Dataset Information

Purification and characterization of a novel β-carotene-9',10'-oxygenase from Saccharomyces cerevisiae ULI3.

ABSTRACT:

Objectives

A novel β-carotene-9,10'-oxygenase (ScBCO2) has been characterized from Saccharomyces cerevisiae ULI3 to convert β-carotene to β-apo-10'-carotenal, which is a precursor of the plant hormone strigolactone.

Results

The ScBCO2 enzyme was purified to homogeneity by ammonium sulfate precipitation, Q sepharose and Superdex-200 chromatography. The molecular mass of the enzyme was ~50 kDa by SDS-PAGE. The purified ScBCO2 enzyme displayed optimal activity at 45 °C and pH 8. Tween 20 (1%, w/v), Trition X-100 (1%, w/v), Mg(2+) (5 mM), Zn(2+) (5 mM), Cu(2+) (5 mM), Ca(2+) (5 mM) or DTT (5 mM) increased in the activity by 3, 7, 14, 17, 23, 26 and 27%, respectively. ScBCO2 only exhibited cleavage activity towards carotenoid substrates containing two β-ionone rings and its catalytic efficiency (kcat/Km) followed the order β-carotene > α-carotene > lutein.

Conclusion

ScBCO2 could be used as a potential candidate for the enzymatic biotransformation of β-carotene to β-apo-10'-carotenal in biotechnological applications.

SUBMITTER: Wei T

PROVIDER: S-EPMC4565880 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Publications

Purification and characterization of a novel β-carotene-9',10'-oxygenase from Saccharomyces cerevisiae ULI3.

Wei Tao T Jia Beilei B Huang Shen S Yang Kunpeng K Jia Chunxiao C Mao Duobin D

Biotechnology letters 20150531 10

<h4>Objectives</h4>A novel β-carotene-9,10'-oxygenase (ScBCO2) has been characterized from Saccharomyces cerevisiae ULI3 to convert β-carotene to β-apo-10'-carotenal, which is a precursor of the plant hormone strigolactone.<h4>Results</h4>The ScBCO2 enzyme was purified to homogeneity by ammonium sulfate precipitation, Q sepharose and Superdex-200 chromatography. The molecular mass of the enzyme was ~50 kDa by SDS-PAGE. The purified ScBCO2 enzyme displayed optimal activity at 45 °C and pH 8. Twee ...[more]

PMID: 26026965

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Project description:β-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment β-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current β-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of β-carotene and its subsequent conversion to β-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on β-carotene accumulation, and later, on β-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For β-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest β-carotene-producing strains, reaching up to 33 mg/L of β-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing β-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Dataset Information

Purification and characterization of a novel β-carotene-9',10'-oxygenase from Saccharomyces cerevisiae ULI3.

Objectives

Results

Conclusion

Publications

Purification and characterization of a novel β-carotene-9',10'-oxygenase from Saccharomyces cerevisiae ULI3.

Similar Datasets

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