Squalene
Squalene is a linear triterpene formed via the MVA or MEP biosynthetic pathway and is widely distributed in bacteria, fungi, algae, plants, and animals. Metabolically, squalene is not only used as a precursor in the synthesis of complex secondary metabolites such as sterols, hormones, and vitamins, but also as a carbon source in aerobic and anaerobic fermentation in micro-organisms. Owing to the increasing roles of squalene as an antioxidant, anticancer, and anti-inflammatory agent, the demand for this chemical is highly urgent. As a result, with the exception of traditional methods of the isolation of squalene from animals (shark liver oil) and plants, biotechnological methods using microorganisms as producers have afforded increased yield
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and productivity, but a reduction in progress. In this study, we first review the biosynthetic routes of squalene and its typical derivatives, particularly the squalene synthase route. Second, typical biotechnological methods for the enhanced production of squalene using microbial factories are summarized and classified. Finally, the outline and discussion of the novel trend in the production of squalene with several updated events to 2015 are presented.
Keywords: squalene; biosynthesis; microbial factory; terpenes; squalene production.
Introduction
Structurally, squalene is a unique 30-carbon, polyunsaturated hydrocarbon of the triterpene group, consisting of six nonconjugated or isoprene units and is consequently an isoprenoid compound (Fig. 1). Several antioxidants such as lycopene and carotene are either isoprenoids or have an isoprenoid tail. The well-known compounds requiring a prenyl group for their synthesis include vitamin A, D, E, K, carotenes, and lycopene, and all have antioxidant properties. In recent years, scientists have been interested in studying the biochemistry and production of squalene owing to its potential pharmaceutical applications. Historically, inhabitants of the Japanese island of Izu used to drink oil that was locally named “Samedawa”, meaning “cure oil” [58]. Squalene was first described by Tsujimoto, a Japanese industrial engineer, and ten years after his initial discovery, Samedawa was found to contain high proportions of a novel highly unsaturated hydrocarbon [66]. It received its name because of the fact that it was first isolated from sea shark (Squalus spp.) liver oil, which was proven to bear a plenty amount of squalene [17]. However, squalene is a naturally-occurring polyprenyl compound widely found in differing amounts in nature such as in olive oil [40], amaranth seeds [2, 44], wheat germ oil, palm oil, and rice bran oil [25]. It is also found in other plant materials, freshwater fish, and human tissue or sebum [41,34] and has various beneficial effects, being useful as a nutrient and as a preventive and therapeutic medicine. The inhibition of cancer risk [51], enhancement of the anti-tumor action of chemotherapeutic agents [47], and efficient improvement of the immune system [52,23] are confirmed as dietary squalene properties. It is a critical agent to protect the skin from short wavelength radiant [65] and is also effective in lowering blood cholesterol [8]. From various studies, it is found that squalene can effectively inhibit induced tumor genesis of the lung, skin, and colon in rodents [59], indicating that it is a high potentially pharmaceutical reagent.
Biochemistry of squalene
General biosynthetic routes of squalene and its derivatives
Isoprenoids are synthesized from the isopentenyl units formed by two different metabolic pathways, leading to isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) [36]. These distinctive pathways utilize nonhomologous enzymes that have evolved independently, to generate the same universal C5 precursors IPP and DMAPP. The classical mevalonate (MVA) pathway was discovered in the 1960s and considered to be the only one source of the precursors IPP and DMAPP [43,27]. The MVA pathway is effective in plants, animals, and fungi, and functions in the water soluble components in cytoplasm to generally supply the precursors for the production of sesquiterpenes and triterpenes such as squalene and its related compounds such as oxidosqualene and bis-oxidosqualene, which are considerd to be the presursers of nearly 200 triterpene skeletons [69], and the more recently discovered 1-deoxyxylulose-5-phosphate (DXP)/methylerythritol phosphate (MEP) pathway [59] primarily found in prokaryotes including E. coli and the plastids of photosynthetic organisms [35, 11]. This pathway, named after the first committed precursor 2-C-methyl-D-erythritol-4-phosphate (MEP; the pathway is also sometimes referred to as the DXP pathway), is plastidial in nature and is used to supply precursors of monoterpenes, diterpenes, and tetraterpenes [66].
Naturally, the synthesis of squalene via the MVA pathway starts with the condensation of one acetyl-CoA with another acetyl-CoA by acetoacetyl-CoA synthase (AAS).
This enzyme is so-called acetyl-CoA transferase functioning in the formation of acetoacetyl-CoA. Reaction chain from acetoacetyl-CoA to mevalonate continues to form 3-hydroxy-3 methylglutaryl-CoA (HMG-CoA) via the condensation in the presence of 3-hydroxy-3 methylglutaryl-CoA synthase (HMGS) [33]. In the presence of HMG-CoA reductase (HMGR) plus NADPH as the cofactor, HMG-CoA is later reduced to mevalonate [21], which is subsequently phosphorylated by mevalonate kinase (MK) in the presence of adenosine triphosphate (ATP) to form mevalonate 5-phosphate and is further phosphorylated by phosphomevalonate kinase (PMK) in the presence of ATP to form mevalonate 5-diphosphate and then decarboxylated by mevalonate 5-diphosphate decarboxylase (PMD) to form isopentenyl diphosphate (IPP), which is used as the chemical base to build most known polyprenyl compounds. IPP is then isomerized to DMAPP by isopentanyl diphosphate isomerase (IDI). The condensation of IPP with DMAPP by farnesyl diphosphate synthase (FPS) results in geranyl diphosphate (GPP), and the subsequent condensation with another IPP to form farnesyl diphosphate (FPP). Finally, squalene synthase has been identified as the enzyme that catalyzes the NADPH-mediated formation of squalene using FPP as the substrate [66] (Fig. 2A). Besides these, …show more content…
glyceraldehyde-3-phosphate (G-3-P) and pyruvate are the initial precursors for the formation of DXP by DXP synthase (DXS). This substrate is consequently reduced into MEP by a reaction using DXP reductoisomerase (DXR) as the biocatalyst. Some bacteria lack DXR but have DRL (DXR-like) enzymes that perform the same reaction [61] (Fig. 2B).
Depending on the species, squalene is continously utilized as a substrate for the synthesis of sterols including cholesterol, and as a typical product or carbon source in eubacteria (Corynebacterium, Pseudomonas or Arthrobacter) to form various metabolites [63,6,70].
Recently, Streptomyces peucetius ATCC27952 originated Spterp25 was successfully heterologously characterized in E. coli. The experimental results show that it is an enzyme functioning as a squalene-hopene cyclase [19] (Fig. 3).
Squalene synthases and its application
Squalene synthase is a very important enzyme catalyzing the first step of sterol/hopanoid biosynthesis in various organisms. Many studies have been reported on SQS such as in plant (Dioscorea zingiberensis (71), Withania somnifera (22), fungi (Candida tropicalis (38)). Detailed characteristics of squalene synthases are described in Table 1.
A Poria cocos – originated SQS was isolated and characterized using degenerate and inverse PCR. Transcriptional level of this SQS gene was increased about four folds by the treatment with P. cocos cell with 300 μM methyl jasmonate, enhancing the content of cellular squalene (128.62 μg/g) after 72 h induction [67].
Recently, directed evolution is a powerful technique to screen and recruit an
appr
and productivity, but a reduction in progress. In this study, we first review the biosynthetic routes of squalene and its typical derivatives, particularly the squalene synthase route. Second, typical biotechnological methods for the enhanced production of squalene using microbial factories are summarized and classified. Finally, the outline and discussion of the novel trend in the production of squalene with several updated events to 2015 are presented.
Keywords: squalene; biosynthesis; microbial factory; terpenes; squalene production.
Introduction
Structurally, squalene is a unique 30-carbon, polyunsaturated hydrocarbon of the triterpene group, consisting of six nonconjugated or isoprene units and is consequently an isoprenoid compound (Fig. 1). Several antioxidants such as lycopene and carotene are either isoprenoids or have an isoprenoid tail. The well-known compounds requiring a prenyl group for their synthesis include vitamin A, D, E, K, carotenes, and lycopene, and all have antioxidant properties. In recent years, scientists have been interested in studying the biochemistry and production of squalene owing to its potential pharmaceutical applications. Historically, inhabitants of the Japanese island of Izu used to drink oil that was locally named “Samedawa”, meaning “cure oil” [58]. Squalene was first described by Tsujimoto, a Japanese industrial engineer, and ten years after his initial discovery, Samedawa was found to contain high proportions of a novel highly unsaturated hydrocarbon [66]. It received its name because of the fact that it was first isolated from sea shark (Squalus spp.) liver oil, which was proven to bear a plenty amount of squalene [17]. However, squalene is a naturally-occurring polyprenyl compound widely found in differing amounts in nature such as in olive oil [40], amaranth seeds [2, 44], wheat germ oil, palm oil, and rice bran oil [25]. It is also found in other plant materials, freshwater fish, and human tissue or sebum [41,34] and has various beneficial effects, being useful as a nutrient and as a preventive and therapeutic medicine. The inhibition of cancer risk [51], enhancement of the anti-tumor action of chemotherapeutic agents [47], and efficient improvement of the immune system [52,23] are confirmed as dietary squalene properties. It is a critical agent to protect the skin from short wavelength radiant [65] and is also effective in lowering blood cholesterol [8]. From various studies, it is found that squalene can effectively inhibit induced tumor genesis of the lung, skin, and colon in rodents [59], indicating that it is a high potentially pharmaceutical reagent.
Biochemistry of squalene
General biosynthetic routes of squalene and its derivatives
Isoprenoids are synthesized from the isopentenyl units formed by two different metabolic pathways, leading to isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) [36]. These distinctive pathways utilize nonhomologous enzymes that have evolved independently, to generate the same universal C5 precursors IPP and DMAPP. The classical mevalonate (MVA) pathway was discovered in the 1960s and considered to be the only one source of the precursors IPP and DMAPP [43,27]. The MVA pathway is effective in plants, animals, and fungi, and functions in the water soluble components in cytoplasm to generally supply the precursors for the production of sesquiterpenes and triterpenes such as squalene and its related compounds such as oxidosqualene and bis-oxidosqualene, which are considerd to be the presursers of nearly 200 triterpene skeletons [69], and the more recently discovered 1-deoxyxylulose-5-phosphate (DXP)/methylerythritol phosphate (MEP) pathway [59] primarily found in prokaryotes including E. coli and the plastids of photosynthetic organisms [35, 11]. This pathway, named after the first committed precursor 2-C-methyl-D-erythritol-4-phosphate (MEP; the pathway is also sometimes referred to as the DXP pathway), is plastidial in nature and is used to supply precursors of monoterpenes, diterpenes, and tetraterpenes [66].
Naturally, the synthesis of squalene via the MVA pathway starts with the condensation of one acetyl-CoA with another acetyl-CoA by acetoacetyl-CoA synthase (AAS).
This enzyme is so-called acetyl-CoA transferase functioning in the formation of acetoacetyl-CoA. Reaction chain from acetoacetyl-CoA to mevalonate continues to form 3-hydroxy-3 methylglutaryl-CoA (HMG-CoA) via the condensation in the presence of 3-hydroxy-3 methylglutaryl-CoA synthase (HMGS) [33]. In the presence of HMG-CoA reductase (HMGR) plus NADPH as the cofactor, HMG-CoA is later reduced to mevalonate [21], which is subsequently phosphorylated by mevalonate kinase (MK) in the presence of adenosine triphosphate (ATP) to form mevalonate 5-phosphate and is further phosphorylated by phosphomevalonate kinase (PMK) in the presence of ATP to form mevalonate 5-diphosphate and then decarboxylated by mevalonate 5-diphosphate decarboxylase (PMD) to form isopentenyl diphosphate (IPP), which is used as the chemical base to build most known polyprenyl compounds. IPP is then isomerized to DMAPP by isopentanyl diphosphate isomerase (IDI). The condensation of IPP with DMAPP by farnesyl diphosphate synthase (FPS) results in geranyl diphosphate (GPP), and the subsequent condensation with another IPP to form farnesyl diphosphate (FPP). Finally, squalene synthase has been identified as the enzyme that catalyzes the NADPH-mediated formation of squalene using FPP as the substrate [66] (Fig. 2A). Besides these, …show more content…
glyceraldehyde-3-phosphate (G-3-P) and pyruvate are the initial precursors for the formation of DXP by DXP synthase (DXS). This substrate is consequently reduced into MEP by a reaction using DXP reductoisomerase (DXR) as the biocatalyst. Some bacteria lack DXR but have DRL (DXR-like) enzymes that perform the same reaction [61] (Fig. 2B).
Depending on the species, squalene is continously utilized as a substrate for the synthesis of sterols including cholesterol, and as a typical product or carbon source in eubacteria (Corynebacterium, Pseudomonas or Arthrobacter) to form various metabolites [63,6,70].
Recently, Streptomyces peucetius ATCC27952 originated Spterp25 was successfully heterologously characterized in E. coli. The experimental results show that it is an enzyme functioning as a squalene-hopene cyclase [19] (Fig. 3).
Squalene synthases and its application
Squalene synthase is a very important enzyme catalyzing the first step of sterol/hopanoid biosynthesis in various organisms. Many studies have been reported on SQS such as in plant (Dioscorea zingiberensis (71), Withania somnifera (22), fungi (Candida tropicalis (38)). Detailed characteristics of squalene synthases are described in Table 1.
A Poria cocos – originated SQS was isolated and characterized using degenerate and inverse PCR. Transcriptional level of this SQS gene was increased about four folds by the treatment with P. cocos cell with 300 μM methyl jasmonate, enhancing the content of cellular squalene (128.62 μg/g) after 72 h induction [67].
Recently, directed evolution is a powerful technique to screen and recruit an
appr