Production of squalene by squalene synthases and their truncated mutants in Escherichia coli

Katabami, Akinori; Li, Ling; Iwasaki, Miki; Furubayashi, Maiko; Saito, Kyoichi; Umeno, Daisuke

doi:10.1016/j.jbiosc.2014.07.013

Cited by 63 publications

(58 citation statements)

References 76 publications

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“…5A). To test these two possibilities, we first engineered Escherichia coli to produce high levels of squalene by introducing a plasmid that expresses the M. alcaliphilum squalene synthase (sqs) gene as well as a second plasmid that increases overall isoprenoid synthesis (40,41). This strain, also expressing M. alcaliphilum ths from a third plasmid, did not demethylate or otherwise modify squalene, nor did it produce tetrahymanol, indicating that Ths was not directly using squalene as a substrate (Fig.…”

Section: Resultsmentioning

confidence: 99%

A distinct pathway for tetrahymanol synthesis in bacteria

Banta

Wei

Welander

2015

Proc. Natl. Acad. Sci. U.S.A.

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Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.gammacerane | tetrahymanol | biomarkers | methanotrophs | sterols

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Section: Resultsmentioning

confidence: 99%

A distinct pathway for tetrahymanol synthesis in bacteria

Banta

Wei

Welander

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…However, previous study has demonstrated that native MEP pathway has a higher theoretical yield of terpenoid than MVA pathway in E. coli by genome-scale in silico modeling (Meng et al, 2011). Katabami et al (2015) used truncated squalene synthases from human (hsqs) in combination with MVA pathways to produce squalene up to 230 mg/L or 55 mg/g-DCW in flask culture, an approximately 55-fold increase as compared to E. coli harboring hsqs alone. In this study, in overexpression of key-enzymes genes dxs, idi and ispA of MEP pathway with double copy of SQS from Y. lipolytica in E. coli BL21(DE3), yield of squalene increased 71 folds, when compared with the strain that only harbor two copies of SQS.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli

Liu¹,

Han²,

Xie³

et al. 2017

Afr. J. Biotechnol.

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2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway has been extensively employed for terpenoids biosynthesis in Escherichia coli.In this study, to obtain key-enzymes of MEP pathway for squalene production, overexpression of different combination of MEP pathway genes were compared. Squalene production in strain YSS12 with overexpressed dxs, idi and ispA of MEP pathway from E. coli was improved by 71-fold when compared with strain YSS3 which only contained double copy SQS. Analysis of transcriptional levels of MEP pathway genes in engineering strains showed that different squalene production can be attributed to changed transcriptional levels of co-overexpressed genes dxs, idi, ispG and ispA in engineering strains. Furthermore, different E. coli expression hosts were compared for squalene production, among which BL21(DE3) was the best squalene producer. These results illustrate that dxs, idi and ispA of the MEP pathway from E. coli were key-enzymes for squalene production in E. coli. These key-enzymes of MEP pathway could also be applied to other terpenoids production in E. coli.

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“…Although further truncation was not investigated for MaSQS, it was predicted that three additional helixes and partial JK-loop removal in the C-terminal regions could lead to the inactivity of SQS due to the loss of NADPH binding (Fig. 2), which influence the dehydrosqualene synthase activity [17]. Moreover, the truncated MaSQSΔC17 was subjected to site-directed mutagenesis and the mutant mMaSQSΔC17 displayed an improved enzyme activity, indicating that the site E186 played a significant role in the catalytic ability.…”

Section: Discussionmentioning

confidence: 99%

Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis

Huang

Yao

Zhang

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 °C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k(cat)/K(m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology.

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Production of squalene by squalene synthases and their truncated mutants in Escherichia coli

Cited by 63 publications

References 76 publications

A distinct pathway for tetrahymanol synthesis in bacteria

A distinct pathway for tetrahymanol synthesis in bacteria

Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli

Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis

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