Biosynthesis of Astaxanthin as a Main Carotenoid in the Heterobasidiomycetous Yeast Xanthophyllomyces dendrorhous
Abstract
:1. Introduction: Xanthophyllomyces dendrorhous and Carotenoids
2. The Biosynthetic Pathway of Astaxanthin in X. dendrorhous
3. Biotechnology-Based Improvement of Astaxanthin Production in X. dendrorhous
4. Omics of X. dendrorhous: Genomics, Transcriptomics, Proteomics, and Metabolomics
5. Conclusions and Future Prospects
Acknowledgments
Conflicts of Interest
References
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Targets | Approach | Result | Ref. |
---|---|---|---|
crtYB gene | Deactivation | No carotenoids | [48] |
crtYB gene | Overexpression | Accumulation of β-carotene and echinenone | [48] |
crtI gene | Overexpression | Increase torulene and HDCO and decrease echinenone, l-carotene and astaxanthin | [48] |
crtR gene | Description of its role | Required together with the crtS gene for the conversion of β-carotene to astaxanthin. | [59] |
double cyp61 genes | Deletion | Enhanced astaxanthin production by 1,4-fold compared with the parental strain | [77] |
crtE gene under Padh4r | Evaluation of promotors | Increase in intracellular astaxanthin by 1.7-fold compared with parental | [78] |
acaT, hmgS and hmgR genes | Triple overexpression | Enhanced volumetric astaxanthin production by 1.4-fold compared with that of the control strain | [23] |
acaT/hmgS/hmgR/crtE/crtS genes | Combined overexpression | Enhanced volumetric astaxanthin production by 2.1-fold higher compared with the control strain | [23] |
Combination of conventional mutagenesis and crtYB gene expression | Combined overexpression | 22 times higher astaxanthin specific production than for the wild type | [75] |
Media | Collection | Disruption | Analysis Method | Ref. |
---|---|---|---|---|
DNA | ||||
YPD medium. 21 °C, 5 days [96] | Culture: 15 mL Suspended: 0.5 mL YPD | Breaking system: 300 μL of glass beads (0.25–0.5 mm diameter). Swing mill (Retsch MM200) at a frequency of 30/s. | Agarose gel and ethidium bromide stain. Fluorescence densitometry measurement. | [19] |
Sample cleaning: Supernatant collected and purified by phenol/chloroform/isoamyl alcohol extraction. DNA precipitation overnight at −20 °C by 100% ice-cold ethanol (2.5 volume) and 1/10 volume of 3 M sodium acetate solution. 70% ice-cold ethanol. Dry at room temperature. DNA pellet resuspension in 30 μL H2O. Store at 4 °C. | ||||
YM medium (100 mL). 22 °C, up to stationary phase | Centrifugation | Breaking system: DNA isolated from protoplasts: 2× wash 50 mM EDTA pH 7.5. Novozyme 234 plus LET buffer (500 mM EDTA, 7.5% 2-mercaptoethanol, 10 mM Tris pH 7.5). 16 h, 37 °C. NDS solution. (2 mg/mL proteinase K in 500 mM EDTA, 1% lauryl sarcosine and 10 mM Tris-HCl, pH 7.5). 24 h, 50 °C. | DNA quantitation: 260/280 ratio (1.7–1.9) and 260/230 ratio (>2) by using a V-630 UV–vis Spectrophotometer. | [85,86] |
Sample cleaning: Phenolic extraction (pH 8.0): 3× wash saturated phenol. 3× phenol: chloroform: isoamyl alcohol (25:24:1). 1× chloroform: isoamyl alcohol (24:1). DNA was precipitated with 98% ethanol and washed with 70% ethanol. Dry DNA resuspended in Tris: EDTA (10:1; pH 8.0) plus 40 μg/mL of RNase A. 37 °C, 30 min. Repeat phenolic extraction | ||||
YM broth (15 mL) at 20 °C, 72 h | Centrifugation | Breaking system: Modified phenol:chloroform:isoamyl alcohol method [97,98]: Resuspend in 500 µL lysis buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA (pH 8.0), 2% Triton X-100, 1% SDS). Add an equal volume of phenol/chloroform (1∶1 v/v). Shake vigorously (Ika-Vibrax VXR shaker) at 1800 rpm, 20 minutes, R/T. Centrifuge at 14,000 rpm, 20 min, 4 °C. | N/A | [88] |
Sample cleaning: Ethanol precipitation. | ||||
RNA | ||||
YPD medium. 21 °C, 5 days [96] | NucleoSpin® RNA Plant kit (MACHEREY-NAGEL GmbH & Co. KG) (Following the manufacturer instructions). | RNA quality by using Nano-Photometer (IMPLEN) 1.5% agarose gel and ethidium bromide stain | [19] | |
Vogel minimal medium (MMv) supplemented with 2% glucose or 2% succinate | Early exponential phase (18 h) Initial stationary phase (72 h) | Breaking system: Mechanical rupture of cell pellets. 0.5 mm glass beads (BioSpec). Vortexing for 10 min. Add Tri-Reagent (Ambion). R/T 10 min. | RNA quantitation: 260/280 ratio (>1.9) by using a V-630 UV–vis Spectrophotometer | [86] |
Sample cleaning: Add 200 μL of chloroform per mL of Tri-Reagent. Mix. Centrifuge: 4000× g, 5 min. Recover supernatant. 2× acidic phenol: chloroform (1:1) extractions. Precipitate: 2 volume of isopropanol. 10 min, R/T. 1× wash 75% ethanol. Resuspend in RNase-free water. RNA samples at a 260/280 ratio >1.9, measured using a V-630 UV–vis Spectrophotometer, were used for next-generation sequencing. | ||||
Proteins | ||||
Minimal medium plus 2% glucose or succinate as carbon sources [99] Preculture: 10 mL Culture: 250 mL in 1-L flask inoculated with 2.5 mL of seed culture. 22 °C, 120 rpm | Centrifugation: 5000× g, 10 min, 4 °C. Pellet washed twice with ice-cold water. Centrifuge: 5000 × g, 10 min, 4 °C. Freeze in liquid N2. Stored at −80 °C | Breaking system: Lyophilise cells. Add an equal volume (±500 μL) of glass beads (500 μm). Add 500 μL of lysis buffer (100 mM sodium. bicarbonate, pH 8.8, 0.5% Triton × 100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors). 15 min in on ice. Shake at 30 s at 4.5 m/s (RiboLyzer). Chill on ice, 1 min between shaking steps. | Bidimensional gel (pI range: 3–10 NL, 17 cm strips) Coomassie brilliant blue Trypsin digestion MALDI-TOF-MS identification | [92,93] |
Sample cleaning: Remove cell debris by centrifugation (15,000 rpm, 20 min, 4 °C. 10% v/v DNase-RNase solution (0.5 M Tris-HCl, pH 7.0, 0.5 M MgCl2, 100 μg/mL RNAse and 2 μL DNase). 1 h, 4 °C. Add water up to 2.5 mL plus 200 μL of 0.5 M Tris (pH 6.8) and 20 μL of 1 M dithiothreitol (DTT). R/T. 30 min. 600 μL of water-saturated phenol. R/T. 30 min. Centrifuge: 5000× g, 10 min, 4 °C Add to supernatant 20 μL of 1 M DTT and 30 μL of 8 M ammonium acetate. R/T. 30 min. Precipitate. 2 mL of cold (−20 °C) methanol. Centrifuge: 13,000 rpm, 4 °C, 30 min. 2× wash: 70% (v/v) cold ethanol at −20 °C. Resuspend pellet: 200 μL of buffer (8 M urea, 2 M thiourea, 2% CHAPS, 0.01% [w/v] bromophenol blue). Store at −80 °C. | ||||
YM medium 20-h old culture (beginning of carotenoid biosynthesis) 20 °C and 150 rpm. | Centrifugation: 5000 rpm, 10 min. | Breaking system: Liquid nitrogen in a mortar Resuspend fine powder in 5 mL of buffer (50 mM Tris–HCl pH 7.4, 0.5 mM PMSF). Add 5 mL of 100 mM sodium carbonate. 1 h on ice. Centrifuge: 6000 rpm, 20 min, 4 °C. Supernatant precipitation: 10% final concentration of trichloroacetic acid (TCA). Centrifuge: 10,000 rpm for 15 min. | Bidimensional gel (pI range: 3–10, 11 cm strips) Colloidal Coomassie [100] | [91] |
Sample cleaning: 1× acetone. 1× 70% ethanol. 200 μL rehydration buffer (7 M urea, 2 M thiourea, 1% CHAPS, 0.5% Triton X-100, 40 mM Tris–HCl, 0.5% ampholytes 3–10, and 0.1% bromophenol blue). Centrifuge: 10,000 rpm, 15 min. Desalt: Micro Bio Spin G-30 columns (Bio-Rad) and rehydration buffer (Bio-Rad, Hercules, CA, USA). | ||||
Seed culture: 20 g/L glucose, 10 g/L yeast extract, 20 g/L peptone. 22 °C, 200 rpm, 48 h. 250-mL flask containing 30 mL Inoculate 3 mL in a 250-mL flask containing 30 mL of seed media. 22 °C, 200 rpm, 24 h Fermentation: (20 g/L glucose, 0.2 g/L yeast extract, 0.5 g/L (NH4)2SO4, 1.0 g/L KH2PO4, 0.1 g/L NaCl, 0.5 g/L MgSO4-7H2O, and 0.1 g/L CaCl2-2H2O). 22 °C, 200 rpm, 144 h | Centrifugation: 8000× g, 10 min, 8 °C, 96 h. | Breaking system: Resuspend pellet in deionised water. 2× 30 kpsi high-pressure disrupter (Constant Systems Limited, Northants, UK). 10,000× g, 15 min, 4 °C. Collect supernatant 0.5 mg dissolved in 500 µL rehydration buffer [8 M urea, 4% CHAPS, 2% IPG buffer and 40 mM DTT (Dithiothreitol)]. | Bidimensional gel (pI range: 4–7) Silver stain Trypsin digestion MALDI-TOF/TOF-MS identification | [94] |
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Barredo, J.L.; García-Estrada, C.; Kosalkova, K.; Barreiro, C. Biosynthesis of Astaxanthin as a Main Carotenoid in the Heterobasidiomycetous Yeast Xanthophyllomyces dendrorhous. J. Fungi 2017, 3, 44. https://doi.org/10.3390/jof3030044
Barredo JL, García-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of Astaxanthin as a Main Carotenoid in the Heterobasidiomycetous Yeast Xanthophyllomyces dendrorhous. Journal of Fungi. 2017; 3(3):44. https://doi.org/10.3390/jof3030044
Chicago/Turabian StyleBarredo, Jose L., Carlos García-Estrada, Katarina Kosalkova, and Carlos Barreiro. 2017. "Biosynthesis of Astaxanthin as a Main Carotenoid in the Heterobasidiomycetous Yeast Xanthophyllomyces dendrorhous" Journal of Fungi 3, no. 3: 44. https://doi.org/10.3390/jof3030044