Ide.pngication of Short Peptides from Oleosin for Lipid Droplet Localization in <i>Xanthophyllomyces dendrorhous</i>

doi:10.13523/j.cb.2302003

China Biotechnology

2023, Vol. 43

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

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

Download:

HTML

PDF(1715KB) HTML
Export: BibTeX | EndNote (RIS)

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

Received: 03 February 2023 Published: 03 August 2023

ZTFLH:

Q819

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Jia-wen LI
	Yu-xuan FAN
	Fu-li LI
	Zhao-hui ZHANG
	Shi-an WANG

Cite this article:

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.

URL:

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

Table 1 Plasmids used in this study

Table 2 Primer sequences for PCR

Table 3 The amino acid sequence of oleosin

Fig.1 The subcellular localization of oleosin in Xanthophyllomyces dendrorhous

Fig.2 Predicting the structure of oleosin and the transmembrane regions (a) The predicted three-dimensional structure of oleosin (b) The predicted transmembrane regions

Fig.3 The model of oleosin and the design of eGFP localizations in LDs (a) The structure of the 15 kDa oleosin in sesame, the numbers denote the position of amino acids (b) The predicted transmembrane structure of fusion protein Ols2_(1-63)-eGFP (c) The predicted transmembrane structure of fusion protein eGFP-Ols3_(64-145)

Fig.4 The subcellular localization of truncated oleosin The standard value of Pearson’s R is -1.0~1.0; the absolute value closer to 1 indicates stronger colocalization of fluorescence

Fig.5 The subcellular localization of truncated oleosin The standard value of Pearson’s R is -1.0~1.0; the absolute value closer to 1 indicates stronger colocalization of fluorescence


[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]	FU Meng-meng, SU Dan-dan, ZUO Kun-lan, WU Zong-zhen, LI Si-si, XU Yan-long, LIU Huan. Biosafety Risks of Synthetic Biology Related to Human Immunity and The Countermeaseures[J]. China Biotechnology, 2023, 43(6): 125-132.

[2]	LI Yu-tong, CUI Tian-qi, ZHANG Hai-lin, YU Guang-le, LUAN Ji, WANG Hai-long. *Research Advances in Tumor-targeting Bacteria Escherichia coli* Nissle 1917 in Cancer Therapy**[J]. China Biotechnology, 2023, 43(6): 54-68.

[3]	LIU Ting-ting, ZHANG Ping, ZHANG Yue. Regulation Role of Light-controlled Expression Systems in Synthetic Biology[J]. China Biotechnology, 2023, 43(4): 92-100.

[4]	NING Jun-tao, ZOU Shi-shi, ZUO Kun-lan, WU Zong-zhen, Li Jing, XU Yan-long, LIU Huan. Biosafety Risks and Countermeasures of Active Substance in Synthesis Biology[J]. China Biotechnology, 2023, 43(2/3): 180-189.

[5]	YANG Yang, YAO Ming-dong, WANG Ying, XIAO Wen-hai. Research Progress of Synthesis of 2'-Fucosyllactose by Yeast[J]. China Biotechnology, 2023, 43(1): 127-138.

[6]	Xue-xia ZENG,Yu DAN,Shao-ming MAO,Jia-hui SUN,Guo-dong LUAN,Xue-feng LV. Research Progress on the Cyanobacterial Photosynthetic Production of Sugars Utilizing Carbon Dioxide[J]. China Biotechnology, 2022, 42(7): 90-100.

[7]	ZHANG Da-lu,GE Qi,FENG Yi-bo,CHEN Wei-gang. Comparison and Analysis on Scientific Research Programs on DNA Data Storage[J]. China Biotechnology, 2022, 42(6): 116-129.

[8]	BAI Song,HOU Zheng-jie,GAO Geng-rong,QIAO Bin,CHENG Jing-sheng. Advances in the Synthesis of Odd-chain Fatty Acids by Microorganisms[J]. China Biotechnology, 2022, 42(6): 76-85.

[9]	LIANG Shi-yu,WAN Li,GUO Xiao-jia,WANG Xue-ying,LV Li-ting,HU Ying-han,ZHAO Zong-bao. *Engineered Rhodosporidium toruloides* Strains Capable of Biosynthesizing a Non-natural Cofactor**[J]. China Biotechnology, 2022, 42(5): 58-68.

[10]	ZHAO Chi-hong,SU Dan-dan,LI Chun,WU Zong-zhen,ZUO Kun-lan,XU Yan-long,LIU Huan. Synthetic Biology Risks and Biosafety Strategies in the View of Overall National Security Concept[J]. China Biotechnology, 2022, 42(12): 120-128.

[11]	Chun-li HAN,Han-jie WANG. Advances of Engineered Live Biotherapeutics in Tumor Immunotherapy[J]. China Biotechnology, 2022, 42(10): 39-50.

[12]	Hui-min LI,Bin JIA,Xia LI,Duo LIU. Advances in Engineering Yeast Chassis for Producing Aromatic Compounds[J]. China Biotechnology, 2022, 42(10): 80-92.

[13]	MA Ning,WANG Han-jie. Advances of Optogenetics in the Regulation of Bacterial Production[J]. China Biotechnology, 2021, 41(9): 101-109.

[14]	HUANG Huan-bang,WU Yang,YANG You-hui,WANG Zhao-guan,QI Hao. Progress in Incorporation of Non-canonical Amino Acid Based on Archaeal Tyrosyl-tRNA Synthetase[J]. China Biotechnology, 2021, 41(9): 110-125.

[15]	GUO Man-man,TIAN Kai-ren,QIAO Jian-jun,LI Yan-ni. Application of Phage Recombinase Systems in Synthetic Biology[J]. China Biotechnology, 2021, 41(8): 90-102.

Viewed

Full text

Abstract

Cited

Shared

Discussed