; 油质蛋白; 红法夫酵母; 合成生物学

Abstract:

Objective: A.pngicial compartmentalization is among the important approaches in metabolic engineering and synthetic biology. Due to the insufficient evaluation of compartmentalization in lipid droplets (LDs), the aim of this work is to ide.pngy efficient LD localization signals in the oleaginous yeast Xanthophyllomyces dendrorhous. Methods: Based on the three-dimensional structure and the predicted transmembrane domain (TMD) of sesame oleosin, LD localization signals were predicted and fused with eGFP to generate a series of expression vectors, which were used to transform X. dendrorhous. Green and orange fluorescence was observed and used to assess the efficiency of the predicted LD localization signals. Results: The intact oleosin protein Ols1_(1-145) and truncated peptides Ols2_(1-63), Ols4_(32-63), and Ols5_(44-63) successfully guided eGFP is localization in LDs, while truncated peptide Ols3_(64-145) was ineffective and most eGFP proteins were expressed in cytoplasm. Conclusion: A short LD localization signal Ols5_(44-63) consisting of 19 amino acids was ide.pngied, which can precisely lead the eGFP to localize inside the LDs.

Key words: Subcellular localization Lipid droplets Oleosin Xanthophyllomyces dendrorhous Synthetic biology

收稿日期: 2023-02-03 出版日期: 2023-08-03

ZTFLH:

Q819

基金资助: 国家重点研发计划(2022YFC2106200)

通讯作者: **电子信箱:zhangzhh@ouc.edu.cn;wangsa@qibebt.ac.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	李佳文
	范雨萱
	李福利
	张朝辉
	王士安

引用本文:

李佳文, 范雨萱, 李福利, 张朝辉, 王士安. 基于油质蛋白oleosin鉴定短序列脂滴定位信号^*[J]. 中国生物工程杂志, 2023, 43(7): 36-43.

Jia-wen LI, Yu-xuan FAN, Fu-li LI, Zhao-hui ZHANG, Shi-an WANG. Ide.pngication of Short Peptides from Oleosin for Lipid Droplet Localization in Xanthophyllomyces dendrorhous. China Biotechnology, 2023, 43(7): 36-43.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2302003 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I7/36

表1 本研究所用质粒

表2 PCR引物序列

表3 Oleosin蛋白的氨基酸序列

图1 完整oleosin蛋白在酵母细胞中的亚细胞定位

图2 油质蛋白oleosin的结构预测及跨膜区预测

图3 油质蛋白oleosin的结构及eGFP在脂滴内部的定位设计

图4 截短的oleosin蛋白在酵母细胞中的亚细胞定位结果

图5 截短的oleosin蛋白在酵母细胞中的亚细胞定位结果

[1]	Shi Y S, Wang D, Li R S, et al. Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides. Metabolic Engineering, 2021, 67: 104-111. doi: 10.1016/j.ymben.2021.06.002 pmid: 34153454
[2]	Chen X, Zhu C X, Na Y T, et al. Compartmentalization of melanin biosynthetic enzymes contributes to self-defense against intermediate compound scytalone in Botrytis cinerea. mBio, 2021, 12(2): e00007-e00021.
[3]	Jaramillo-Madrid A C, Lacchini E, Goossens A. Within and beyond organelle engineering: strategies for increased terpene production in yeasts and plants. Current Opinion in Green and Sustainable Chemistry, 2022, 33: 100572. doi: 10.1016/j.cogsc.2021.100572
[4]	Hammer S K, Avalos J L. Harnessing yeast organelles for metabolic engineering. Nature Chemical Biology, 2017, 13(8): 823-832. doi: 10.1038/nchembio.2429 pmid: 28853733
[5]	Avalos J L, Fink G R, Stephanopoulos G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnology, 2013, 31(4): 335-341. doi: 10.1038/nbt.2509 pmid: 23417095
[6]	Cao X, Yang S, Cao C Y, et al. Harnessing sub-organelle metabolism for biosynthesis of isoprenoids in yeast. Synthetic and Systems Biotechnology, 2020, 5(3): 179-186. doi: 10.1016/j.synbio.2020.06.005 pmid: 32637671
[7]	Plegaria J S, Kerfeld C A. Engineering nanoreactors using bacterial microcompartment architectures. Current Opinion in Biotechnology, 2018, 51: 1-7. doi: S0958-1669(17)30137-4 pmid: 29035760
[8]	Zhao E M, Suek N, Wilson M Z, et al. Light-based control of metabolic flux through assembly of synthetic organelles. Nature Chemical Biology, 2019, 15(6): 589-597. doi: 10.1038/s41589-019-0284-8 pmid: 31086330
[9]	Li T P, Jiang Q Y, Huang J F, et al. Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature Communications, 2020, 11(1): 5448. doi: 10.1038/s41467-020-19280-0 pmid: 33116131
[10]	Dusséaux S, Wajn W T, Liu Y X, et al. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(50): 31789-31799.
[11]	Kim J E, Jang I S, Son S H, et al. Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway. Metabolic Engineering, 2019, 56: 50-59. doi: 10.1016/j.ymben.2019.08.013
[12]	Thodey K, Galanie S, Smolke C D. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nature Chemical Biology, 2014, 10(10): 837-844. doi: 10.1038/nchembio.1613 pmid: 25151135
[13]	Yu Y, Rasool A, Liu H R, et al. Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool. Metabolic Engineering, 2020, 62: 72-83. doi: 10.1016/j.ymben.2020.08.010
[14]	Zhang J L, Bai Q Y, Peng Y Z, et al. High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components. Biotechnology for Biofuels, 2020, 13:133. doi: 10.1186/s13068-020-01773-1
[15]	Ma T, Shi B, Ye Z L, et al. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metabolic Engineering, 2019, 52: 134-142. doi: 10.1016/j.ymben.2018.11.009
[16]	Yang K X, Qiao Y G, Li F, et al. Subcellular engineering of lipase dependent pathways directed towards lipid related organelles for highly effectively compartmentalized biosynthesis of triacylglycerol derived products in Yarrowia lipolytica. Metabolic Engineering, 2019, 55: 231-238. doi: 10.1016/j.ymben.2019.08.001
[17]	Sadre R, Kuo P, Chen J X, et al. Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves. Nature Communications, 2019, 10: 853. doi: 10.1038/s41467-019-08515-4 pmid: 30787273
[18]	Bhatla S C, Kaushik V, Yadav M K. Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications. Biotechnology Advances, 2010, 28(3): 293-300. doi: 10.1016/j.biotechadv.2010.01.001 pmid: 20067829
[19]	Li M, Murphy D J, Lee K H K, et al. Purification and structural characterization of the central hydrophobic domain of oleosin. Journal of Biological Chemistry, 2002, 277(40): 37888-37895. doi: 10.1074/jbc.M202721200 pmid: 12124381
[20]	Cao H P. Genome-wide analysis of oleosin gene family in 22 tree species: an accelerator for metabolic engineering of bioFuel crops and agrigenomics industrial applications. OMICS: A Journal of Integrative Biology, 2015, 19(9): 521-541. doi: 10.1089/omi.2015.0073
[21]	Welte M A, Gould A P. Lipid droplet functions beyond energy storage. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2017, 1862(10): 1260-1272. doi: 10.1016/j.bbalip.2017.07.006
[22]	Tai S S K, Chen M C M, Peng C C, et al. Gene family of oleosin isoforms and their structural stabilization in sesame seed oil bodies. Bioscience, Biotechnology, and Biochemistry, 2002, 66(10): 2146-2153. pmid: 12450125
[23]	Frandsen G I, Mundy J, Tzen J T C. Oil bodies and their associated proteins, oleosin and caleosin. Physiologia Plantarum, 2001, 112(3): 301-307. doi: 10.1034/j.1399-3054.2001.1120301.x
[24]	Jolivet P, Aymé L, Giuliani A, et al. Structural proteomics: topology and relative accessibility of plant lipid droplet associated proteins. Journal of Proteomics, 2017, 169: 87-98. doi: S1874-3919(17)30320-2 pmid: 28918933
[25]	Mussagy C U, Remonatto D, Picheli F P, et al. A look into Phaffia rhodozyma biorefinery: from the recovery and fractionation of carotenoids, lipids and proteins to the sustainable manufacturing of biologically active bioplastics. Bioresource Technology, 2022, 362: 127785. doi: 10.1016/j.biortech.2022.127785
[26]	Zhang L L, Reyes A, Wang X D. The role of mitochondria-targeted antioxidant MitoQ in neurodegenerative disease. Molecular and Cellular Therapies, 2018, 6(1): 1-8.
[27]	Gómez M, Campusano S, Gutiérrez M S, et al. Sterol regulatory element-binding protein Sre 1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous. Journal of Lipid Research, 2020, 61(12): 1658-1674. doi: 10.1194/jlr.RA120000975
[28]	Alesci A, Salvo A, Lauriano E R, et al. Production and extraction of astaxanthin from Phaffia rhodozyma and its biological effect on alcohol-induced renal hypoxia in Carassius auratus. Natural Product Research, 2015, 29(12): 1122-1126. doi: 10.1080/14786419.2014.979417
[29]	Davidi L, Levin Y, Ben-Dor S, et al. Proteome analysis of cytoplasmatic and plastidic β-carotene lipid droplets in Dunaliella bardawil. Plant Physiology, 2015, 167(1): 60-79. doi: 10.1104/pp.114.248450 pmid: 25404729
[30]	Van de Linde S. Single-molecule localization microscopy analysis with ImageJ. Journal of Physics D: Applied Physics, 2019, 52(20): 203002. doi: 10.1088/1361-6463/ab092f
[31]	Ling H. Oleosin fusion expression systems for the production of recombinant proteins. Biologia, 2007, 62(2): 119-123. doi: 10.2478/s11756-007-0041-4
[32]	Lv X M, Wang F, Zhou P P, et al. Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nature Communications, 2016, 7: 12851. doi: 10.1038/ncomms12851
[33]	Zhang W, Du L, Qu Z P, et al. Compartmentalized biosynthesis of mycophenolic acid. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(27): 13305-13310.
[34]	Sheng J Y, Stevens J, Feng X Y. Pathway compartmentalization in peroxisome of Saccharomyces cerevisiae to produce versatile medium chain fatty alcohols. Scie.pngic Reports, 2016, 6: 26884.
[35]	Grewal P S, Samson J A, Baker J J, et al. Peroxisome compartmentalization of a toxic enzyme improves alkaloid production. Nature Chemical Biology, 2021, 17(1): 96-103. doi: 10.1038/s41589-020-00668-4 pmid: 33046851
[36]	Van Rossum H M, Kozak B U, Pronk J T, et al. Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: pathway stoichiometry, free-energy conservation and redox-cofactor balancing. Metabolic Engineering, 2016, 36: 99-115. doi: 10.1016/j.ymben.2016.03.006
[37]	Zhou Y J, Buijs N A, Zhu Z W, et al. Harnessing yeast peroxisomes for biosynthesis of fatty-acid-derived biofuels and chemicals with relieved side-pathway competition. Journal of the American Chemical Society, 2016, 138(47): 15368-15377. pmid: 27753483
[38]	Gao S L, Tong Y Y, Zhu L, et al. Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production. Metabolic Engineering, 2017, 41: 192-201. doi: 10.1016/j.ymben.2017.04.004
[39]	Liu G S, Li T, Zhou W, et al. The yeast peroxisome: a dynamic storage depot and subcellular factory for squalene overproduction. Metabolic Engineering, 2020, 57: 151-161. doi: 10.1016/j.ymben.2019.11.001
[40]	Zhu Z W, Zhang S F, Liu H W, et al. A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nature Communications, 2012, 3: 112.
[41]	Deng J N, Liu S X, Zou L Q, et al. Lipolysis response to endoplasmic reticulum stress in adipose cells. The Journal of Biological Chemistry, 2012, 287(9): 6240-6249. doi: 10.1074/jbc.M111.299115
[42]	Yang L L, Liang J J, Lam S M, et al. Neuronal lipolysis participates in PUFA-mediated neural function and neurodegeneration. EMBO Reports, 2020, 21(11): e50214. doi: 10.15252/embr.202050214

[1]	付萌萌, 苏丹丹, 左锟澜, 吴宗震, 李思思, 徐雁龙, 刘欢. 人体免疫相关的合成生物学生物安全风险和应对策略研究^*[J]. 中国生物工程杂志, 2023, 43(6): 125-132.
[2]	李雨桐, 崔天琦, 张海林, 于广乐, 栾霁, 王海龙. 肿瘤靶向细菌Escherichia coli Nissle 1917在癌症治疗中的研究进展^*[J]. 中国生物工程杂志, 2023, 43(6): 54-68.
[3]	刘亭亭, 张平, 张悦. 光控表达系统在合成生物学中的调控作用^*[J]. 中国生物工程杂志, 2023, 43(4): 92-100.
[4]	宁峻涛, 邹诗施, 左锟澜, 吴宗震, 李晶, 徐雁龙, 刘欢. 合成生物活性物质的生物安全风险和应对策略研究^*[J]. 中国生物工程杂志, 2023, 43(2/3): 180-189.
[5]	杨洋, 姚明东, 王颖, 肖文海. 酵母合成2'-岩藻糖基乳糖的研究进展^*[J]. 中国生物工程杂志, 2023, 43(1): 127-138.
[6]	曾雪霞,但玉,毛绍名,孙佳慧,栾国栋,吕雪峰. 蓝藻光驱固碳合成糖类物质的技术研究进展^*[J]. 中国生物工程杂志, 2022, 42(7): 90-100.
[7]	张大璐,葛奇,冯一博,陈为刚. DNA数据存储的科研概况国际对比与分析[J]. 中国生物工程杂志, 2022, 42(6): 116-129.
[8]	白松,侯正杰,高庚荣,乔斌,程景胜. 微生物合成奇数链脂肪酸研究进展^*[J]. 中国生物工程杂志, 2022, 42(6): 76-85.
[9]	梁世玉,万里,郭潇佳,王雪颖,吕力婷,胡英菡,赵宗保. 构建可合成非天然辅酶的圆红冬孢酵母工程菌^*[J]. 中国生物工程杂志, 2022, 42(5): 58-68.
[10]	赵赤鸿,苏丹丹,厉春,吴宗震,左锟澜,徐雁龙,刘欢. 总体国家安全观下合成生物学风险和应对策略研究^*[J]. 中国生物工程杂志, 2022, 42(12): 120-128.
[11]	韩春丽,王汉杰. 工程生物活药在肿瘤免疫治疗中的应用[J]. 中国生物工程杂志, 2022, 42(10): 39-50.
[12]	李慧敏,贾斌,李霞,刘夺. 合成芳香族化合物的酵母底盘改造策略^*[J]. 中国生物工程杂志, 2022, 42(10): 80-92.
[13]	马宁,王汉杰. 光遗传学在细菌生产调控中的应用进展[J]. 中国生物工程杂志, 2021, 41(9): 101-109.
[14]	黄焕邦,吴洋,杨友辉,王兆官,齐浩. 基于古菌酪氨酰tRNA合成酶非天然氨基酸插入的研究进展[J]. 中国生物工程杂志, 2021, 41(9): 110-125.
[15]	郭曼曼,田开仁,乔建军,李艳妮. 噬菌体重组酶系统在合成生物学中的应用^*[J]. 中国生物工程杂志, 2021, 41(8): 90-102.

Viewed

Full text

Abstract

Cited

Shared

Discussed