CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes

Gábor Nagy, Amanda Grace Vaz, Csilla Szebenyi, Miklós Takó, Eszter J. Tóth, Árpád Csernetics, Ottó Bencsik, A. Szekeres, Mónika Homa, F. Ayaydin, László Galgóczy, C. Vágvölgyi, T. Papp

Research output: Article

1 Citation (Scopus)

Abstract

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the β-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

Original languageEnglish
Pages (from-to)30-39
Number of pages10
JournalFungal Genetics and Biology
Volume129
DOIs
Publication statusPublished - aug. 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Hydroxymethylglutaryl CoA Reductases
Mucor
Terpenes
Ergosterol
Mevalonic Acid
Carotenoids
Enzymes
Genes
Biological Phenomena
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Sterols
Coenzyme A
Germination
Spores
Fluorescence Microscopy
Confocal Microscopy
Endoplasmic Reticulum
Hydrogen Peroxide
Proteins

ASJC Scopus subject areas

  • Microbiology
  • Genetics

Cite this

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes. / Nagy, Gábor; Vaz, Amanda Grace; Szebenyi, Csilla; Takó, Miklós; Tóth, Eszter J.; Csernetics, Árpád; Bencsik, Ottó; Szekeres, A.; Homa, Mónika; Ayaydin, F.; Galgóczy, László; Vágvölgyi, C.; Papp, T.

In: Fungal Genetics and Biology, Vol. 129, 01.08.2019, p. 30-39.

Research output: Article

Nagy, Gábor ; Vaz, Amanda Grace ; Szebenyi, Csilla ; Takó, Miklós ; Tóth, Eszter J. ; Csernetics, Árpád ; Bencsik, Ottó ; Szekeres, A. ; Homa, Mónika ; Ayaydin, F. ; Galgóczy, László ; Vágvölgyi, C. ; Papp, T. / CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes. In: Fungal Genetics and Biology. 2019 ; Vol. 129. pp. 30-39.
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AU - Takó, Miklós

AU - Tóth, Eszter J.

AU - Csernetics, Árpád

AU - Bencsik, Ottó

AU - Szekeres, A.

AU - Homa, Mónika

AU - Ayaydin, F.

AU - Galgóczy, László

AU - Vágvölgyi, C.

AU - Papp, T.

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AB - Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the β-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

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