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A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene.

ABSTRACT: Hopene is an important precursor for synthesizing bioactive hopanoids with great commercial value. However, the chemical methods for synthesizing hopene are not efficient to date. Hopene is commonly obtained by extracting from plants or bacteria like other terpenoids, but the complicated extraction process is inefficient and unfriendly to the environment. Hopene can be biological synthesized by squalene-hopene cyclase (SHC) from squalene. However, hopene production by SHC remained at a low level until now. In this work, we found a novel SHC named OUC-SaSHC from Streptomyces albolongus ATCC 27414. An easy procedure for expression and purification of OUC-SaSHC was established. The conditions for OUC-SaSHC to convert squalene into hopene are optimized as in 100 mM sodium phosphate buffer (pH 7.0) containing 0.5% Tween 80, 20 mM squalene and 0.14 mg/mL OUC-SaSHC at 30°C. In the scale-up reaction with the final volume of 100 mL, the yield of squalene could be up to 99% at 36 h, and 8.07 mg/mL hopene was produced. Our work showed a great potential of OUC-SaSHC as biocatalyst on scale-up production of hopene, hence improves the SHC-catalyzing enzyme synthesis of hopene from laboratory level to application level.

SUBMITTER: Liu Z

PROVIDER: S-EPMC7232578 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene.

Liu Zhen Z Zhang Yinan Y Sun Jianan J Huang Wen-Can WC Xue Changhu C Mao Xiangzhao X

Frontiers in bioengineering and biotechnology 20200505

Hopene is an important precursor for synthesizing bioactive hopanoids with great commercial value. However, the chemical methods for synthesizing hopene are not efficient to date. Hopene is commonly obtained by extracting from plants or bacteria like other terpenoids, but the complicated extraction process is inefficient and unfriendly to the environment. Hopene can be biological synthesized by squalene-hopene cyclase (SHC) from squalene. However, hopene production by SHC remained at a low level ...[more]

PMID: 32478051

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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

A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene.

Publications

A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene.

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