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De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Frank Koopman123, Jules Beekwilder245, Barbara Crimi123, Adele van Houwelingen4, Robert D Hall245, Dirk Bosch245, Antonius JA van Maris13, Jack T Pronk123 and Jean-Marc Daran123*

Author Affiliations

1 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, the Netherlands

2 Platform for Green Synthetic Biology, P.O. Box 5057, 2600 GA, Delft, The Netherlands

3 Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA, Delft, The Netherlands

4 Plant Research International (PRI), P.O. Box 16, 6700 AA, Wageningen, The Netherlands

5 Centre for Biosystems Genomics, PO Box 98, 6700 AB, Wageningen, The Netherlands

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Microbial Cell Factories 2012, 11:155  doi:10.1186/1475-2859-11-155

Published: 8 December 2012

Abstract

Background

Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules.

Result

Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 μM). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached.

Conclusion

The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.

Keywords:
Saccharomyces cerevisiae; Naringenin; de novo; Flavonoids; Metabolic engineering