Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae

E.J. Jongedijk, K. Cankar, J. Ranzijn, A.R. van der Krol, H.J. Bouwmeester, M.J. Beekwilder

Research output: Contribution to journalArticleAcademicpeer-review

28 Citations (Scopus)

Abstract

Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
LanguageEnglish
Pages159-171
JournalYeast
Volume32
Issue number1
DOIs
Publication statusPublished - 2015

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Monoterpenes
Alkenes
Yeast
Olefins
Saccharomyces cerevisiae
Perilla
Enzymes
Citrus
Terpenes
Fragrances
Recovery
Sorting
Fermentation
Culture Media
Genes
Perilla frutescens
Flavoring Agents
Yeasts
Proteins
Molecules

Keywords

  • monoterpene biosynthesis
  • escherichia-coli
  • synthase
  • precursor

Cite this

Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; van der Krol, A.R. ; Bouwmeester, H.J. ; Beekwilder, M.J. / Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae. In: Yeast. 2015 ; Vol. 32, No. 1. pp. 159-171.
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Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae. / Jongedijk, E.J.; Cankar, K.; Ranzijn, J.; van der Krol, A.R.; Bouwmeester, H.J.; Beekwilder, M.J.

In: Yeast, Vol. 32, No. 1, 2015, p. 159-171.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae

AU - Jongedijk, E.J.

AU - Cankar, K.

AU - Ranzijn, J.

AU - van der Krol, A.R.

AU - Bouwmeester, H.J.

AU - Beekwilder, M.J.

PY - 2015

Y1 - 2015

N2 - Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).

AB - Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).

KW - monoterpene biosynthesis

KW - escherichia-coli

KW - synthase

KW - precursor

U2 - 10.1002/yea.3038

DO - 10.1002/yea.3038

M3 - Article

VL - 32

SP - 159

EP - 171

JO - Yeast

T2 - Yeast

JF - Yeast

SN - 0749-503X

IS - 1

ER -