Engineering de novo anthocyanin production in Saccharomyces cerevisiae

Mark Levisson, Constantinos Patinios, Sascha Hein, Philip A. de Groot, Jean M. Daran, Robert D. Hall, Stefan Martens, Jules Beekwilder

Research output: Contribution to journalArticleAcademicpeer-review

9 Citations (Scopus)

Abstract

Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 μmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 μM [5 mg/L] and 150 μM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

LanguageEnglish
Article number103
JournalMicrobial Cell Factories
Volume17
Issue number1
DOIs
Publication statusPublished - 3 Jul 2018

Fingerprint

Anthocyanins
Yeast
Saccharomyces cerevisiae
Glucosides
Yeasts
Genes
Food Coloring Agents
Health
Plant extracts
Glucosidases
Flavonoids
Chassis
Plant Extracts
Batch reactors
Polyphenols
Fruits
pelargonidin
Arabidopsis
Pigments
Fermentation

Keywords

  • Anthocyanin
  • Flavonoids
  • Metabolic engineering
  • Natural products
  • Pelargonidin
  • Plant secondary metabolites
  • Saccharomyces cerevisiae

Cite this

@article{97a061528d73445c8f082030a621877f,
title = "Engineering de novo anthocyanin production in Saccharomyces cerevisiae",
abstract = "Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 μmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 μM [5 mg/L] and 150 μM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.",
keywords = "Anthocyanin, Flavonoids, Metabolic engineering, Natural products, Pelargonidin, Plant secondary metabolites, Saccharomyces cerevisiae",
author = "Mark Levisson and Constantinos Patinios and Sascha Hein and {de Groot}, {Philip A.} and Daran, {Jean M.} and Hall, {Robert D.} and Stefan Martens and Jules Beekwilder",
year = "2018",
month = "7",
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language = "English",
volume = "17",
journal = "Microbial Cell Factories",
issn = "1475-2859",
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Engineering de novo anthocyanin production in Saccharomyces cerevisiae. / Levisson, Mark; Patinios, Constantinos; Hein, Sascha; de Groot, Philip A.; Daran, Jean M.; Hall, Robert D.; Martens, Stefan; Beekwilder, Jules.

In: Microbial Cell Factories, Vol. 17, No. 1, 103, 03.07.2018.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Engineering de novo anthocyanin production in Saccharomyces cerevisiae

AU - Levisson, Mark

AU - Patinios, Constantinos

AU - Hein, Sascha

AU - de Groot, Philip A.

AU - Daran, Jean M.

AU - Hall, Robert D.

AU - Martens, Stefan

AU - Beekwilder, Jules

PY - 2018/7/3

Y1 - 2018/7/3

N2 - Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 μmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 μM [5 mg/L] and 150 μM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

AB - Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 μmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 μM [5 mg/L] and 150 μM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

KW - Anthocyanin

KW - Flavonoids

KW - Metabolic engineering

KW - Natural products

KW - Pelargonidin

KW - Plant secondary metabolites

KW - Saccharomyces cerevisiae

U2 - 10.1186/s12934-018-0951-6

DO - 10.1186/s12934-018-0951-6

M3 - Article

VL - 17

JO - Microbial Cell Factories

T2 - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

IS - 1

M1 - 103

ER -