Yarrowia lipolytica as a Platform for Punicic Acid Production
Abstract
:1. Introduction
2. Results
2.1. Lipid and Fatty Acid Profiling of Y. lipolytica Cells Grown in Medium Supplemented with Pomegranate Seed Oil
2.2. Pomegranate Seed Oil Supplementation Leads to the Accumulation of Lipid Bodies in Y. lipolytica
2.3. Neosynthesis of PuA in Y. lipolytica
2.4. Recombinant Strains Contain PuA in Polar and Neutral Lipids
3. Discussion
4. Materials and Methods
4.1. Media Composition and Culture Conditions
4.2. Plasmid and Strain Construction
4.3. Verification of the Presence of the DGA2 Gene
4.4. Fluorescence Microscopy Analysis
4.5. Lipid Extraction
4.6. TLC Analysis
4.7. Fatty Acid Analysis
4.8. Data Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | PgFADX Promoter | DCW (g/L) | TFA/DCW (%) | PuA (µg/mg DCW) | PuA (mg/L) |
---|---|---|---|---|---|
JMY8381 obese | - | 19.1 ± 0.1 | 34.2 ± 1.9 | - | - |
JMY8395 obese + PgFADX | pTEF | 18.6 ± 0.6 | 33.5 ± 1.4 | 0.9 ± 0.0 | 17.3 ± 1.3 |
JMY8710 obese + Δeyk1 | - | 14.4 ± 0.3 | 25.5 ± 1.4 | - | - |
JMY8716 obese+ Δeyk1 + PgFADX | pEYK1 4AB | 18.3 ± 0.3 | 30.9 ± 3.6 | 1.0 ± 0.1 | 18.9 ± 1.9 |
JMY8719 obese + Δeyk1 + PgFADX | pEYD1 | 19.2 ± 0.6 | 32.1 ±1.4 | 0.2 ± 0.1 | 4.7 ± 1.9 |
JMY8721 obese + Δeyk1 + PgFADX | pEYK1 4AB-coreTEF | 19.9 ± 0.3 | 31.6 ± 0.4 | 1.8 ± 0.1 | 36.6 ± 2.4 |
Strain | JMY8381 | JMY8395 | JMY8710 | JMY8716 | JMY8719 | JMY8721 |
---|---|---|---|---|---|---|
Obese | Obese + PgFADX | Obese + Δeyk1 | Obese + Δeyk1 + PgFADX | Obese + Δeyk1 + PgFADX | Obese + Δeyk1 + PgFADX | |
Promoter | pTEF | pEYK1 4AB | pEYD1 | pEYK1 4AB-coreTEF | ||
% of esterified fatty acids | ||||||
PuA | - | 0.4 ± 0.2 | - | 0.3 ± 0.0 | 0.1 ± 0.0 | 0.5 ± 0.1 |
C18:2 | 2.9 ± 0.1 | 2.7 ± 0.3 | 5.1 ± 0.1 | 2.4 ± 0.1 | 2.6 ± 0.1 | 2.9 ± 1.0 |
C18:1 | 43.9 ± 0.6 | 50.4 ± 2.7 | 42.1 ± 0.2 | 46.9 ± 0.6 | 44.9 ± 1.5 | 46.9 ± 4.8 |
C18:0 | 11.9 ± 1.1 | 9.3 ± 1.6 | 9.0 ± 0.2 | 12.4 ± 0.1 | 10.4.0 ± 1.0 | 11.3 ± 2.0 |
C16:1 | 5.6 ± 0.2 | 6.1 ± 0.2 | 7.4 ± 0.1 | 5.3 ± 0.0 | 5.8 ± 0.2 | 5.3 ± 0.5 |
C16:0 | 30.2 ± 0.6 | 25.9 ± 1.2 | 31.7 ± 0.2 | 27.4 ± 0.5 | 31.5 ± 0.4 | 27.6 ± 1.2 |
others | 5.6 ± 0.5 | 5.3 ± 0.7 | 4.7 ± 0.2 | 5.3 ± 0.2 | 4.7 ± 0.4 | 5.5 ± 1.4 |
Host | Carbon Source | Mechanism | Promoter | Gene | Fatty Acid Produced | PuA Level (% TFA) | PuA Yield (mg/L) | References |
---|---|---|---|---|---|---|---|---|
S. cerevisiae | Glucose + galactose | Neosynthesis | pGAL1 | PgFADX (PuFADX) | C18:2 | n.d. | ND | [16] |
S. cerevisiae | Glucose + galactose + C18:2 | Bioconversion | pGAL1 | PgFADX (PuFADX) | PuA | 1.6 | ND | [16] |
S. cerevisiae | Galactose + C18:2 | Bioconversion | pGAL10 | TkFADX (TkFac) | C18:2, PuA | 0.1 | ND | [17] |
S. cerevisiae | Galactose + C18:2 | Bioconversion | pGAL10 | PgFADX (PgFac) | C18:2, PuA | 0.8 | ND | [17] |
S. cerevisiae Δsnf2 | Galactose + C18:2 | Bioconversion | pGAL10 | PgFADX, PgPDAT, PgPDCT/PgLPCAT | PuA | 3.4 | 7.2 | [43] |
S. pombe | Glucose | Neosynthesis | pNMT1 | PgFADX | C18:2, PuA | 19.6 | 38.7 | [18] |
S. pombe | Glucose | Neosynthesis | pNMT1 | PgFADX, PgFAD2 | C18:2, PuA | 25.1 | 34.3 | [18] |
A. thaliana | Neosynthesis | CaMV | TkFADX (TkFac) | PuA | ~0.4 | [17] | ||
A. thaliana | Neosynthesis | CaMV | PgFADX (PgFac) | PuA | ~0.4 | [17] | ||
A. thaliana | Neosynthesis | napin | TkFADX (TkFac) | PuA | 10.2 | [17] | ||
A. thaliana | Neosynthesis | napin | PgFADX (PgFac) | PuA | 4.4 | [17] | ||
A. thaliana fad3 fae1 | Neosynthesis | napin | PgFADX | PuA | 11.5 | [53] | ||
A. thaliana fad3 fae1 | Neosynthesis | napin | PgFADX, PgFAD2 | PuA | 21.2 | [53] | ||
A. thaliana fad3 fae1 | Neosynthesis | napin | PgFADX, PgFAD2, PgDGAT2 | PuA | 24.8 | [19] | ||
A. thaliana fad3 fae1 | Neosynthesis | napin | PgFADX | PuA | 9.2 | [49] | ||
A. thaliana fad3 fae1 | Neosynthesis | phaseolin | PgFADX | PuA | 9.1 | [49] | ||
A. thaliana fad3 fae1 | Neosynthesis | linin | PgFADX | PuA | 13.2 | [49] | ||
A. thaliana fad3 fae1 | Neosynthesis | conlinin | PgFADX | PuA | 10.3 | [49] | ||
Brassica napus | Neosynthesis | napin | TkFADX (TkFac) | PuA | 2.5 | [50] | ||
B. napus | Neosynthesis | napin | PgFADX, PgFAD2 | PuA | 11.1 | [51] |
Strain | Genotype | Reference |
---|---|---|
E. coli. | ||
JME5213 | GGV-URA3 ex-pTEF-PgFADX | This study |
JME5215 | GGV-URA3 ex-pTEF-TkFADX | This study |
JME5334 | GGV-URA3 ex-pEYK1 4AB-PgFADX | This study |
JME5335 | GGV-URA3 ex-pEYD1-PgFADX | This study |
JME5336 | GGV-URA3 ex-pEYK1 4AB-coreTEF-PgFADX | This study |
Y. lipolytica | ||
JMY3820 | MATa ura3-302 leu2-270 xpr2-322 Δpox1-6 Δtgl4 + pTEF-DGA2 + pTEF-GPD1 | [39] |
JMY8381 | JMY3820 + URA3 | This study |
JMY8393 JMY8394 JMY8395 | JMY3820 + URA3 ex-pTEF-PgFADX | This study |
JMY8385 | JMY3820 + URA3 ex-pTEF-TkFADX | This study |
JMY8709 | JMY3820 Δeyk1 | This study |
JMY8710 | JMY3820 Δeyk1 + URA3 | This study |
JMY8714 JMY8715 JMY8716 | JMY3820 Δeyk1 + URA3 ex-Peyk1 4AB-PgFADX | This study |
JMY8717 JMY8718 JMY8719 | JMY3820 Δeyk1 + URA3 ex-pEYD1-PgFADX | This study |
JMY8720 JMY8721 JMY8722 | JMY3820 Δeyk1 + URA3 ex-pEYK1 4AB-coreTEF-PgFADX | This study |
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Urbanikova, V.; Park, Y.-K.; Krajciova, D.; Tachekort, M.; Certik, M.; Grigoras, I.; Holic, R.; Nicaud, J.-M.; Gajdos, P. Yarrowia lipolytica as a Platform for Punicic Acid Production. Int. J. Mol. Sci. 2023, 24, 8823. https://doi.org/10.3390/ijms24108823
Urbanikova V, Park Y-K, Krajciova D, Tachekort M, Certik M, Grigoras I, Holic R, Nicaud J-M, Gajdos P. Yarrowia lipolytica as a Platform for Punicic Acid Production. International Journal of Molecular Sciences. 2023; 24(10):8823. https://doi.org/10.3390/ijms24108823
Chicago/Turabian StyleUrbanikova, Veronika, Young-Kyoung Park, Daniela Krajciova, Mehdi Tachekort, Milan Certik, Ioana Grigoras, Roman Holic, Jean-Marc Nicaud, and Peter Gajdos. 2023. "Yarrowia lipolytica as a Platform for Punicic Acid Production" International Journal of Molecular Sciences 24, no. 10: 8823. https://doi.org/10.3390/ijms24108823
APA StyleUrbanikova, V., Park, Y. -K., Krajciova, D., Tachekort, M., Certik, M., Grigoras, I., Holic, R., Nicaud, J. -M., & Gajdos, P. (2023). Yarrowia lipolytica as a Platform for Punicic Acid Production. International Journal of Molecular Sciences, 24(10), 8823. https://doi.org/10.3390/ijms24108823