Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production

J. Beekwilder, H.M. Rossum, F. Koopman, F. Sonntag, M. Buchhaupt, J. Schrader, R.D. Hall, H.J. Bosch, J.T. Pronk, A.J.A. van Maris, J.M. Daran

Research output: Contribution to journalArticleAcademicpeer-review

45 Citations (Scopus)

Abstract

The flavour and fragrance compound ß-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ß-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ß-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ß-carotene. To enable ß-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ß-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ß-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.
LanguageEnglish
Pages383-392
JournalJournal of Biotechnology
Volume192
Issue numberpartB
DOIs
Publication statusPublished - 2014

Fingerprint

Norisoprenoids
Biosynthetic Pathways
Carotenoids
Yeast
Saccharomyces cerevisiae
Proteins
Dioxygenases
Fruits
Glucose
Fruit
Flavor compounds
Synthetic Biology
Fragrances
Ascomycota
Flavors
Biosynthesis
Viruses
Genes
Western Blotting

Keywords

  • cleavage dioxygenase
  • yeast
  • genes
  • sequences
  • transformation
  • translation
  • polyprotein
  • versatile
  • genome
  • strain

Cite this

Beekwilder, J. ; Rossum, H.M. ; Koopman, F. ; Sonntag, F. ; Buchhaupt, M. ; Schrader, J. ; Hall, R.D. ; Bosch, H.J. ; Pronk, J.T. ; van Maris, A.J.A. ; Daran, J.M. / Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production. In: Journal of Biotechnology. 2014 ; Vol. 192, No. partB. pp. 383-392.
@article{e5933f84230d44d7ac4c14f07efeb281,
title = "Polycistronic expression of a {\ss}-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to {\ss}-ionone production",
abstract = "The flavour and fragrance compound {\ss}-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for {\ss}-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three {\ss}-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of {\ss}-carotene. To enable {\ss}-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the {\ss}-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, {\ss}-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.",
keywords = "cleavage dioxygenase, yeast, genes, sequences, transformation, translation, polyprotein, versatile, genome, strain",
author = "J. Beekwilder and H.M. Rossum and F. Koopman and F. Sonntag and M. Buchhaupt and J. Schrader and R.D. Hall and H.J. Bosch and J.T. Pronk and {van Maris}, A.J.A. and J.M. Daran",
year = "2014",
doi = "10.1016/j.jbiotec.2013.12.016",
language = "English",
volume = "192",
pages = "383--392",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier",
number = "partB",

}

Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production. / Beekwilder, J.; Rossum, H.M.; Koopman, F.; Sonntag, F.; Buchhaupt, M.; Schrader, J.; Hall, R.D.; Bosch, H.J.; Pronk, J.T.; van Maris, A.J.A.; Daran, J.M.

In: Journal of Biotechnology, Vol. 192, No. partB, 2014, p. 383-392.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production

AU - Beekwilder, J.

AU - Rossum, H.M.

AU - Koopman, F.

AU - Sonntag, F.

AU - Buchhaupt, M.

AU - Schrader, J.

AU - Hall, R.D.

AU - Bosch, H.J.

AU - Pronk, J.T.

AU - van Maris, A.J.A.

AU - Daran, J.M.

PY - 2014

Y1 - 2014

N2 - The flavour and fragrance compound ß-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ß-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ß-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ß-carotene. To enable ß-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ß-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ß-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.

AB - The flavour and fragrance compound ß-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ß-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ß-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ß-carotene. To enable ß-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ß-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ß-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.

KW - cleavage dioxygenase

KW - yeast

KW - genes

KW - sequences

KW - transformation

KW - translation

KW - polyprotein

KW - versatile

KW - genome

KW - strain

U2 - 10.1016/j.jbiotec.2013.12.016

DO - 10.1016/j.jbiotec.2013.12.016

M3 - Article

VL - 192

SP - 383

EP - 392

JO - Journal of Biotechnology

T2 - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - partB

ER -