Squalene is lipotoxic to yeast cells defective in lipid droplet biogenesis

Valachovič, Martin; Garaiová, Martina; Holic̆, Roman; Hapala, Ivan

doi:10.1016/j.bbrc.2015.12.050

Cited by 46 publications

(40 citation statements)

References 35 publications

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“…However, the genetically engineered squalene content in transgenic T. brevicorniculatum roots by simultaneous overexpression of ACL, AACT and HMGR is within an industrially relevant range. Interestingly, the large amount of squalene present in the lipid droplets in the T. brevicorniculatum root does not appear to be toxic, which is consistent with experiments in S. cerevisiae showing that squalene accumulation is toxic only when lipid droplet formation is impaired [49]. The similar levels of 2,3-oxidosqualene in wild-type and transgenic plants despite the substantial differences in squalene levels indicate that the formation of 2,3-oxidosqualene catalyzed by squalene epoxidase might act as the main bottleneck between the MVA pathway and downstream isoprenoid biosynthesis.…”

Section: Resultssupporting

confidence: 81%

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

et al. 2017

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BackgroundLatex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex.ResultsThe spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections.ConclusionsWe could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1036-0) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 81%

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

et al. 2017

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“…We transformed this strain with the expression plasmid containing Bme TC‐D373C and tested for triterpenoid production. However, we observed a clear decrease in growth in SD‐Ura medium by about 50% whereas addition of terbinafine completely diminished growth, the latter observation being consistent with the findings of Valachovic, Garaiova, Holic, and Hapala (), although rich medium was used in that study. In our analyses, only trace amounts of 8α‐hydroxypolypoda‐13,17,21‐triene and neither 3‐deoxyachilleol nor (+)‐ambrein could be detected (data not shown).…”

Section: Resultssupporting

confidence: 93%

“…whereas addition of terbinafine completely diminished growth, the latter observation being consistent with the findings of Valachovic, Garaiova, Holic, and Hapala (2016), although rich medium was used in that study. In our analyses, only trace amounts of 8αhydroxypolypoda-13,17,21-triene and neither 3-deoxyachilleol nor (+)-ambrein could be detected (data not shown).…”

Section: Product Analysis Of Strain Erg9-thmg Expressing Bmetc-d373csupporting

confidence: 89%

Engineering of Saccharomyces cerevisiae for the production of (+)‐ambrein

Moser

Leitner

Plocek³

et al. 2019

Yeast

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The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the Nterminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.

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“…These neutral lipids are likely to partition into the droplet core, although this has only been shown thus far in a few cases [42, 46]. For the hydrocarbon precursor of cholesterol, squalene, it has been shown in yeast that its sequestration in LDs is essential for preventing toxicity [47]. Natural rubber ( cis -1,4-polyisoprene) is a commercially important hydrocarbon produced by laticifer cells of the rubber tree Hevea brasiliensis , but also made in varying amounts by thousands of other plant species.…”

Section: 11 What's Inside a Lipid Droplet?mentioning

confidence: 99%

Lipid droplet functions beyond energy storage

Welte

Gould

2017

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

458

358

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Lipid droplets are cytoplasmic organelles that store neutral lipids and are critically important for energy metabolism. Their function in energy storage is firmly established and increasingly well characterized. However, emerging evidence indicates that lipid droplets also play important and diverse roles in the cellular handling of lipids and proteins that may not be directly related to energy homeostasis. Lipid handling roles of droplets include the storage of hydrophobic vitamin and signaling precursors, and the management of endoplasmic reticulum and oxidative stress. Roles of lipid droplets in protein handling encompass functions in the maturation, storage, and turnover of cellular and viral polypeptides. Other potential roles of lipid droplets may be connected with their intracellular motility and, in some cases, their nuclear localization. This diversity highlights that lipid droplets are very adaptable organelles, performing different functions in different biological contexts.

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Squalene is lipotoxic to yeast cells defective in lipid droplet biogenesis

Cited by 46 publications

References 35 publications

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

Engineering of Saccharomyces cerevisiae for the production of (+)‐ambrein

Lipid droplet functions beyond energy storage

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