CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae

Kanchana Rueksomtawin Kildegaard, Larissa Ribeiro Ramos Tramontin, Ksenia Chekina, Mingji Li, Tobias Justus Goedecke, Mette Kristensen, Irina Borodina*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The yeast Saccharomyces cerevisiaeis widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction offuture cell factories.
Original languageEnglish
JournalYeast
Volume36
Issue number5
Pages (from-to)237-247
ISSN0749-503X
DOIs
Publication statusPublished - 2019

Keywords

  • CRISPR/Cas9
  • RNA interference
  • Saccharomyces cerevisiae
  • cis,cis-muconic acid
  • genome editing
  • metabolic engineering

Cite this

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title = "CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae",
abstract = "The yeast Saccharomyces cerevisiaeis widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction offuture cell factories.",
keywords = "CRISPR/Cas9, RNA interference, Saccharomyces cerevisiae, cis,cis-muconic acid, genome editing, metabolic engineering",
author = "Kildegaard, {Kanchana Rueksomtawin} and {Ribeiro Ramos Tramontin}, Larissa and Ksenia Chekina and Mingji Li and Goedecke, {Tobias Justus} and Mette Kristensen and Irina Borodina",
year = "2019",
doi = "10.1002/yea.3390",
language = "English",
volume = "36",
pages = "237--247",
journal = "Yeast",
issn = "0749-503X",
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}

CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae. / Kildegaard, Kanchana Rueksomtawin; Ribeiro Ramos Tramontin, Larissa; Chekina, Ksenia; Li, Mingji; Goedecke, Tobias Justus; Kristensen, Mette; Borodina, Irina.

In: Yeast, Vol. 36, No. 5, 2019, p. 237-247.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae

AU - Kildegaard, Kanchana Rueksomtawin

AU - Ribeiro Ramos Tramontin, Larissa

AU - Chekina, Ksenia

AU - Li, Mingji

AU - Goedecke, Tobias Justus

AU - Kristensen, Mette

AU - Borodina, Irina

PY - 2019

Y1 - 2019

N2 - The yeast Saccharomyces cerevisiaeis widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction offuture cell factories.

AB - The yeast Saccharomyces cerevisiaeis widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction offuture cell factories.

KW - CRISPR/Cas9

KW - RNA interference

KW - Saccharomyces cerevisiae

KW - cis,cis-muconic acid

KW - genome editing

KW - metabolic engineering

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SN - 0749-503X

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