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

Abstract

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.

Pathway Engineering, Re-targeting, and Synthetic Scaffolding Improve the Production of Squalene in Plants

“…Although these are not directly comparable without creating IDI fusions with other variants, these fusions may be worth further investigation in future engineering. While the Ma SQS CΔ17 E186K variant previously demonstrated an increased catalytic efficiency ( k cat / K m ) in vitro , it did not increase squalene yields with either truncation variant in this system (Figure c). Ma SQS CΔ17 showed squalene yields 63% higher than the mean yield of other variants (Figure c) and was chosen as the SQS to use for development of lipid droplet scaffolding, along with At FDPS.…”

“…Additionally, a mutant of MaSQS E186K was created, which was previously shown to improve the catalytic efficiency 3.4-fold in in vitro studies. 40 Finally, two truncated SQS variants from the diatom Haslea ostrearia ( HoIDISQS ), which is a native fusion gene encoding an isopentenyl diphosphate isomerase (IDI) fused to the N-terminus of the SQS, were included. Both protein domains of Ho IDISQS were shown to be functional, 41 and previous studies have shown that co-expression of the gene encoding IDI, which catalyzes interconversion of IDP and DMADP, increases terpene yields.…”

“…benthamiana from the following organisms: Amaranthus hybridus ( AhSQS ), Botryococcus braunii ( BbSQS ), Euphorbia lathyris ( ElSQS ), Ganoderma lucidum ( GlSQS ), Mortierella alpina ( MaSQS ), and Saccharomyces cerevisiae ( ScERG9 ), which are all species that can accumulate large amounts of squalene-related compounds. Additionally, a mutant of MaSQS E186K was created, which was previously shown to improve the catalytic efficiency 3.4-fold in in vitro studies . Finally, two truncated SQS variants from the diatom Haslea ostrearia ( HoIDISQS ), which is a native fusion gene encoding an isopentenyl diphosphate isomerase (IDI) fused to the N-terminus of the SQS, were included.…”