Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Culture Condition
2.2. Batch Fermentation
2.3. Genetic Techniques
2.4. Analytical Methods
2.4.1. Dry Biomass
2.4.2. Glycerol and Citric Acid Concentrations
2.4.3. Qualitative and Quantitative Analysis of Lipids
3. Results
3.1. Comparative Time-Course Study
3.1.1. Glycerol Consumption
3.1.2. Biomass Production
3.1.3. Citric Acid Production
3.1.4. Lipid Production
3.2. Fermentation Study
3.3. Comparative Study of LEU2 Expression and Leucine Addition
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Y. Lipolytica Strain Names | Strain Genotypes | Gene Configurations | Reference |
---|---|---|---|
H222 (H) | MatA mating type | [27] | |
H222ΔP leu+ ura− (HP-U) | MATA ura3-302::SUC2 ΔPOX1–6 | [27] | |
H222ΔP leu+ ura+ (HP) | HP-U, ΔPOX3::URA3 | loxR-URA3-loxP flanked by POX3 homologous up/down stream sequences | This study |
H222ΔP ΔL + DGA1 DGA2 leu− ura+ (HPDD) | HP, ΔLEU2 + DGA1 + DGA2::URA3 | loxR-URA3-loxP flanked by LEU2 homologous upstream and pFBA-DGA1-tLip1 pTEF-DGA2-tXPR2 LEU2 homologous downstream sequences | This study |
H222ΔP ΔL + DGA1 DGA2 ΔSNF1 leu− ura+ (HPDDS) | HPDD, ΔSNF1::URA3 | loxR-URA3-loxP flanked by SNF1 up/down homologous stream sequences | This study |
Vector Names | Features |
---|---|
Cre-recombinase (CR) | Shuttle vector carrying leucine marker, Cre recombinase flanked by TEFin promoter and Xpr2 terminator |
pGR12 (L) | Shuttle empty vector carrying leucine marker, FBA promoter and lip1 terminator, used for study of leucine biosynthesis |
POX3 Ura (PU) | Uracil selection marker flanked by POX3 upstream and downstream homologous sequences, used for construction of HP strain |
LEU2 Ura (LU) | Uracil selection marker flanked by LEU2 upstream and downstream homologous sequences, used for construction of LDD vector |
SNF1 Ura (SU) | Uracil selection marker flanked by SNF1 upstream and downstream homologous sequences, used for construction of HPDDS strain |
pGR12 DGA1 (D1) | Single gene centromeric shuttle replicative vector with leucine selection marker, DGA1 gene cloned between FBA promoter and lip1 terminator, used for double gene expression cassette construction |
pJN44 DGA2 (D2) | Single gene centromeric shuttle replicative vector with leucine selection marker, DGA2 gene cloned between TEFin promoter and xpr2 terminator, used for double gene expression cassette construction |
DGA1 DGA2 (DD) | Double gene centromeric shuttle replicative vector with leucine selection marker, used for construction of LDD vector |
LEU2 DGA1 DGA2 (LDD) | Uracil selection marker flanked by LEU2 homologous upstream sequence and combination of double gene expression cassettes and LEU2 homologous downstream sequence, used for construction of HPDD strain |
No. | Name | Sequence (5′―›3′, Underlined Restriction Site) |
---|---|---|
1 | POX3 up F ApaI | CTATAGGGCCCCTGGGCTGTTCGGTCGA |
2 | POX3 up R XbaI | GATCCTCTAGAAGGACGCACAACGCC |
3 | POX3 down F SpeI | CTGGACTAGTCGCTCCCATTGGAAACTACGA |
4 | POX3 down R NdeI | CCTCACATATGTCTCTTCGCTGTGGTCTAGG |
5 | POX3 F Ura | GTCTCTACTTGTAGTTCTGTAGACAGACT |
6 | POX3 Ura R | GAAGAATGTATCGTCAAAGTGATCCAAG |
7 | POX3 Ura F | TGACTTGTGTATGACTTATTCTCAACTACA |
8 | POX3 R Ura | AGATGCGTGATAGATTACTTGGATTTAGT |
9 | DGA1 F HindIII | GAGCGAAAGCTTATGACTATCGACTCACAATACTACAAGT |
10 | DGA1 R SalI | GTTCAAGTCGACTTACTCAATCATTCGGAACTCTGGG |
11 | DGA2 F HindIII | GCAAGGAAGCTTATGGAAGTCCGACGACGA |
12 | DGA2 R PstI | ATGCTACTGCAGCTACTGGTTCTGCTTGTAGTTGT |
13 | LEU2 up F ApaI | CTATAGGGCCC ACCGGCAAGATCTCGTTAAGACAC |
14 | LEU2 up R XbaI | GATCCTCTAGATGTGTGTGGTTGTATGTGTGATGTGG |
15 | LEU2 down F SpeI | CTGGACTAGTCTCTATAAAAAGGGCCCAGCCCTG |
16 | LEU2 down R NdeI | CCTCACATATG GACAGCCTTGACAACTTGGTTGTTG |
17 | LEU2 F Ura | TACAGTTGTAACTATGGTGCTTATCTGGG |
18 | LEU2 Ura R | CCTTGGGAACCACCACCGT |
19 | LEU2 Ura F | ACTTCCTGGAGGCAGAAGAACTT |
20 | LEU2 R Ura | ATAGCAAATTTAGTCGTCGAGAAAGGGTC |
21 | SNF1 up F ApaI | CAATTGGGCCCGTGATCAAAGCATGAGATACTGTCAAGG |
22 | SNF1 up R XbaI | GATCCTCTAGAGAGGTGGTGGAAGGAGTGGTATGTAGTC |
23 | SNF1 down F SpeI | CTGGACTAGT TCATTAATACGTTTCCCTGGTG |
24 | SNF1 down R NdeI | CCTCACATATGGGAATTCGTGCAGAAGAACA |
25 | SNF1 F Ura | GCGGGAAATCAAGATTGAGA |
26 | SNF1 Ura R | CGGTCCATTTCTCACCAACT |
27 | SNF1 Ura F | CCTGGAGGCAGAAGAACTTG |
28 | SNF1 R Ura | ACTACTGGCGGACTTTGTGG |
Strain and Culture Medium | Residual Glycerol | DCW (g/L) | Citric Acid (g/L) | Lipid (g/L) |
---|---|---|---|---|
HPDDS ura+ leu−, YNB-Ura | 4.93 ± 0.74 a | 7.75 ± 0.89 a | 28.36 ± 4.36 a | 3.6 ± 0.18 a |
HPDDS ura+ pGR12-leucine, YNB-Leu | 2.84 ± 0.45 b | 6.73 ± 0.24 a | 14.21 ± 1.12 b | 1.99 ± 0.15 b |
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Abghari, A.; Chen, S. Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid. Fermentation 2017, 3, 34. https://doi.org/10.3390/fermentation3030034
Abghari A, Chen S. Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid. Fermentation. 2017; 3(3):34. https://doi.org/10.3390/fermentation3030034
Chicago/Turabian StyleAbghari, Ali, and Shulin Chen. 2017. "Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid" Fermentation 3, no. 3: 34. https://doi.org/10.3390/fermentation3030034