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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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Product formation by S. cerevisiae IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑). Formation of naringenin (○),coumaric acid (●) and phloretic acid (□). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in triplicate. Error bars denote standard deviation.
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Figure 4: Product formation by S. cerevisiae IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑). Formation of naringenin (○),coumaric acid (●) and phloretic acid (□). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in triplicate. Error bars denote standard deviation.

Mentions: In S. cerevisiae, phenylethanol is produced via the Ehrlich pathway. Decarboxylation of phenylpyruvate, the 2-oxo acid associated to phenylalanine, yields phenylacetaldehyde, which is subsequently reduced into phenylethanol and/or oxidized to phenylacetate[41-43]. Decarboxylation of phenylpyruvate can be catalyzed by four different thiamine pyrophosphate-dependent 2-oxo acid decarboxylases encoded by ARO10, PDC1, PDC5, and PDC6[43,44]. Recent work in our group has demonstrated that, among the four decarboxylases capable of phenylpyruvate decarboxylase, Pdc1 and Pdc6 showed a much lower affinity and decarboxylation rate of phenylpyruvate than Pdc5 and, in particular, Aro10[44]. Since absence of all three pyruvate decarboxylase genes (PDC1, PDC5 and PDC6) abolishes growth on glucose in synthetic media, strains only retaining PDC1 (aro10Δ, pcd5Δ, pdc6Δ) were constructed[45]. The intermediate strain IMX185 (ARO4G226S, aro3Δ, pdc5Δ, pdc6Δ) did not show a reduced phenylethanol titer compared to its ancestor IMX183 (ARO4G226S, aro3Δ) (Figure3B). In contrast, the naringenin producing strain IMX106 (ARO4G226S, aro3Δ, pdc5Δ, pdc6Δ, aro10Δ) that also carries the ARO10 deletion exhibited a 22-fold lower concentration of extracellular phenylethanol (44 μM) compared to both ancestor strains IMX183 and IMX185 (955 μM). This strong reduction in phenylethanol production coincided with a 3-fold increase of extracellular naringenin (up to 46.5 μM) (Figure3A), indicating that reduction of the flux through the Ehrlich pathway had indeed led to substantial improvement in naringenin production. Analysis of culture supernatants revealed that strain IMX106 also produced coumaric acid up to a concentration of 200 μM (Figure4). Moreover, this strain showed increased production of another aromatic metabolite that, based on HPLC spectra and LC-MS analysis, was identified as phloretic acid (Figure4). Production of phloretic acid was only observed in cultures that also produced coumaric acid and which expressed 4CL3 (data not shown). This suggests that phloretic acid is most likely produced from coumaroyl-CoA (Figure4).


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)

Product formation by S. cerevisiae IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑). Formation of naringenin (○),coumaric acid (●) and phloretic acid (□). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in triplicate. Error bars denote standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Product formation by S. cerevisiae IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑). Formation of naringenin (○),coumaric acid (●) and phloretic acid (□). Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose. All cultures were performed in triplicate. Error bars denote standard deviation.
Mentions: In S. cerevisiae, phenylethanol is produced via the Ehrlich pathway. Decarboxylation of phenylpyruvate, the 2-oxo acid associated to phenylalanine, yields phenylacetaldehyde, which is subsequently reduced into phenylethanol and/or oxidized to phenylacetate[41-43]. Decarboxylation of phenylpyruvate can be catalyzed by four different thiamine pyrophosphate-dependent 2-oxo acid decarboxylases encoded by ARO10, PDC1, PDC5, and PDC6[43,44]. Recent work in our group has demonstrated that, among the four decarboxylases capable of phenylpyruvate decarboxylase, Pdc1 and Pdc6 showed a much lower affinity and decarboxylation rate of phenylpyruvate than Pdc5 and, in particular, Aro10[44]. Since absence of all three pyruvate decarboxylase genes (PDC1, PDC5 and PDC6) abolishes growth on glucose in synthetic media, strains only retaining PDC1 (aro10Δ, pcd5Δ, pdc6Δ) were constructed[45]. The intermediate strain IMX185 (ARO4G226S, aro3Δ, pdc5Δ, pdc6Δ) did not show a reduced phenylethanol titer compared to its ancestor IMX183 (ARO4G226S, aro3Δ) (Figure3B). In contrast, the naringenin producing strain IMX106 (ARO4G226S, aro3Δ, pdc5Δ, pdc6Δ, aro10Δ) that also carries the ARO10 deletion exhibited a 22-fold lower concentration of extracellular phenylethanol (44 μM) compared to both ancestor strains IMX183 and IMX185 (955 μM). This strong reduction in phenylethanol production coincided with a 3-fold increase of extracellular naringenin (up to 46.5 μM) (Figure3A), indicating that reduction of the flux through the Ehrlich pathway had indeed led to substantial improvement in naringenin production. Analysis of culture supernatants revealed that strain IMX106 also produced coumaric acid up to a concentration of 200 μM (Figure4). Moreover, this strain showed increased production of another aromatic metabolite that, based on HPLC spectra and LC-MS analysis, was identified as phloretic acid (Figure4). Production of phloretic acid was only observed in cultures that also produced coumaric acid and which expressed 4CL3 (data not shown). This suggests that phloretic acid is most likely produced from coumaroyl-CoA (Figure4).

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