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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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Naringenin production inS. cerevisiae.A) Increase in heterologous production of naringenin. B) Phenylethanol production in engineered strains. (○) IMU011 (atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (●) IMX183 (aro3Δ ARO4G226SatPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (□) IMX185 (aro3Δ ARO4G226Spdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (■) IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (Δ) non-naringenin producing reference strain CEN.PK2-1C. Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose and appropriate growth factors to supplement the auxotrophic requirements of the strains. All cultures were performed in triplicate. Error bars denote standard deviation.
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Figure 3: Naringenin production inS. cerevisiae.A) Increase in heterologous production of naringenin. B) Phenylethanol production in engineered strains. (○) IMU011 (atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (●) IMX183 (aro3Δ ARO4G226SatPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (□) IMX185 (aro3Δ ARO4G226Spdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (■) IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (Δ) non-naringenin producing reference strain CEN.PK2-1C. Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose and appropriate growth factors to supplement the auxotrophic requirements of the strains. All cultures were performed in triplicate. Error bars denote standard deviation.

Mentions: To enable naringenin production in S. cerevisiae, one episomal and one integrative expression vector were constructed which together, harbor the five flavonoid biosynthetic genes. Additionally, activation of the cytochrome P450 C4H requires a cytochrome P450 reductase (CPR). To choose the best candidate gene, the two A. thaliana CPR variants (CPR1 or CPR2) were separately included in the pathway engineering strategy. First, the centromeric episomal plasmid pUDE172, carrying PAL1 and yeast codon-optimized versions of the A. thaliana C4H (coC4H) and CPR1 (coCPR1) genes was constructed. In a same manner, separate construct, PAL1 and coC4H were combined with the coCPR2 gene. Subsequently, the integration plasmid pUDI065 was constructed, which carried the non yeast optimized A. thaliana genes at4CL3, atCHS1 and atCHI1. Only the, C4H and CPR1 in the naringenin production pathway were codon optimized since these genes hold the majority of rare codons for yeast (8), compared to the other genes (PAL1, 4CL3, CHS3 and CHI1) (3). When coCPR2 was expressed instead of coCPR1, significantly lower naringenin titers were observed (data not shown); coCPR2 was therefore not used in further experiments. Introduction of the coCPR1 version of the centromeric expression vector and the integration vector yielded the S. cerevisiae strain IMU011. In shake-flask cultures containing synthetic medium and with glucose as the sole carbon source, this strain produced naringenin to a concentration of 5.4 μM (Figure3A). Interestingly, naringenin was measured extracellularly, although its export mechanism has not yet been elucidated.


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)

Naringenin production inS. cerevisiae.A) Increase in heterologous production of naringenin. B) Phenylethanol production in engineered strains. (○) IMU011 (atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (●) IMX183 (aro3Δ ARO4G226SatPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (□) IMX185 (aro3Δ ARO4G226Spdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (■) IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (Δ) non-naringenin producing reference strain CEN.PK2-1C. Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose and appropriate growth factors to supplement the auxotrophic requirements of the strains. 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
getmorefigures.php?uid=PMC3539886&req=5

Figure 3: Naringenin production inS. cerevisiae.A) Increase in heterologous production of naringenin. B) Phenylethanol production in engineered strains. (○) IMU011 (atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (●) IMX183 (aro3Δ ARO4G226SatPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (□) IMX185 (aro3Δ ARO4G226Spdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (■) IMX106 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,at4CL3↑), (Δ) non-naringenin producing reference strain CEN.PK2-1C. Cultures were grown in shake flasks on synthetic medium containing 20 g·l-1 glucose and appropriate growth factors to supplement the auxotrophic requirements of the strains. All cultures were performed in triplicate. Error bars denote standard deviation.
Mentions: To enable naringenin production in S. cerevisiae, one episomal and one integrative expression vector were constructed which together, harbor the five flavonoid biosynthetic genes. Additionally, activation of the cytochrome P450 C4H requires a cytochrome P450 reductase (CPR). To choose the best candidate gene, the two A. thaliana CPR variants (CPR1 or CPR2) were separately included in the pathway engineering strategy. First, the centromeric episomal plasmid pUDE172, carrying PAL1 and yeast codon-optimized versions of the A. thaliana C4H (coC4H) and CPR1 (coCPR1) genes was constructed. In a same manner, separate construct, PAL1 and coC4H were combined with the coCPR2 gene. Subsequently, the integration plasmid pUDI065 was constructed, which carried the non yeast optimized A. thaliana genes at4CL3, atCHS1 and atCHI1. Only the, C4H and CPR1 in the naringenin production pathway were codon optimized since these genes hold the majority of rare codons for yeast (8), compared to the other genes (PAL1, 4CL3, CHS3 and CHI1) (3). When coCPR2 was expressed instead of coCPR1, significantly lower naringenin titers were observed (data not shown); coCPR2 was therefore not used in further experiments. Introduction of the coCPR1 version of the centromeric expression vector and the integration vector yielded the S. cerevisiae strain IMU011. In shake-flask cultures containing synthetic medium and with glucose as the sole carbon source, this strain produced naringenin to a concentration of 5.4 μM (Figure3A). Interestingly, naringenin was measured extracellularly, although its export mechanism has not yet been elucidated.

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