Engineered Reversal of Function in Glycolytic Yeast Promoters

Arun S. Rajkumar, Emre Özdemir, Alicia V. Lis, Konstantin Schneider, Jiufu Qin, Michael K. Jensen*, Jay D. Keasling

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

Original languageEnglish
JournalACS Synthetic Biology
Volume8
Issue number6
Pages (from-to)1462-1468
Number of pages7
ISSN2161-5063
DOIs
Publication statusPublished - 2019

Keywords

  • Gluconeogenesis
  • Glycolysis
  • Synthetic promoter
  • Transcriptome
  • Vanillin

Cite this

Rajkumar, Arun S. ; Özdemir, Emre ; Lis, Alicia V. ; Schneider, Konstantin ; Qin, Jiufu ; Jensen, Michael K. ; Keasling, Jay D. / Engineered Reversal of Function in Glycolytic Yeast Promoters. In: ACS Synthetic Biology. 2019 ; Vol. 8, No. 6. pp. 1462-1468.
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abstract = "Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.",
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Engineered Reversal of Function in Glycolytic Yeast Promoters. / Rajkumar, Arun S.; Özdemir, Emre; Lis, Alicia V.; Schneider, Konstantin; Qin, Jiufu; Jensen, Michael K.; Keasling, Jay D.

In: ACS Synthetic Biology, Vol. 8, No. 6, 2019, p. 1462-1468.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Engineered Reversal of Function in Glycolytic Yeast Promoters

AU - Rajkumar, Arun S.

AU - Özdemir, Emre

AU - Lis, Alicia V.

AU - Schneider, Konstantin

AU - Qin, Jiufu

AU - Jensen, Michael K.

AU - Keasling, Jay D.

PY - 2019

Y1 - 2019

N2 - Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

AB - Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

KW - Gluconeogenesis

KW - Glycolysis

KW - Synthetic promoter

KW - Transcriptome

KW - Vanillin

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