Observations on squalene accumulation in Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6

Mantzouridou, Fani; Tsimidou, Maria Z.

doi:10.1111/j.1567-1364.2010.00645.x

Cited by 55 publications

(51 citation statements)

References 37 publications

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“…These two considerations may account for the post-transcriptional regulation on Erg9p observed in the current study. Supporting this idea, the fluorescence pattern we observed is consistent with dynamic squalene accumulation observed in an MVA pathway-enhanced strain, which showed a decrease in squalene content in the ethanol growth phase relative to in the exponental growth phase (Mantzouridou and Tsimidou, 2010). These data suggest that squalene synthase is down-regulated as cell growth slows down during the diauxic shift.…”

Section: Erg9p Signal Peptidesupporting

confidence: 88%

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

123

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A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae, Metabolic Engineering, http://dx.doi.org/10. 1016/j.ymben.2016.12.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractSesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1 % biomass; moreover, the specific growth rate declined. In order to redirect carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. 2A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86 % to ~100 mg L -1 . In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

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Section: Erg9p Signal Peptidesupporting

confidence: 88%

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

123

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“…The results suggest that increased ganoderic acid accumulation may be due to the increased supply of precursors. Similar observations were also reported for S. cerevisiae, in which the overexpression of tHMGR gene also resulted in more accumulation of squalene and lanosterol (6,21,25). Constitutive expression of HMGR gene in tobacco led to a Ͼ100-fold increase in the level of cycloartenol, the analogue of lanosterol in fungi (3).…”

Section: Discussionsupporting

confidence: 71%

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Zhong

2012

Appl Environ Microbiol

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Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.

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“…However, few studies report reduced growth rates in yeast from squalene overproduction (Mantzouridou F, 2010 ). Thus, the toxicity of squalene on yeast growth and how much is lethal is still debatable.…”

Section: Squalene Overproduction By Engineering the Squalene Biosynthmentioning

confidence: 99%

Engineering of the terpenoid pathway in Saccharomyces cerevisiae co-overproduces squalene and the non-terpenoid compound oleic acid

Rasool

Zhang

et al. 2016

Chemical Engineering Science

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Observations on squalene accumulation in Saccharomyces cerevisiae due to the manipulation of HMG2 and ERG6

Cited by 55 publications

References 37 publications

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Engineering of the terpenoid pathway in Saccharomyces cerevisiae co-overproduces squalene and the non-terpenoid compound oleic acid

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