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Cited by 121Plant biochemistry and biosynthesisMicrobial Metabolic Engineering and BioproductionMicrobial Natural Products and Biosynthesis

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Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

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Gohil, Nisarg, Bhattacharjee, Gargi, Khambhati, Khushal, Braddick, Darren, Singh, Vijai·2019·Frontiers in Bioengineering and Biotechnology
DOI 10.3389/fbioe.2019.00050PMC PMC6439483
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Abstract

The triterpene squalene is a natural compound that has demonstrated an extraordinary diversity of uses in pharmaceutical, nutraceutical, and personal care industries. Emboldened by this range of uses, novel applications that can gain profit from the benefits of squalene as an additive or supplement are expanding, resulting in its increasing demand. Ever since its discovery, the primary source has been the deep-sea shark liver, although recent declines in their populations and justified animal conservation and protection regulations have encouraged researchers to identify a novel route for squalene biosynthesis. This renewed scientific interest has profited from immense developments in synthetic biology, which now allows fine-tuning of a wider range of plants, fungi, and microorganisms for improved squalene production. There are numerous naturally squalene producing species and strains; although they generally do not make commercially viable yields as primary shark liver sources can deliver. The recent advances made toward improving squalene output from natural and engineered species have inspired this review. Accordingly, it will cover in-depth knowledge offered by the studies of the natural sources, and various engineering-based strategies that have been used to drive the improvements in the pathways toward large-scale production. The wide uses of squalene are also discussed, including the notable developments in anti-cancer applications and in augmenting influenza vaccines for greater efficacy.

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Figure 1 Potential natural sources of squalene. Figure depicts the possible squalene sources ranging from unicellular microbes such as yeast and other bacterial cells to multicellular fungi, plants, and deep sea sharks. All these sources can produce squalene through mevalonate pathway. DCW, Dry cell weight.
Figure 1 Potential natural sources of squalene. Figure depicts the possible squalene sources ranging from unicellular microbes such as yeast and other bacterial cells to multicellular fungi, plants, and deep sea sharks. All these sources can produce squalene through mevalonate pathway. DCW, Dry cell weight.CC BY · PubMed Central
Figure 2 Squalene biosynthetic pathways and production strategies. (A) Squalene biosynthesis via MVA pathway in yeast, fungi, and algae. The engineering strategies for enhanced squalene production are as follows: overexpression of HMGR (Polakowski et al., 1998; Tokuhiro et al., 2009; Mantzouridou and Tsimidou, 2010; Dai et al., 2012, 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b; Kwak et al., 2017; Paramasivan and Mutturi, 2017; Han et al., 2018; Huang et al., 2018; Wei et al., 2018) and SQS (Dai et al., 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b), downregulation of SQE (Garaiová et al., 2014; Hull et al., 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b; Han et al., 2018) in yeast; downregulation of SQE in algae (Kajikawa et al., 2015). (B) Squalene biosynthesis via MEP pathway in bacteria. The engineering strategies for the enhanced squalene production are as follows: overexpression of DXS (Ghimire et al., 2009), IDI (Ghimire et al., 2009; Katabami et al., 2015) and FPS (Katabami et al., 2015), introduction of heterologous squalene producing enzyme(s) (Ghimire et al., 2009; Furubayashi et al., 2014a; Katabami et al., 2015; Pan et al., 2015) in E. coli; overexpression of DXS and IDI, and the introduction of FPS into Synechococcus elongatus (Choi et al., 2016); deletion of SHC in Synechocystis (Englund et al., 2014) and Rhodopseudomonas palustris (Xu et al., 2016). MVA, mevalonate; MEP, 2-C-methyl-d-erythritol 4-phosphate; AACT, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; DMAPP, dimethylallyl diphosphate; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; FPS, farnesyl pyrophosphate synthase; GPP, geranyl diphosphate; FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; GA3P,glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; DLR, DXR-like enzyme; HMBPP, 4-hydroxy-3-methyl-but-2-enylpyrophosphate; IspH,IPP/DMAPP synthase; SHC, squalene hopene cyclase.
Figure 2 Squalene biosynthetic pathways and production strategies. (A) Squalene biosynthesis via MVA pathway in yeast, fungi, and algae. The engineering strategies for enhanced squalene production are as follows: overexpression of HMGR (Polakowski et al., 1998; Tokuhiro et al., 2009; Mantzouridou and Tsimidou, 2010; Dai et al., 2012, 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b; Kwak et al., 2017; Paramasivan and Mutturi, 2017; Han et al., 2018; Huang et al., 2018; Wei et al., 2018) and SQS (Dai et al., 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b), downregulation of SQE (Garaiová et al., 2014; Hull et al., 2014; Zhuang and Chappell, 2015; Rasool et al., 2016a,b; Han et al., 2018) in yeast; downregulation of SQE in algae (Kajikawa et al., 2015). (B) Squalene biosynthesis via MEP pathway in bacteria. The engineering strategies for the enhanced squalene production are as follows: overexpression of DXS (Ghimire et al., 2009), IDI (Ghimire et al., 2009; Katabami et al., 2015) and FPS (Katabami et al., 2015), introduction of heterologous squalene producing enzyme(s) (Ghimire et al., 2009; Furubayashi et al., 2014a; Katabami et al., 2015; Pan et al., 2015) in E. coli; overexpression of DXS and IDI, and the introduction of FPS into Synechococcus elongatus (Choi et al., 2016); deletion of SHC in Synechocystis (Englund et al., 2014) and Rhodopseudomonas palustris (Xu et al., 2016). MVA, mevalonate; MEP, 2-C-methyl-d-erythritol 4-phosphate; AACT, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; DMAPP, dimethylallyl diphosphate; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; FPS, farnesyl pyrophosphate synthase; GPP, geranyl diphosphate; FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; GA3P,glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; DLR, DXR-like enzyme; HMBPP, 4-hydroxy-3-methyl-but-2-enylpyrophosphate; IspH,IPP/DMAPP synthase; SHC, squalene hopene cyclase.CC BY · PubMed Central
Figure 3 Various engineering strategies employed for enhanced production of squalene in E. coli. (A) Ghimire et al. (2009) expressed hopD and hopAB from Streptomyces peucetius followed by overexpression of dxs and idi, (B) Pan et al. (2015) expressed hpnC, hpnD, and hpnE from Zymomonas mobilis or Rhodopseudomonas palustris (C) Furubayashi et al. (2014a) expressed hSQS, (D) Katabami et al. (2015) expressed hSQS or tSQS from Thermosynechococcus elongates, atoB, HMGS, HMGR, HK, PMK, PMD from S. cerevisiae followed by overexpression of idi and ispA. GA3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; dxs, DXP synthase; IPP, isopentenyl pyrophosphate; idi, isopentenyl diphosphate isomerase; DMAPP, dimethylallyl diphosphate; ispA, farnesyl pyrophosphate synthase; GPP, geranyl diphosphate; FPP, isopentenyl pyrophosphate; hopD, farnesyl diphosphate synthase; hopAB, squalene/phytoene synthases; hpnC, hydroxyl squalene synthase; hpnD, presqualene diphosphate synthase; hpnE, hydroxysqualene dehydroxylase; hSQS, human squalene synthase; tSQS, Thermosynechococcus SQS; atoB, acetyl-CoA acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; HK, mevalonate kinase; PMK, phosphomevalonate kinase; PMD, mevalonate diphosphate decarboxylase.
Figure 3 Various engineering strategies employed for enhanced production of squalene in E. coli. (A) Ghimire et al. (2009) expressed hopD and hopAB from Streptomyces peucetius followed by overexpression of dxs and idi, (B) Pan et al. (2015) expressed hpnC, hpnD, and hpnE from Zymomonas mobilis or Rhodopseudomonas palustris (C) Furubayashi et al. (2014a) expressed hSQS, (D) Katabami et al. (2015) expressed hSQS or tSQS from Thermosynechococcus elongates, atoB, HMGS, HMGR, HK, PMK, PMD from S. cerevisiae followed by overexpression of idi and ispA. GA3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; dxs, DXP synthase; IPP, isopentenyl pyrophosphate; idi, isopentenyl diphosphate isomerase; DMAPP, dimethylallyl diphosphate; ispA, farnesyl pyrophosphate synthase; GPP, geranyl diphosphate; FPP, isopentenyl pyrophosphate; hopD, farnesyl diphosphate synthase; hopAB, squalene/phytoene synthases; hpnC, hydroxyl squalene synthase; hpnD, presqualene diphosphate synthase; hpnE, hydroxysqualene dehydroxylase; hSQS, human squalene synthase; tSQS, Thermosynechococcus SQS; atoB, acetyl-CoA acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; HK, mevalonate kinase; PMK, phosphomevalonate kinase; PMD, mevalonate diphosphate decarboxylase.CC BY · PubMed Central
Figure 4 Potential applications of squalene for treatment of cancer, as detoxifier, use in cosmetics, drug and vaccine adjuvants.
Figure 4 Potential applications of squalene for treatment of cancer, as detoxifier, use in cosmetics, drug and vaccine adjuvants.CC BY · PubMed Central

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Research works printed but not linked: 6

  1. Bhattacharjee, 2003, Extraction of squalene from yeast by supercritical carbon dioxide, World J. Microbiol. Biotechnol., 19, 605, 10.1023/A:1025146132281 · DOI
  2. Panchasara, 2018, Biosynthetic pathway for production of renewable biofuels, 63
  3. Popa, 2014, Squalene - natural resources and applications, Farmacia, 62, 840
  4. Pora, 2014, Method for the Preparation and Extraction of Squalene From Microalgae.
  5. Tsujiwaki, 1995, Manufacture of squalene with Candida famata, Jpn. Kokai Tokkyo Koho JP, 7, 272
  6. Uragami, 1986, Bacterial production of squalene, Jpn. Kokai Tokkyo Koho JP, 61, 212

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Metadata

Authors
Gohil, N., Bhattacharjee, G., Khambhati, K., Braddick, D., & Singh, V.
Affiliations
  • Indian Institute of Advanced Research (IN)
  • Genopole (France) (FR)
Corresponding author
Singh, Vijai
ORCID
Venue
Frontiers in Bioengineering and Biotechnology
Publisher
Frontiers Media SA
Published
2019-03-22
Volume
7
Pages
114-114
ISSN
2296-4185 (ISSN-L)
DOI
10.3389/fbioe.2019.00050
PMC
PMC6439483
OpenAlex
W2924306915
Cited by
121
References
199
Citations by year
2026: 15 · 2025: 15 · 2024: 20 · 2023: 21 · 2022: 19 · 2021: 15 · 2020: 14 · 2019: 2
FWCI
1.10
Type
journal article
Language
en
Update notices
corrigendum (2019-05-28)
Open access
Licence
Locations
  • Frontiers in Bioengineering and Biotechnology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
Keywords
squalene, triterpenoids, synthetic biology, influenza vaccine adjuvants, nutraceuticals, pharmaceutical applications, industrial biotechnology

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