Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Culture Conditions
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 Fatty Acids and LCDCAs
3. Results
3.1. Screening Process
3.2. Preliminary Shake Flask Experiments for Fatty Acid and LCDCA Production
3.3. Batch Fermentation
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Y. lipolytica Strains Names | Strain Genotypes | Gene Configurations | Reference |
---|---|---|---|
H222 (W) [wild type] | MatA | [42] | |
H222ΔP (HP-U) [leu+, ura−] | MatA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 | [42] | |
H222ΔPΔLΔSΔF (F) [leu−, ura+] | Same as HP-U, +Δleu2 Δsnf1 Δfaa1 Δsnf1::URA3 | loxR-URA3-loxP flanked by SNF1 upstream and downstream | This study |
H222ΔPΔLΔSΔF ΔL +ALK5 YlCPR YlFAO1 (P) [leu−, ura+] | Same as F, +Δleu2::URA3 YlALK5 YlCPR YFAO1 | loxR-URA3-loxP flanked by SNF1 upstream and downstream | This study |
H222ΔPΔLΔSΔF ΔL +ALK5 YlCPR YlFAO1 ++ALK5 (M) [leu−, ura+] | Same as P, +YlALK5 | Multiple-copy integration of YlALK5 using zeta based integrative vector pINA1291(this strain was selected after screening of 10–20 transformants for their growth and production capacities) | This study |
Vector Names | Map | Features |
---|---|---|
Cre (CR) | Cre recombinase flanked by TEFin promoter and Xpr2 terminator | |
pGR12 YlALK5 (5) | Centromeric (CEN) replicative vector, low-copy CEN plasmids (1–2 copies/cell~1.6 plasmid copies/cell), YlALK5 controlled by PFBA-Tlip1, leucine selection marker | |
pGR51 YlCPR (C) | Centromeric (CEN) replicative vector, YlCPR controlled by PGPM-Tcyc1 | |
pJN44 YlFAO (F) | Centromeric (CEN) replicative vector, YlFAO1 controlled by PTEFin-Txpr2 | |
ALK5 CPR FAO (5CF) | CEN replicative vector, PFBA-YlALK5 Tlip1 PGPM-YlCPR Tcyc1 PTEFin-YlFAO1 Txpr2 | |
Leu 5cf (L5CF) | Uracil selection marker flanked by LEU2 upstream and PFBA-YlALK5 Tlip1 PGPM-YlCPR Tcyc1 PTEFin-YlFAO1 Txpr2 LEU2 downstream sequences | |
pINA1291 ALK5 (Z5) | Integrative vector, hp4d- YlALK5 Tlip2, ura3d4 defective marker, zeta region for multi-copy integration | |
LEU (LU) | Uracil selection marker flanked by LEU2 upstream and downstream sequences | |
SNF (SU) | Uracil selection marker flanked by SNF1 upstream and downstream sequences | |
FAA (FU) | Uracil selection marker flanked by FAA1 upstream and downstream sequences |
No. | Name | Sequence (5′→›3′, Underlined Restriction Site) |
---|---|---|
1 | Alk 5 F HindIII | GAGCGAAAGCTTATGCTACAACTCTTTGGCGTCC |
2 | Alk 5 R PstI | CTTAGA CTGCAG CTACGCCTTCTCACCCTTATACA |
3 | Alk 5 Zeta F A | AATGCTACAACTCTTTGGCGTCC |
4 | Alk 5 Zeta KpnI R | TTGCAAGGTACCCTACGCCTTCTCACCCTTATACATCT |
5 | YlCPR F HindIII | GAGCGAAAGCTT ATGGCTCTACTCGAC TCTC |
6 | YlCPR R SmaI | GTTAT CCCGGG CTACCACACATCTTCCTGG |
7 | FAO1F NdeI | CCTCA CATATGATGTCTGACGACAAGCACACT |
8 | FAO1R SmaI | GTTAT CCCGGG AGGATCTCCGACCTCGAATC |
9 | LEU2 up F ApaI | CTATAGGGCCC ACCGGCAAGATCTCGTTAAGACAC |
10 | LEU2 up R XbaI | GATCCTCTAGATGTGTGTGGTTGTATGTGTGATGTGG |
11 | LEU2 down F SpeI | CTGGACTAGTCTCTATAAAAAGGGCCCAGCCCTG |
12 | LEU2 down R NdeI | CCTCACATATG GACAGCCTTGACAACTTGGTTGTTG |
13 | LEU2 F Ura | TACAGTTGTAACTATGGTGCTTATCTGGG |
14 | LEU2 Ura R | CCTTGGGAACCACCACCGT |
15 | LEU2 Ura F | ACTTCCTGGAGGCAGAAGAACTT |
16 | LEU2 R Ura | ATAGCAAATTTAGTCGTCGAGAAAGGGTC |
17 | SNF1 up F ApaI | CAATTGGGCCCGTGATCAAAGCATGAGATACTGTCAAGG |
18 | SNF1 up R XbaI | GATCCTCTAGAGAGGTGGTGGAAGGAGTGGTATGTAGTC |
19 | SNF1 down F SpeI | CTGGACTAGT TCATTAATACGTTTCCCTGGTG |
20 | SNF1 down R NdeI | CCTCACATATGGGAATTCGTGCAGAAGAACA |
21 | SNF1 F Ura | GCGGGAAATCAAGATTGAGA |
22 | SNF1Ura R | CGGTCCATTTCTCACCAACT |
23 | SNF1 Ura F | CCTGGAGGCAGAAGAACTTG |
24 | SNF1 R Ura | ACTACTGGCGGACTTTGTGG |
25 | FAA1 up F ApaI | CAATT GGGCCC CCAGGTCTCAGTTGCACTTGC |
26 | FAA1 up R XbaI | GATCC TCTAGA CAAATTATACCCCTCATCTCTCTAGGACA |
27 | FAA1 down F SpeI | CT GGACTAGTTTGGTGAGCCCACCGC |
28 | FAA1 down R NdeI | CCTCACATATGAACCTCCAGCAGACTAACTAGAACA |
29 | FAA1 F Ura | ACTGTAGCTAGATGGGTGCC |
30 | FAA1 Ura R | CGGTCCATTTCTCACCAACT |
31 | FAA1 Ura F | CCTGGAGGCAGAAGAACTTG |
32 | FAA1 R Ura | ACCAGCCCAGCCGG |
33 | LEU2 up F Ura | TCATGTTCGTGGAGGGGAG |
34 | LEU2 up Ura R | AAAACGCAGCTGTCAGACC |
35 | LEU2 down F FAO | GGAGTCCAAGCCCTTCGA |
36 | LEU2 down R | CACAAGACGTCAACTAAAGCGT |
DCW (g/L) | Citric Acid (g/L) | LCDCA Titer (g/L) | LCDCA Yield (g/g) | LCDCA Productivity (g/L·h) |
---|---|---|---|---|
8.58 ± 0.23 | 39.2 ± 3.5 | 3.49 ± 0.14 | 0.06 | 0.04 |
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Abghari, A.; Madzak, C.; Chen, S. Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids. Fermentation 2017, 3, 40. https://doi.org/10.3390/fermentation3030040
Abghari A, Madzak C, Chen S. Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids. Fermentation. 2017; 3(3):40. https://doi.org/10.3390/fermentation3030040
Chicago/Turabian StyleAbghari, Ali, Catherine Madzak, and Shulin Chen. 2017. "Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids" Fermentation 3, no. 3: 40. https://doi.org/10.3390/fermentation3030040