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De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae.

Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall RD, Bosch D, van Maris AJ, Pronk JT, Daran JM - Microb. Cell Fact. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.

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.

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Stepwise increase in naringenin formation byS cerevisiae. Formation of A) naringenin and B) phloretic acid in the engineered strains IMX106 (○)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), IMX197 (●)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑, at4CL3↑, cotal1↑) and IMX198 (□) (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in duplicate. Error bar denotes deviation of the mean.
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Figure 5: Stepwise increase in naringenin formation byS cerevisiae. Formation of A) naringenin and B) phloretic acid in the engineered strains IMX106 (○)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), IMX197 (●)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑, at4CL3↑, cotal1↑) and IMX198 (□) (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in duplicate. Error bar denotes deviation of the mean.

Mentions: Naringenin chalcone synthase, which catalyzes the formation of chalcone by condensing coumaroyl-CoA with three molecules of malonyl-CoA, is known to be an enzyme with low catalytic activity[46]. The transient accumulation and later reconsumption of coumaric acid in shake flask cultures suggested that reactions downstream of coumaric acid were limiting naringenin production. To test whether the capacity of chalcone synthase was indeed controlling flux through the pathway, two additional copies of the coCHS3 gene were introduced into strain IMX106 on an episomal plasmid (pUDE188), yielding strain IMX197. The two additional copies resulted in a 2.5 fold increase in naringenin accumulation (134.5 μM) in shake flask cultures (Figure5A), indicating that coCHS3 was indeed a limiting step in the naringenin production. Additional coCHS3 copies also caused a decreased production of phloretic acid (Figure5B), consistent with the hypothesis that phloretic acid production occurs when coumaroyl-CoA cannot be efficiently converted to naringenin.


De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae.

Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall RD, Bosch D, van Maris AJ, Pronk JT, Daran JM - Microb. Cell Fact. (2012)

Stepwise increase in naringenin formation byS cerevisiae. Formation of A) naringenin and B) phloretic acid in the engineered strains IMX106 (○)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), IMX197 (●)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑, at4CL3↑, cotal1↑) and IMX198 (□) (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in duplicate. Error bar denotes deviation of the mean.
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Related In: Results  -  Collection

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Figure 5: Stepwise increase in naringenin formation byS cerevisiae. Formation of A) naringenin and B) phloretic acid in the engineered strains IMX106 (○)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), IMX197 (●)(aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑, at4CL3↑, cotal1↑) and IMX198 (□) (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in duplicate. Error bar denotes deviation of the mean.
Mentions: Naringenin chalcone synthase, which catalyzes the formation of chalcone by condensing coumaroyl-CoA with three molecules of malonyl-CoA, is known to be an enzyme with low catalytic activity[46]. The transient accumulation and later reconsumption of coumaric acid in shake flask cultures suggested that reactions downstream of coumaric acid were limiting naringenin production. To test whether the capacity of chalcone synthase was indeed controlling flux through the pathway, two additional copies of the coCHS3 gene were introduced into strain IMX106 on an episomal plasmid (pUDE188), yielding strain IMX197. The two additional copies resulted in a 2.5 fold increase in naringenin accumulation (134.5 μM) in shake flask cultures (Figure5A), indicating that coCHS3 was indeed a limiting step in the naringenin production. Additional coCHS3 copies also caused a decreased production of phloretic acid (Figure5B), consistent with the hypothesis that phloretic acid production occurs when coumaroyl-CoA cannot be efficiently converted to naringenin.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus