Dataset Information

In silico characterization and over-expression of squalene hopene cyclase from Pseudomonas mendocina.

ABSTRACT: Pseudomonas mendocina was identified as a novel endophytic isolate of Murraya koenigii with squalene cyclase activity. The PCR amplification of squalene hopene cyclase (shc) gene from the isolate Pseudomonas mendocina with the primers PA1/PA2 showed a band at 1980 bp specific for the enzyme squalene hopene cyclase. The in silico translation of the squalene hopene cyclase gene showed 96% sequence similarity with squalene hopene cyclase of Pseudomonas agarici (WP-060782422). Docking studies of the template and the modeled protein with the ligand squalene showed that the main interacting residues were Asp376 and Asp377. Squalene hopene cyclase template 1 sqc.1A sequence from Alicyclobacillus acidocaldaruis was used as the template for docking experiments. The gene coding for squalene hopene cyclase from Pseudomonas mendocina has been cloned in pET-28a vector to produce recombinant vector and was expressed in E.coli BL21 (DE3) expression system. Squalene hopene cyclase enzyme was isolated, purified and the molecular weight was confirmed by SDS-PAGE as 75 KDa.

SUBMITTER: Nair IM

PROVIDER: S-EPMC6776573 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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In silico characterization and over-expression of squalene hopene cyclase from Pseudomonas mendocina.

Nair Indu M IM Kochupurakal Jayachandran J

3 Biotech 20191003 11

Pseudomonas mendocina was identified as a novel endophytic isolate of Murraya koenigii with squalene cyclase activity. The PCR amplification of squalene hopene cyclase (shc) gene from the isolate Pseudomonas mendocina with the primers PA1/PA2 showed a band at 1980 bp specific for the enzyme squalene hopene cyclase. The in silico translation of the squalene hopene cyclase gene showed 96% sequence similarity with squalene hopene cyclase of Pseudomonas agarici (WP ...[more]

PMID: 31588405

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Project description:Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.IMPORTANCE The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.

Dataset Information

In silico characterization and over-expression of squalene hopene cyclase from Pseudomonas mendocina.

Publications

In silico characterization and over-expression of squalene hopene cyclase from <i>Pseudomonas mendocina</i>.

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