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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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Schematic representation of the engineered naringenin production pathway inS. cerevisiae. Six A. thaliana genes were overexpressed: PAL1 (phenylalanine ammonia lyase), C4H (Cinnamate 4-hydroxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase) and CHI1 (chalcone isomerase), and one gene from Rhodobacter capsulatus; Tal1 (tyrosine ammonia lyase). Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cerevisiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A. thaliana[22]. Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdc1, 5, 6; pyruvate decarboxylases, Aro10; phenylpyruvate decarboxylase.
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Figure 1: Schematic representation of the engineered naringenin production pathway inS. cerevisiae. Six A. thaliana genes were overexpressed: PAL1 (phenylalanine ammonia lyase), C4H (Cinnamate 4-hydroxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase) and CHI1 (chalcone isomerase), and one gene from Rhodobacter capsulatus; Tal1 (tyrosine ammonia lyase). Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cerevisiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A. thaliana[22]. Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdc1, 5, 6; pyruvate decarboxylases, Aro10; phenylpyruvate decarboxylase.

Mentions: In response to the poor production efficiency from plants and chemical synthesis, research groups have directed their attention to the heterologous production of flavonoids in microorganisms such as Escherichia coli and Saccharomyces cerevisiae, using metabolic engineering and synthetic biology[14,17-20]. In plants, formation of the central metabolite naringenin involves six steps catalyzed by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and its associated cytochrome P450 reductase (CPR), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) (Figure1)[21,22]. Alternatively, a tyrosine ammonia lyase (TAL) can convert tyrosine directly to coumaric acid and circumvent the use of membrane bound P450 related enzymes, which may pose challenges in E. coli[14,18]. Additionally, in plants, these enzymes and their corresponding genes are often represented by several isoforms, which may differ in substrate preference or kinetic properties[23]. Moreover, the different isoforms are proposed to be organized in one or more enzyme complexes that may promote substrate channeling. In Arabidopsis thaliana, interaction between CHS, CHI and an enzyme downstream of naringenin, flavonol 3-hydroxylase were demonstrated based on affinity chromatography and immunoprecipitation assays[24-26].


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)

Schematic representation of the engineered naringenin production pathway inS. cerevisiae. Six A. thaliana genes were overexpressed: PAL1 (phenylalanine ammonia lyase), C4H (Cinnamate 4-hydroxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase) and CHI1 (chalcone isomerase), and one gene from Rhodobacter capsulatus; Tal1 (tyrosine ammonia lyase). Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cerevisiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A. thaliana[22]. Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdc1, 5, 6; pyruvate decarboxylases, Aro10; phenylpyruvate decarboxylase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of the engineered naringenin production pathway inS. cerevisiae. Six A. thaliana genes were overexpressed: PAL1 (phenylalanine ammonia lyase), C4H (Cinnamate 4-hydroxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase) and CHI1 (chalcone isomerase), and one gene from Rhodobacter capsulatus; Tal1 (tyrosine ammonia lyase). Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cerevisiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A. thaliana[22]. Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdc1, 5, 6; pyruvate decarboxylases, Aro10; phenylpyruvate decarboxylase.
Mentions: In response to the poor production efficiency from plants and chemical synthesis, research groups have directed their attention to the heterologous production of flavonoids in microorganisms such as Escherichia coli and Saccharomyces cerevisiae, using metabolic engineering and synthetic biology[14,17-20]. In plants, formation of the central metabolite naringenin involves six steps catalyzed by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and its associated cytochrome P450 reductase (CPR), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) (Figure1)[21,22]. Alternatively, a tyrosine ammonia lyase (TAL) can convert tyrosine directly to coumaric acid and circumvent the use of membrane bound P450 related enzymes, which may pose challenges in E. coli[14,18]. Additionally, in plants, these enzymes and their corresponding genes are often represented by several isoforms, which may differ in substrate preference or kinetic properties[23]. Moreover, the different isoforms are proposed to be organized in one or more enzyme complexes that may promote substrate channeling. In Arabidopsis thaliana, interaction between CHS, CHI and an enzyme downstream of naringenin, flavonol 3-hydroxylase were demonstrated based on affinity chromatography and immunoprecipitation assays[24-26].

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