TABLE 1.
Oligonucleotides used in this studya
| No. | Oligonucleotide | Sequence (5′→3′) |
|---|---|---|
| 1 | desA-a2 | CCGTTCTGCAGGTTTCTTGGCGCAAGGGTTACAGCTTCC |
| 2 | desA-a1 | GCAACCATGGGAAACCCAACGCAAGG |
| 3 | desA-b2 | CCGTTGGATCCGTACGCTTCCATACCATGTTCACCAATATCG |
| 4 | desA-b1 | CCTCACAGGTTCGGCCTACAGTGG |
| 5 | desB-a2 | CCGTTCTGCAGCTTTACAACCCCTAATCCGCCTTTATTCATTTCC |
| 6 | desB-a1 | CAGATCGAGGGGAACCTGGTTTGCG |
| 7 | desB-b2 | CCGTTGGATCCCAACAACGCCTTGCAGAAAATCCCCAGC |
| 8 | desB-b1 | CCAGTTTTAACAGACCTTGGGTAAAGGCTTC |
| 9 | desC-a2 | CCGTTCTGCAGCCTTGTCACCTACGGCGAAGGTTGG |
| 10 | desC-a1 | GGAATACACTGACGAATACCGCGATGGG |
| 11 | desC-b2 | CCGTTGGATCCGGTAATTCCAATGCCGCCGGTAATCCAGT |
| 12 | desC-b1 | GGTTTCTGGGCGAATTGCGATCTTGAGG |
| 13 | desE-a2 | CCGTTCTGCAGATTGTTGTCCCCAAAGGGAAATATCTATTCCG |
| 14 | desE-a1 | GACCCAGAAAACCCCGTAGAATAATCACG |
| 15 | desE-b2 | CCGTTGGATCCAATGCTTTCCTAACGAGTTGAGAATATCTTCTATG |
| 16 | desE-b1 | CGTCGATTTTGCCTCATTAATTTAGTTAAAGCAGC |
| 17 | desF-a2 | CCGTTCTGCAGGAATAAATTTGCGTTTGATCATTACCGCCAATTC |
| 18 | desF-a1 | GGTTGAAACCATTTAGGGAAACCCATACTG |
| 19 | desF-b2 | CCGTTGGATCCGAATTCTCAAACAATAGAACAAGACAAAGGGGAATATC |
| 20 | desF-b1 | CGAGGCAGGTTTTGAGAGCGTCAAC |
| 21 | desE-US-Fw | TATGCACATATGCGTCGATTTTGCCTCATTAATTTAGTTAAAGCAGC |
| 22 | desE-US-Rv | GCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGCTTTCCTAACGAGTTGAGAATATCTTCTATGAAACCG |
| 23 | KM-Fw | TTAGAAAAACTCATCGAGCATCAAATGAAACTGC |
| 24 | KM-Rv | GGACTCTTCTCTACAGGTGGGTATAGATTTGTTAAGCTTTGGCAGGATCCGGCTGCTAACAAA |
| 25 | Cpc-prom-Fw | CTTAACAAATCTATACCCACCTGTAGAGAAGAGTCC |
| 26 | Cpc-prom-Rv | GGGTCAAGAACGTTGCTGTAATGCGTCATGGAATTAATCTCCTACTTGACTTTATGAGTTGGG |
| 27 | desE-DS-Fw | ACGCATTACAGCAACGTTCTTGACCC |
| 28 | desE-DS-Rv | CTGCCGCCAGGCAAATTCTGTTTTATCCATATGCTAGGGATTGGCCGCGTTTTGTAGATC |
| 29 | pBAD18-Fw | GATAAAACAGAATTTGCCTGGCGGCAG |
| 30 | pBAD18-desE-USRv | GCTGCTTTAACTAAATTAATGAGGCAAAATCGACGCATATGTGCATAGGAGAAACAGTAGAGAGTTGCGATAAAAAGCG |
| 31 | pBAD18-NdeI-Fw | CATATGGATAAAACAGAATTTGCCTGGCGGCAG |
| 32 | pBAD18-A-acsRv | TAGGAGGTTACGGGGAAAAGCCAATAGGCATATGTGCATAGGAGAAACAGTAGAGAGTTGCGATAAAAAGCG |
| 33 | A-acsA-Fw | CCTATTGGCTTTTCCCCGTAACCTCCTA |
| 34 | A-acsA-Rv | GCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACCTCGGCAGCAAAGTCTGGTG |
| 35 | KM-desE-Rv | GTTTCGGTGGTGACAGTTTCTGGGCTTTGGCAGGATCCGGCTGCTAACAAA |
| 36 | desE-comp-Fw | CCCAGAAACTGTCACCACCGAAAC |
| 37 | desE-comp-Rv | GTGTCGCCCACAATTTCCTGACCCCCAGGGCATCGTTTTAGCAACG |
| 38 | B-acs-Fw | GGTCAGGAAATTGTGGGCGACAC |
| 39 | B-acs-Rv | CTGCCGCCAGGCAAATTCTGTTTTATCCATATGCCAACAAGCCTTTGCCGCTGATC |
| 40 | AAS675-a1 | TATGCACATATGCCTTCCATCACGTCGGCAGTAATTTC |
| 41 | AAS675-a2 | GCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACAAGCCGAAATCATGGCTACAATCCTAC |
| 42 | KM-675-Rv | GAAGATTCCGCCATTCGGATCGCCTTTGGCAGGATCCGGCTGCTAACAAA |
| 43 | AAS675-b1 | GCGATCCGAATGGCGGAATCTTC |
| 44 | AAS675-b2 | CTGCCGCCAGGCAAATTCTGTTTTATCCATATGCACTGAGGCCACATCCGTCAAAATC |
| 45 | pBAD18-675Rv | GAAATTACTGCCGACGTGATGGAAGGCATATGTGCATAGGAGAAACAGTAGAGAGTTGCGATAAAAAGCG |
| 46 | AAS1977-a1 | TATGCACATATGGCAGTTCTGTAAGGCCCTACTAGAGG |
| 47 | AAS1977-a2 | GCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGTTTTCGCAGAATGGGTCATGGTGG |
| 48 | KM-1977-Rv | GCAATTTTCATCGCCACCCTTTAGAGGCTTTGGCAGGATCCGGCTGCTAACAAA |
| 49 | AAS1977-b1 | CCTCTAAAGGGTGGCGATGAAAATTGC |
| 50 | AAS1977-b2 | CTGCCGCCAGGCAAATTCTGTTTTATCCATATGCATTGACCCCAGGCTCAATCAGATTCC |
| 51 | pBAD18-1977Rv | CCTCTAGTAGGGCCTTACAGAACTGCCATATGTGCATAGGAGAAACAGTAGAGAGTTGCGATAAAAAGCG |
a
Restriction sites are underlined. Oligonucleotides 1 to 20 were used to construct integration cassettes for creating the desaturase knockouts (a1 and a2 primers were used for the upstream region of each gene, b1 and b2 for the downstream region). Oligonucleotides 21 to 30 were used to construct integration cassettes for creating the desE-up strain. Oligonucleotides 31 to 39 and 23 were used to amplify DNA fragments for subsequent Gibson assembly of an integration cassette used to create the ΔdesE+ complementation strain. Oligonocleotides 40 to 51 as well as 24 and 29 were used to construct integration cassettes for creating Δaas strains.