Assay of chalcone synthase activity by high-performance liquid chromatography

Zuurbier, Karin W.M.; Fung, Suen‐Ying; Scheffer, J. J. C.; Verpoorte, Robert

doi:10.1016/0031-9422(91)80005-l

Cited by 19 publications

(8 citation statements)

References 19 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

et al. 2012

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BackgroundFlavonoids 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.ResultSaccharomyces 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.ConclusionThe 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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Section: Resultsmentioning

confidence: 99%

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

et al. 2012

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“…Chalcone synthase activity was measured according to Zuurbier (1993). Hairy roots were ground in the presence of liquid nitrogen, sand and 10% (w/w) PVPP.…”

Section: Chalcone Synthase Activity Assaymentioning

confidence: 99%

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Lozovaya

Lygin

Zernova

et al. 2006

Planta

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Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen.

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“…Indeed, when the reaction products of HmC, P. lobata CHS and A. hypogaea STS were extracted without acidification, the detected amount of bis-noryangonin was reduced drastically, CTAL could not be detected at all (data not shown), and the chromatograms looked as though naringenin or resveratrol was produced as the sole product. Only one research group, Zuurbier et al [17], was aware of the presence of a hydrophilic product in the CHS reaction mixture, which could only be detected when the reaction mixture was acidified before extraction. Although its structure has not been reported, the above circumstantial evidence suggests that this hydrophilic product is CTAL.…”

Section: P-coumaroyltriacetic Acid Synthasementioning

confidence: 99%

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

Akiyama

Shibuya

Liu

et al. 1999

European Journal of Biochemistry

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Chalcone synthase and stilbene synthase are plant-specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.

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Assay of chalcone synthase activity by high-performance liquid chromatography

Cited by 19 publications

References 19 publications

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

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