Isolation of a 螖5 Desaturase Gene from Euglena gracilis and Functional Dissection of Its HPGG and HDASH Motifs
详细信息 查看全文
- 作者：Dana Walters Pollak (1)
Michael W. Bostick (1)
Hyeryoung Yoon (1)
Jamie Wang (1)
Dieter H. Hollerbach (1)
Hongxian He (1)
Howard G. Damude (1)
Hongxiang Zhang (1)
Narendra S. Yadav (1)
Seung-Pyo Hong (1)
Pamela Sharpe (1)
Zhixiong Xue (1)
Quinn Zhu (1) quinn.zhu@usa.dupont.com - 关键词：Δ ; 5 desaturase – ; HPGG motif – ; HDASH motif – ; Double mutant – ; Fatty acid biosynthesis – ; Yarrowia lipolytica
- 刊名：Lipids
- 出版年：2012
- 出版时间：September 2012
- 年：2012
- 卷：47
- 期：9
- 页码：913-926
- 全文大小：933.5 KB
- 参考文献：1. Riediger ND, Othman RA, Suh M, Moghadasian MH (2009) A systemic review of the roles of n-3 fatty acids in health and disease. J Am Diet Assoc 109:668–679
2. Freeman LM (2010) Beneficial effects of omega-3 fatty acids in cardiovascular disease. J Small Anim Pract 51:462–470
3. Mourek J, Mourek J Jr (2011) Developmentally dependent and different roles of fatty acids OMEGA-6 and OMEGA-3. Prague Med Rep 112:81–92
4. Mayurasakorn K, Williams JJ, Ten VS, Deckelbaum RJ (2011) Docosahexaenoic acid: brain accretion and roles in neuroprotection after brain hypoxia and ischemia. Curr Opin Clin Nutr Metab Care 14:158–167
5. Uddin M, Levy BD (2011) Resolvins: natural agonists for resolution of pulmonary inflammation. Prog Lipid Res 50:75–88
6. Serhan CN (2007) Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways. Annu Rev Immunol 25:101–137
7. Kiso Y (2011) Pharmacology in health foods: effects of arachidonic acid and docosahexaenoic acid on the age-related decline in brain and cardiovascular system function. J Pharmacol Sci 115:471–475
8. Weitz D, Weintraub H, Fisher E, Schwartzbard AZ (2010) Fish oil for the treatment of cardiovascular disease. Cardiol Rev 18:258–263
9. Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K (2007) Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet 369:1090–1098
10. F茅art C, Peuchant E, Letenneur L, Samieri C, Montagnier D, Fourrier-Reglat A, Barberger-Gateau P (2008) Plasma eicosapentaenoic acid is inversely associated with severity of depressive symptomatology in the elderly: data from the Bordeaux sample of the three-city study. Am J Clin Nutr 87:1156–1162
11. Costa LG (2007) Contaminants in fish: risk-benefit considerations. Arh Hig Rada Toksikol 58:567–574
12. Streekstra H (2010) Arachidonic acid: fermentative production by Mortierella fungi. In: Cohen Z, Ratledge C (eds) Single cell oils, 2nd edn. AOCS Press, Urbana
13. Barclay W, Weaver C, Metz J, Hansen J (2010) Development of a docosahexaenoic acid production technology using Schizochytrium: historical perspective and update. In: Cohen Z, Ratledge C (eds) Single cell oils, 2nd edn. AOCS Press, Urbana
14. Zhu Q, Xue Z, Yadav N, Damude H, Pollak D, Rupert R, Seip J, Hollerbach D, Macool D, Zhang H (2010) Metabolic engineering of an oleaginous yeast for the production of omega-3 fatty acids. In: Cohen Z, Ratledge C (eds) Single cell oils, 2nd edn. AOCS Press, Urbana
15. Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M, Valentine R, Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293:290–293
16. Wallis JG, Browse J (1999) The Δ8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys 365:307–316
17. Pereira SL, Leonard AE, Mukerji P (2003) Recent advances in the study of fatty acid desaturases from animals and lower eukaryotes. Prostaglandins Leukot Essent Fatty Acids 68:97–106
18. Qiu X, Hong H, MacKenzie SL (2001) Identification of a delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. J Biol Chem 276:31561–31566
19. Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler JJ, Napier A, Stobart AK, Lazarus CM (2004) Production of very long chain polysaturated omega-3 and omega-6 fatty acids in plants. Nat Biotechnol 22:739–745
20. Kajikawa M, Yamato KT, Kohzu Y, Nojiri M, Sakuradani E, Shimizu S, Sakai Y, Fukuzawa H, Ohyama K (2004) Isolation and characterization of delta(6)-desaturase, an ELO-like enzyme and delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Plant Mol Biol 54:335–352
21. Damude HG, Kinney AJ (2008) Engineering oilseeds to produce nutritional fatty acids. Physiol Plant 132:1–10
22. Venegas-Caler贸n M, Sayanova O, Napier JA (2010) An alternative to fish oils: metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids. Prog Lipid Res 49:108–119
23. Tavares S, Grotkj忙r T, Obsen T, Haslam RP, Napier JA, Gunnarsson N (2011) Metabolic engineering of Saccharomyces cerevisiae for production of eicosapentaenoic acid, using a novel {delta}5-desaturase from Paramecium tetraurelia. Appl Environ Microbiol 77:1854–1861
24. Michaelson LV, Lazarus CM, Griffiths G, Napier JA, Stobart AK (1998) Isolation of a delta5-fatty acid desaturase gene from Mortierella alpina. J Biol Chem 273:19055–19059
25. Knutzon DS, Thurmond JM, Huang YS, Chaudhary S, Bobik EG Jr, Chan GM, Kirchner SJ, Mukerji P (1998) Identification of delta5-desaturase from Mortierella alpina by heterologous expression in bakers’ yeast and canola. J Biol Chem 273:29360–29366
26. Meesapyodsuk D, Qiu X (2012) The front-end desaturase: structure, function, evolution and biotechnological use. Lipids 47:227–237
27. Sperling P, Heinz E (2001) Desaturases fused to their electron donor. Eur J Lipid Sci Technol 103:158–180
28. Napier JA, Michaelson LV, Sayanova O (2003) The role of cytochrome b 5 fusion desaturases in the synthesis of polyunsaturated fatty acids. Prostaglandins Leukot Essent Fatty Acids 68:135–143
29. Schenkman JB, Jansson I (2003) The many roles of cytochrome b5. Pharmacol Ther 97:139–152
30. Stukey JE, McDonough VM, Martin CE (1990) The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J Biol Chem 265:20144–20149
31. Shanklin J, Guy JE, Mishra G, Lindqvist Y (2009) Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes. J Biol Chem 284:18559–18563
32. Lin YW, Ying TL, Liao LF (2009) Dynamic consequences of mutating the typical HPGG motif of apocytochrome b 5 revealed by computer simulation. Chin Chem Let 200:631–634
33. Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33:12787–12794
34. Knutzon D, Mukerji P, Huang YS, Thurmond J, Chaudhary S (1999) Methods and compositions for synthesis of long chain poly-unsaturated fatty acids. US Patent 5,972,664
35. Yadav NS, Zhu Q, Zhang H (2009) Δ12 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous yeast. US Patent 7,504,259
36. Macool, DJ, Xue Z, Zhu Q (2008) A Mortierella alpina c16/18 fatty acid elongase. US Patent 7,470,532
37. Damude HG, McGonigle B, Zhu Q, Xue Z (2011) Delta-9 elongases and their use in making polyunsaturated fatty acids. US Patent 8,049,062
38. Damude HG, He H, Liao D-I, Zhu Q (2011) Mutant delta-8 desaturase genes engineered by targeted mutagenesis and their use in making polyunsaturated fatty acids. US Patent 8,026,089
39. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
40. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2010) Current protocols in molecular biology. Wiley, New York
41. Fournier P, Abbas A, Chasles M, Kudla B, Ogrydziak D, Yaver D, Xuan JW, Peito A, Ribe AM, He F, Gaillardin CC (1993) Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci USA 90:4912–4916
42. Hong S-Y, Seip J, Walters-Pollak D, Rupert R, Jackson R, Xue Z, Zhu Q (2011) Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter. Yeast 29:59–72
43. Chen DC, Beckerich JM, Gaillardin C (1997) One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 48:232–235
44. Cahoon EB, Ripp KG, Hall SE, Kinney AL (2001) Formation of conjugated Δ8, Δ10-double bonds by Δ12-oleic acid desaturase-related enzymes: biosynthetic origin of calendic acid. J Biol Chem 276:2637–2643
45. Meyer A, Cirpus P, Ott C, Schlecker R, Zahringer U, Heinz E (2003) Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a delta4-fatty acyl group desaturase. J Biochem 42:9779–9788
46. Hong H, Datla N, MacKenzie SL, Qiu X (2002) Isolation and characterization of a Δ5 FA desaturase from Pythium irregulare by heterologous expression in Saccharomyces cerevisiae and oilseed crops. Lipids 37:863–868
47. Domergue F, Lerchl J, Zahringer U, Heinz E (2002) Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem 269:4105–4113
48. Damude H, Zhu Q (2007) Delta-8 desaturase and its use in making polyunsaturated fatty acids. US Patent 7,943,823
49. Kobayashi T, Sakaguchi K, Matsuda T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M (2011) Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid {delta}5 desaturase gene. Appl Environ Microbiol 77:3870–3876
50. Lederer F (1994) The cytochrome b4-fold: an adaptable module. Biochimie 76:674–692
51. Sayanova O, Shewry PR, Napier JA (1999) Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity. Plant Physiol 121:641–646
52. Guillou H, D’Andrea S, Rioux V, Barnouin R, Dalaine S, Pedrono F, Jan S, Legrand P (2004) Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Δ6-desaturase activity. J Lipid Res 45:32–40
53. Hongsthong A, Subudhi S, Sirijuntarut M, Kurdrid P, Cheevadhanarak S, Tanticharoen M (2006) Revealing the complementation of ferredoxin by cytochrome b5 in the Spirulina-Δ6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b5 domain and Spirulina-Δ6-acyl-lipid desaturase. Appl Microbiol Biotechnol 72:1192–1201
54. Gostincar C, Turk M, Gunde-Cimerman N (2010) The evolution of fatty acid desaturases and cytochrome b5 in eukaryotes. J Membr Biol 233:63–72
55. Ratledge C (2010) Single cell oils for the 21st century. In: Cohen Z, Ratledge C (eds) Single cell oils, 2nd edn. AOCS Press, Urbana
56. Beopoulos A, Nicaud JM, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206
57. Sabirova JS, Haddouche R, Van Bogaert IN, Mulaa F, Verstraete W, Timmis KN, Schmidt-Dannert C, Nicaud JM, Soetaert W (2011) The ‘LipoYeasts’ project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils into high-value products. Microb Biotechnol 4:47–54
58. Kudla G, Murray AW, Tollervey D, Plotkin JB (2009) Coding sequence determinants of gene expression in Escherichia coli. Science 324:255–258
59. Kahali B, Ahmad S, Ghosh TC (2011) Selective constraints in yeast genes with differential expressivity: codon pair usage and mRNA stability perspectives. Gene 481:76–82 - 作者单位：1. Biochemical Sciences and Engineering, Central Research and Development, E. I. du Pont de Nemours and Company, Wilmington, DE 19880, USA
- ISSN：1558-9307
文摘
Delta (Δ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the “desaturation and elongation” pathways. A full length Δ5 desaturase gene from Euglena gracilis (EgΔ5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5′ and 3′ rapid amplification of cDNA ends. The whole coding region of EgΔ5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that EgΔ5D has about 39 % identity with a Δ5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, EgΔ5D had strong Δ5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of EgΔ5D. A double mutant EgΔ5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type EgΔ5D. Codon optimization of the N-terminal region of EgΔ5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.