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Production of squalene by microbes: an update

Abstract

Squalene, a naturally occurring linear triterpene formed via MVA or MEP biosynthetic pathway, is widely distributed in microorganisms, plants and animals. At present, squalene is used extensively in the food, cosmetic and medicine industries because of its antioxidant, antistatic and anti-carcinogenic properties. Increased consumer demand has led to the development of microbial bioprocesses for the commercial production of squalene, in addition to the traditional methods of isolating squalene from the liver oils of deep-sea sharks and plant seed oils. As knowledge of the biosynthetic enzymes and of regulatory mechanisms modulating squalene production increases, opportunities arise for the genetic engineering of squalene production in hosts. In this review, we present the various strategies used up to date to improve and/or engineer squalene production in microbes and analyze yields.

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References

  1. Aasen IM, Ertesvåg H, Heggeset TMB, Liu B, Brautaset T, Vadstein O, Ellingsen TE (2016) Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids. Appl Microbiol Biotechnol 100:4309–4321

  2. Ackman R, Addison R, Eaton C (1968) Unusual occurrence of squalene in a fish, the Eulachon Thaleichthys pacificus. Nature 220:1033–1034

  3. Aguilera Y, Dorado ME, Prada FA, Martínez JJ, Quesada A, Ruiz-Gutiérrez V (2005) The protective role of squalene in alcohol damage in the chick embryo retina. Exp Eye Res 80:535–543

  4. Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Pontén T, Alsmark UCM, Podowski RM, Näslund AK, Eriksson A-S, Winkler HH, Kurland CG (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133–140

  5. Banerjee A, Sharma R, Chisti Y, Banerjee U (2002) Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 22:245–279

  6. Berger A, Gremaud G, Baumgartner M, Rein D, Monnard I, Kratky E, Geiger W, Burri J, Dionisi F, Allan M (2003) Cholesterol-lowering properties of amaranth grain and oil in hamsters. Int J Vitam Nutr Res 73:39–47

  7. Bhattacharjee P, Shukla V, Singhal R, Kulkarni P (2001) Studies on fermentative production of squalene. World J Microbiol Biotechnol 17:811–816

  8. Budiyanto A, Ahmed NU, Wu A, Bito T, Nikaido O, Osawa T, Ueda M, Ichihashi M (2000) Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice. Carcinogenesis 21:2085–2090

  9. Chan P, Tomlinson B, Lee CB, Lee YS (1996) Effectiveness and safety of low-dose pravastatin and squalene, alone and in combination, in elderly patients with hypercholesterolemia. J Clin Pharmacol 36:422–427

  10. Chang MH, Kim HJ, Jahng KY, Hong SC (2008) The isolation and characterization of Pseudozyma sp. JCC 207, a novel producer of squalene. Appl Microbiol Biotechnol 78:963–972

  11. Chen G, Fan K-W, Lu F-P, Li Q, Aki T, Chen F, Jiang Y (2010) Optimization of nitrogen source for enhanced production of squalene from thraustochytrid Aurantiochytrium sp. New Biotechnol 27:382–389

  12. De Leonardis A, Macciola V, De Felice M (1998) Rapid determination of squalene in virgin olive oils using gas–liquid chromatography. Ital J Food Sci 10:75–80

  13. Dellas N, Thomas ST, Manning G, Noel JP (2013) Discovery of a metabolic alternative to the classical mevalonate pathway. Elife 2:e00672

  14. Dessi MA, Deiana M, Day BW, Rosa A, Banni S, Corongiu FP (2002) Oxidative stability of polyunsaturated fatty acids: effect of squalene. Eur J Lipid Sci Technol 104:506–512

  15. Drozdíková E, Garaiová M, Csáky Z, Obernauerová M, Hapala I (2015) Production of squalene by lactose-fermenting yeast Kluyveromyces lactis with reduced squalene epoxidase activity. Lett Appl Microbiol 61:77–84

  16. Englund E, Pattanaik B, Ubhayasekera SJK, Stensjö K, Bergquist J, Lindberg P (2014) Production of squalene in Synechocystis sp. PCC 6803. PLoS One 9:e90270

  17. Epstein W, Rilling H (1970) Studies on the mechanism of squalene biosynthesis the structure of presqualene pyrophosphate. J Biol Chem 245:4597–4605

  18. Fan KW, Aki T, Chen F, Jiang Y (2010) Enhanced production of squalene in the thraustochytrid Aurantiochytrium mangrovei by medium optimization and treatment with terbinafine. World J Microbiol Biotechnol 26:1303–1309

  19. Fang H (2014) Frontier and future development of information technology in medicine and education. Lect Notes Electr Eng 269:1699–1705

  20. Fraser CM, Gocayne JD, White O, Adams MD (1995) The minimal gene complement of Mycoplasma genitalium. Science 270:197

  21. Garaiová M, Zambojová V, Šimová Z, Griač P, Hapala I (2014) Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae. FEMS Yeast Res 14:310–323

  22. Gershbein LL, Singh EJ (1969) Hydrocarbons of dogfish and cod livers and herring oil. J Am Oil Chem Soc 46:554–557

  23. Ghimire GP, Lee HC, Sohng JK (2009) Improved squalene production via modulation of the methylerythritol 4-phosphate pathway and heterologous expression of genes from Streptomyces peucetius ATCC 27952 in Escherichia coli. Appl Environ Microbiol 75:7291–7293

  24. Ghimire GP, Thuan NH, Koirala N, Sohng JK (2016) Advances in biochemistry and microbial production of squalene and its derivatives. J Microbiol Biotechnol 26:441–451

  25. He H-P, Cai Y, Sun M, Corke H (2002) Extraction and purification of squalene from amaranthus grain. J Agric Food Chem 50:368–372

  26. Hernández-Pérez M, Gallego RMR, Alayón PJP, Hernández AB (1997) Squalene content in livers from deep-sea sharks caught in Canary Island waters. Mar Freshw Res 48:573–576

  27. Hoang TMH, Nguyen CH, Le TT, Hoang THQ, Ngo THT, Hoang TLA, Dang DH (2016) Squalene isolated from Schizochytrium mangrovei is a peroxisome proliferator-activated receptor-α agonist that regulates lipid metabolism in HepG2 cells. Biotechnol Lett 38:1065–1071

  28. Huang Z-R, Lin Y-K, Fang J-Y (2009) Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Molecules 14:540–554

  29. Hull CM, Loveridge EJ, Rolley NJ, Donnison IS, Kelly SL, Kelly DE (2014) Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock. Biotechnol Biofuels 7:1

  30. Jennings SM, Tsay YH, Fisch TM, Robinson GW (1991) Molecular cloning and characterization of the yeast gene for squalene synthetase. Proc Natl Acad Sci 88:6038–6042

  31. Jiang Y, Fan K-W, Tsz-Yeung Wong R, Chen F (2004) Fatty acid composition and squalene content of the marine microalga Schizochytrium mangrovei. J Agric Food Chem 52:1196–1200

  32. Kajikawa M, Kinohira S, Ando A, Shimoyama M, Kato M, Fukuzawa H (2015) Accumulation of squalene in a microalga Chlamydomonas reinhardtii by genetic modification of squalene synthase and squalene epoxidase genes. PLoS One 10:e0120446

  33. Kamimura N, Hidaka M, Masaki H, Uozumi T (1994) Construction of squalene-accumulating Saccharomyces cerevisiae mutants by gene disruption through homologous recombination. Appl Microbiol Biotechnol 42:353–357

  34. Kasai H, Katsuta A, Sekiguchi H, Matsuda S, Adachi K, Shindo K, Yoon J, Yokota A, Shizuri Y (2007) Rubritalea squalenifaciens sp. nov., a squalene-producing marine bacterium belonging to subdivision 1 of the phylum ‘Verrucomicrobia’. Int J Syst Evol Microbiol 57:1630–1634

  35. Katabami A, Li L, Iwasaki M, Furubayashi M, Saito K, Umeno D (2015) Production of squalene by squalene synthases and their truncated mutants in Escherichia coli. J Biosci Bioeng 119:165–171

  36. Kaya K, Nakazawa A, Matsuura H, Honda D, Inouye I, Watanabe MM (2011) Thraustochytrid Aurantiochytrium sp. 18W-13a accummulates high amounts of squalene. Biosci Biotechnol Biochem 75:2246–2248

  37. Kelly GS (1999) Squalene and its potential clinical uses. Altern Med Rev J Clin Ther 4:29–36

  38. Kim S-J, Kim M-D, Choi J-H, Kim S-Y, Ryu Y-W, Seo J-H (2006) Amplification of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q (10) production in recombinant Escherichia coli. Appl Microbiol Biotechnol 72:982–985

  39. Kohno Y, Egawa Y, Itoh S, S-i Nagaoka, Takahashi M, Mukai K (1995) Kinetic study of quenching reaction of singlet oxygen and scavenging reaction of free radical by squalene in n-butanol. Biochim Biophys Acta (BBA) Lipids Lipid Metab 1256:52–56

  40. KopiCoVá Z, VaVreiNoVá S (2007) Occurrence of squalene and cholesterol in various species of Czech freshwater fish. Czech J Food Sci 25:195

  41. Kuzuyama T (2002) Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Biosci Biotechnol Biochem 66:1619–1627

  42. Lack M, Sant G (2009) Trends in global shark catch and recent developments in management. In: TRAFFIC International, vol 33

  43. Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci 97:13172–13177

  44. Lee S, Poulter CD (2008) Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1. J Bacteriol 190:3808–3816

  45. Lee J-K, Oh D-K, Kim S-Y (2007) Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens. J Biotechnol 128:555–566

  46. Lee PY, Yong VC, Rosli R, Gam LH, Chong PP (2014) Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris. Protein Expr Purif 94:15–21

  47. Li Q, Chen G-Q, Fan K-W, Lu F-P, Aki T, Jiang Y (2009) Screening and characterization of squalene-producing thraustochytrids from Hong Kong mangroves. J Agric Food Chem 57:4267–4272

  48. Liu G, Ahrens E, Schreibman PH, Crouse JR (1976) Measurement of squalene in human tissues and plasma: validation and application. J Lipid Res 17:38–45

  49. Maguire L, O’sullivan S, Galvin K, O’connor T, O’brien N (2009) Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut. Int J Food Sci Nutr 55:171–178

  50. Mantzouridou F, Tsimidou MZ (2010) Observations on squalene accumulation in Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6. FEMS Yeast Res 10:699–707

  51. Mantzouridou F, Naziri E, Tsimidou MZ (2009) Squalene versus ergosterol formation using Saccharomyces cerevisiae: combined effect of oxygen supply, inoculum size, and fermentation time on yield and selectivity of the bioprocess. J Agric Food Chem 57:6189–6198

  52. Martirosyan DM, Miroshnichenko LA, Kulakova SN, Pogojeva AV, Zoloedov VI (2007) Amaranth oil application for coronary heart disease and hypertension. Lipids Health Dis 6:1

  53. Maury J, Asadollahi MA, Møller K, Clark A, Nielsen J (2005) Microbial isoprenoid production: an example of green chemistry through metabolic engineering. Biotechnology for the Future. Springer, Berlin, pp 19–51

  54. Miettinen TA, Vanhanen H (1994) Serum concentration and metabolism of cholesterol during rapeseed oil and squalene feeding. Am J Clin Nutr 59:356–363

  55. Nakashima T, Inoue T, Oka A, Nishino T, Osumi T, Hata S (1995) Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase. Proc Natl Acad Sci 92:2328–2332

  56. Nakazawa A, Matsuura H, Kose R, Kato S, Honda D, Inouye I, Kaya K, Watanabe MM (2012) Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production. Bioresour Technol 109:287–291

  57. Nakazawa A, Kokubun Y, Matsuura H, Yonezawa N, Kose R, Yoshida M, Tanabe Y, Kusuda E, Van Thang D, Ueda M (2014) TLC screening of thraustochytrid strains for squalene production. J Appl Phycol 26:29–41

  58. Naziri E, Mantzouridou F, Tsimidou MZ (2011) Enhanced squalene production by wild-type Saccharomyces cerevisiae strains using safe chemical means. J Agric Food Chem 59:9980–9989

  59. Obulesu T, Anandan R, Mathew S, Ganesan B, Krishna G, Lakra W, Asha K (2015) Antioxidant defence of dietary squalene supplementation on n-3 poly unsaturated fatty acids (PUFA)-mediated oxidative stress in young and aged rats. Fish Technol 52:48–52

  60. Ohkuma T, Otagiri K, Tanaka S, Ikekawa T (1983) Intensification of host’s immunity by squalene in sarcoma 180 bearing ICR mice. J Pharmacobiodyn 6:148–151

  61. Ohtake K, Saito N, Shibuya S, Kobayashi W, Amano R, Hirai T, Sasaki S, Nakano C, Hoshino T (2014) Biochemical characterization of the water-soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD (E) D motifs and the highly conserved aromatic amino acid residues. FEBS J 281:5479–5497

  62. Orihara N, Kuzuyama T, Takahashi S, Furihata K, Seto H (1998) Studies on the biosynthesis of terpenoid compounds produced by actinomycetes. 3. Biosynthesis of the isoprenoid side chain of novobiocin via the non-mevalonate pathway in Streptomyces niveus. J Antibiot 51:676–678

  63. Pan J-J, Solbiati JO, Ramamoorthy G, Hillerich BS, Seidel RD, Cronan JE, Almo SC, Poulter CD (2015) Biosynthesis of squalene from farnesyl diphosphate in bacteria: three steps catalyzed by three enzymes. ACS Cent Sci 1:77–82

  64. Ravi Kumar S, Yamauchi I, Narayan B, Katsuki A, Hosokawa M, Miyashita K (2016) Squalene modulates fatty acid metabolism: enhanced EPA/DHA in obese/diabetic mice (KK-Ay) model. Eur J Lipid Sci Technol. doi:10.1002/ejlt.201600006

  65. Ren LJ, Ji XJ, Huang H, Qu L, Feng Y, Tong QQ, Ouyang P-K (2010) Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. Appl Microbiol Biotechnol 87:1649–1656

  66. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H (1993) Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J 295:517–524

  67. Ronco AL, De Stéfani E (2013) Squalene: a multi-task link in the crossroads of cancer and aging. Funct Foods Health Dis 3:462–476

  68. Salvador M, Aranda F, Gomez-Alonso S, Fregapane G (2003) Influence of extraction system, production year and area on Cornicabra virgin olive oil: a study of five crop seasons. Food Chem 80:359–366

  69. Sánchez-Fidalgo S, Villegas I, Rosillo MÁ, Aparicio-Soto M, de la Lastra CA (2015) Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways. Mol Nutr Food Res 59:284–292

  70. Sangari FJ, Pérez-Gil J, Carretero-Paulet L, García-Lobo JM, Rodríguez-Concepción M (2010) A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria. Proc Natl Acad Sci 107:14081–14086

  71. Shechter I, Klinger E, Rucker M, Engstrom R, Spirito J, Islam M, Boettcher B, Weinstein D (1992) Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. J Biol Chem 267:8628–8635

  72. Singhal RS, Kulkarni P (1990) Effect of puffing on oil characteristics of Amaranth (Rajgeera) seeds. J Am Oil Chem Soc 67:952–954

  73. Socaciu C, Faye M, Salin F, Pauly G, Gleizes M (1995) In vitro yeast (Saccharomyces cerevisiae) presqualene and squalene synthesis related to substrate and cofactor availability. C rendus de l’Acad Sci Ser III Sci de la vie 318:919–926

  74. Spanova M, Daum G (2011) Squalene-biochemistry, molecular biology, process biotechnology, and applications. Eur J Lipid Sci Technol 113:1299–1320

  75. Storm HM, Oh SY, Kimler BF, Norton S (1993) Radioprotection of mice by dietary squalene. Lipids 28:555–559

  76. Thompson JF, Danley DE, Mazzalupo S, Milos PM, Lira ME, Harwood HJ (1998) Truncation of human squalene synthase yields active, crystallizable protein. Arch Biochem Biophys 350:283–290

  77. Tsujimoto M (1916) A highly unsaturated hydrocarbon in shark liver oil. Ind Eng Chem 8:889–896

  78. Tsujiwaki G, Yamamoto H, Minami K (1995) Manufacture of squalene with Candida famata. Japan Kokai Tokkyo Koho JP 07, 289, 272 (C.A.-124: 230184)

  79. Uragami S, Koga H (1986) Bacterial production of squalene. Japan Kokai Tokkyo Koho JP 61:212–290

  80. Welander PV, Hunter RC, Zhang L, Sessions AL, Summons RE, Newman DK (2009) Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. J Bacteriol 191:6145–6156

  81. Wyre C, Overton TW (2014) Use of a stress-minimisation paradigm in high cell density fed-batch Escherichia coli fermentations to optimise recombinant protein production. J Ind Microbiol Biotechnol 41:1391–1404

  82. Xiao H, Yao Z, Peng Q, Ni F, Sun Y, Zhang C, Zhong Z (2016) Extraction of squalene from camellia oil by silver ion complexation. Sep Purif Technol 169:196–201

  83. Xu W, Yang S, Zhao J, Su T, Zhao L, Liu J (2014) Improving coenzyme Q (8) production in Escherichia coli employing multiple strategies. J Ind Microbiol Biotechnol 41:1297–1303

  84. Xu W, Chai C, Shao L, Yao J, Wang Y (2016) Metabolic engineering of Rhodopseudomonas palustris for squalene production. J Ind Microbiol Biotechnol 43:719–725

  85. Yoon J, Matsuo Y, Matsuda S, Adachi K, Kasai H, Yokota A (2008) Rubritalea sabuli sp. nov., a carotenoid-and squalene-producing member of the family Verrucomicrobiaceae, isolated from marine sediment. Int J Syst Evol Microbiol 58:992–997

  86. Yue C-J, Jiang Y (2009) Impact of methyl jasmonate on squalene biosynthesis in microalga Schizochytrium mangrovei. Process Biochem 44:923–927

  87. Zahiri HS, Yoon SH, Keasling JD, Lee SH, Kim SW, Yoon SC, Shin YC (2006) Coenzyme Q (10) production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway. Metab Eng 8:406–416

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Acknowledgments

We thank Professor Arno Müllbacher (Australian National University) for critical reading of the manuscript. This paper was supported by the Shaanxi Science and Technology Innovation Project 2016KTCQ03-07 (to Y. Wang). X. Ma was supported by a Research Support Grant (2015NQ03) from the Xi’an Medical University.

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Correspondence to Yang Wang.

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Xu, W., Ma, X. & Wang, Y. Production of squalene by microbes: an update. World J Microbiol Biotechnol 32, 195 (2016). https://doi.org/10.1007/s11274-016-2155-8

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Keywords

  • Biosynthesis
  • Fermentation
  • Microbial production
  • Metabolic engineering
  • Squalene