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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 of the naringenin-producing strainS. cerevisiaeIMX198 in bioreactors. Growth and extracellular metabolite formation were studied in aerobic and pH controlled (pH 5.0) batch cultures of IMX198 on glucose. The results shown are from a single representative experiment. A) Concentrations of glucose (○), ethanol (●), acetate (□), glycerol (■) and optical density (OD660) (Δ). B) Concentrations of naringenin (▲), coumaric acid (♦) and phloretic acid (◊).
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Figure 6: Product formation of the naringenin-producing strainS. cerevisiaeIMX198 in bioreactors. Growth and extracellular metabolite formation were studied in aerobic and pH controlled (pH 5.0) batch cultures of IMX198 on glucose. The results shown are from a single representative experiment. A) Concentrations of glucose (○), ethanol (●), acetate (□), glycerol (■) and optical density (OD660) (Δ). B) Concentrations of naringenin (▲), coumaric acid (♦) and phloretic acid (◊).

Mentions: To further characterize strain IMX198 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑) under controlled conditions, this strain was cultured in a 2L batch bioreactor with 20 g·l-1 glucose at pH 5.0 (Figure6A, B). When S. cerevisiae is grown aerobically in batch cultures on glucose, alcoholic fermentation is the predominant mode of glucose metabolism[49,50] and is characterized by a diauxic growth profile. During the glucose consumption phase the specific growth rate of IMX198 was 0.2 h-1, which is approximately 50% of the specific growth rate of the reference strain S. cerevisiae CEN.PK113-7D under the same conditions[51]. Besides the expected formation of ethanol, a specific naringenin production rate of 12.545 ± 0.333 μmol.g-1CDWh-1 was obtained. After complete consumption of glucose, a naringenin titer 148.06 ± 5.67 μM at a naringenin yield on glucose of 0.002 ± 0.000 (g·g-1) was obtained. When all the glucose was consumed, ethanol, acetate and glycerol that were produced during the first phase were subsequently consumed (Figure6A). During this reconsumption phase, naringenin titers increased to 414.63 ± 1.60 μM, indicating that most naringenin is produced during this second phase (Figure6B). However, it must be taken into account that during this reconsumption phase, naringenin production is also facilitated by the presence of available coumarate that was previously formed during the glucose consumption phase and also by the higher amount of biomass, compared to the glucose phase. When only calculating the product yield over the total glucose and ethanol consumption phase, a yield of 0.006 ± 0.000 (g·g-1) was obtained, which is approximately triple of what is obtained during solely the glucose consumption phase.


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 of the naringenin-producing strainS. cerevisiaeIMX198 in bioreactors. Growth and extracellular metabolite formation were studied in aerobic and pH controlled (pH 5.0) batch cultures of IMX198 on glucose. The results shown are from a single representative experiment. A) Concentrations of glucose (○), ethanol (●), acetate (□), glycerol (■) and optical density (OD660) (Δ). B) Concentrations of naringenin (▲), coumaric acid (♦) and phloretic acid (◊).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539886&req=5

Figure 6: Product formation of the naringenin-producing strainS. cerevisiaeIMX198 in bioreactors. Growth and extracellular metabolite formation were studied in aerobic and pH controlled (pH 5.0) batch cultures of IMX198 on glucose. The results shown are from a single representative experiment. A) Concentrations of glucose (○), ethanol (●), acetate (□), glycerol (■) and optical density (OD660) (Δ). B) Concentrations of naringenin (▲), coumaric acid (♦) and phloretic acid (◊).
Mentions: To further characterize strain IMX198 (aro3Δ ARO4G226Saro10Δ, pdc5Δ, pdc6Δ, atPAL1↑, coC4H↑, coCPR1↑, atCHI1↑, atCHS3↑,coCHS3↑, at4CL3↑, cotal1↑) under controlled conditions, this strain was cultured in a 2L batch bioreactor with 20 g·l-1 glucose at pH 5.0 (Figure6A, B). When S. cerevisiae is grown aerobically in batch cultures on glucose, alcoholic fermentation is the predominant mode of glucose metabolism[49,50] and is characterized by a diauxic growth profile. During the glucose consumption phase the specific growth rate of IMX198 was 0.2 h-1, which is approximately 50% of the specific growth rate of the reference strain S. cerevisiae CEN.PK113-7D under the same conditions[51]. Besides the expected formation of ethanol, a specific naringenin production rate of 12.545 ± 0.333 μmol.g-1CDWh-1 was obtained. After complete consumption of glucose, a naringenin titer 148.06 ± 5.67 μM at a naringenin yield on glucose of 0.002 ± 0.000 (g·g-1) was obtained. When all the glucose was consumed, ethanol, acetate and glycerol that were produced during the first phase were subsequently consumed (Figure6A). During this reconsumption phase, naringenin titers increased to 414.63 ± 1.60 μM, indicating that most naringenin is produced during this second phase (Figure6B). However, it must be taken into account that during this reconsumption phase, naringenin production is also facilitated by the presence of available coumarate that was previously formed during the glucose consumption phase and also by the higher amount of biomass, compared to the glucose phase. When only calculating the product yield over the total glucose and ethanol consumption phase, a yield of 0.006 ± 0.000 (g·g-1) was obtained, which is approximately triple of what is obtained during solely the glucose consumption phase.

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