Improving isobutanol production with the yeast Saccharomyces cerevisiae by successively blocking competing metabolic pathways as well as ethanol and glycerol formation

Wess, Johannes; Brinek, Martin; Boles, Eckhard

doi:10.1186/s13068-019-1486-8

Biotechnology for Biofuels and Bioproducts

Table 4 List of primers used in this work

From: Improving isobutanol production with the yeast Saccharomyces cerevisiae by successively blocking competing metabolic pathways as well as ethanol and glycerol formation

Primer name	Sequence	Explanation
pRCC2_Fw	TGTTGTCTGACATTTTGAGAGTTAACACCGAAATTACCAAGGCTC	Primer for pRCCK/pRCCN amplification
pRCC1_Rv	CTTGGTGGTGTTCGTCGTATCTCTTAATCATAGAAGCAGACAATGGAG	Primer for pRCCK/pRCCN amplification
CC-Bdh1_Fw	GAAAATCTATGTACCCACGCGTTTTAGAGCTAGAAATAGCAAGTTAAA ATAAGG	BDH1 target for pRCCK
CC-Bdh1_Rv	GCGTGGGTACATAGATTTTCGATCATTTATCTTTCACTGCGGAG	BDH1 target for pRCCK
JWP001-DR-BDH1/2	GCAATAAGAATAACAATAAATTCATTGAACATATTTCAGATGACAAAAT AATATTTGGGGCCCCTCGCGGCTCATTTGTA	Donor DNA for ∆bdh1
JWP002-DR-BDH1/2c	TACAAATGAGCCGCGAGGGGCCCCAAATATTATTTTGTCATCTGAAAT ATGTTCAATGAATTTATTGTTATTCTTATTGC	Donor DNA for ∆bdh1
A1-Bdh2	TGACTGTGTTTGTGGTTCTC	PCR control of ∆bdh1 and ∆bdh2
A4-Bdh1	TCGTCTTTGTTCCCACATTC	PCR control of ∆bdh1 and ∆bdh2
CC-Leu4-9_Fw	ACAATGACCGTGGTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATA AGG	LEU4/9 target for pRCCK
CC-Leu4-9_Rv	CAACCACGGTCATTGTGACAGATCATTTATCTTTCACTGCGGAG	LEU4/9 target for pRCCK
DR-Leu4	TACTGTAGACTTTTTCCTTACAAAAAGACAAGGAACAATCGAACTTTTC TGTATTTCAGGACTTATTCGCTTCTATTTAT	Donor DNA for ∆leu4
JWP003-DR-Leu4c	ATAAATAGAAGCGAATAAGTCCTGAAATACAGAAAAGTTCGATTGTTC CTTGTCTTTTTGTAAGGAAAAAGTCTACAGTA	Donor DNA for ∆leu4
A1-Leu4	TTGTACAGTAACGGCCAGTC	PCR control of ∆leu4
A4-Leu4	TTCGTCACTAACCGCCAAAC	PCR control of ∆leu4
DR-Leu9	GGATAATACTATCGGCACATTATCATTTAGCCGCGTAGCCTAGAAAGG AGTAGCTTATGATTACTCATGTTATATATATA	Donor DNA for ∆leu9
JWP004-DR-Leu9c	TATATATATAACATGAGTAATCATAAGCTACTCCTTTCTAGGCTACGCG GCTAAATGATAATGTGCCGATAGTATTATCC	Donor DNA for ∆leu9
A1-Leu9	GGTAACGGTCGTAGTGAATG	PCR control of ∆leu9
A4-Leu9	TGTTCTCCCTTCACAAAGTC	PCR control of ∆leu9
CC-Ecm31_Fw	GAAGAACTGTGCTCCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA GG	ECM31 target for pRCCN
CC-Ecm31_Rv	GGAGCACAGTTCTTCAATGATCATTTATCTTTCACTGCGGAG	ECM31 target for pRCCN
DR-Ecm31	ATTAGCTTGCCATAAAATTAGGGAAATTTTTACTCACAATAATATATAGA TAAAAATCACTGCATAGGGAAAAAAACTTT	Donor DNA for ∆ecm31
JWP005_DR-Ecm31c	AAAGTTTTTTTCCCTATGCAGTGATTTTTATCTATATATTATTGTGAGTA AAAATTTCCCTAATTTTATGGCAAGCTAAT	Donor DNA for ∆ecm31
A1-Ecm31	ATGTACACGACAGACATTCC	PCR control of ∆ecm31
A4-Ecm31	TATTATAAAGCGGCCAGCTC	PCR control of ∆ecm31
CC-Ilv1_Fw	TACTTTACCCGACGTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGG	ILV1 target for pRCCK
CC-Ilv1_Rv	GACGTCGGGTAAAGTAACGATCATTTATCTTTCACTGCGGAG	ILV1 target for pRCCK
DR-Ilv1	CAAGCCACATTTAAACTAAGTCAATTACACAAAGTTAGTGAACCGACA ATTTACTTTATAAATTTACGCAACAACTTGTT	Donor DNA for ∆ilv1
JWP006-DR-ilv1c	AACAAGTTGTTGCGTAAATTTATAAAGTAAATTGTCGGTTCACTAACT TTGTGTAATTGACTTAGTTTAAATGTGGCTTG	Donor DNA for ∆ilv1
A1-Ilv1	AATTCACTAGCGGCTCCTTG	PCR control of ∆ilv1
A4-Ilv1	ATGGCTATGTGGAAGAAGTC	PCR control of ∆ilv1
CC-Bat12_Fw	ACAAGAGCTTGGCCAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGG	BAT1 and BAT2 target for pRCCK
CC-Bat1_Rv	CCTGGCCAAGCTCTTGTAGCGATCATTTATCTTTCACTGCGGAG	BAT1 target for pRCCK
CC-Bat2_Rv	CCTGGCCAAGCTCTTGTGGCGATCATTTATCTTTCACTGCGGAG	BAT2 target for pRCCK
DR-Bat1	TATAAACGCAAAATCAGCTAGAACCTTAGCATACTAAAACTGATAA TGAAGGTAAACATCCCCTCCCCCCCCAAAAAAAA	Donor DNA for ∆bat1
JWP007-DR-Bat1c	TTTTTTTTGGGGGGGGAGGGGATGTTTACCTTCATTATCAGTTTTAG TATGCTAAGGTTCTAGCTGATTTTGCGTTTATA	Donor DNA for ∆bat1
A1-Bat1	TTTAATGGCCCATCCGATCC	PCR control of ∆bat1
A4-Bat1	AAGTCCAGCGAGATACCTTG	PCR control of ∆bat1
DR-Bat2	AAATTTAAGGGAAAGCATCTCCACGAGTTTTAAGAACGATAGTATCG CTATTGCTACGTAAAGTAATTAAAAGTTAAAAA	Donor DNA for ∆bat2
JWP008-DR-Bat2c	TTTTTAACTTTTAATTACTTTACGTAGCAATAGCGATACTATCGTTCT TAAAACTCGTGGAGATGCTTTCCCTTAAATTT	Donor DNA for ∆bat2
A1-Bat2	GTGAGAGGAGATCCGAAATGAG	PCR control of ∆bat2
A4-Bat2	TCCACCGACATTACGGAAAC	PCR control of ∆bat2
JWP051-CC-Pdc1_Fw(4)	TGTTCCAGACACGACGTCAGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGG	PDC1 target for pRCCK
JWP052-CC-Pdc1_Rv(4)	CTGACGTCGTGTCTGGAACAGATCATTTATCTTTCACTGCGGAG	PDC1 target for pRCCK
DR_PDC1-Fw	TCTCAATTATTATCTTCTACTCATAACCTCACGCAAAATAACACAGT CAAATCAATCAAAGCGATTTAATCTCTAATTATTAGTTAAAGTTTTA TAAGCATTTTTATGTAACGAAAAATA	Donor DNA for ∆pdc1
DR_PDC1-Rv	TATTTTTCGTTACATAAAAATGCTTATAAAACTTTAACTAATAATTA GAGATTAAATCGCTTTGATTGATTTGACTGTGTTATTTTGCGTGAG GTTATGAGTAGAAGATAATAATTGAGA	Donor DNA for ∆pdc1
A1-PDC1	GAAATCAGCTTGTGGGTATTGTTCAGAG	PCR control of ∆pdc1
A4-PDC1	CCTGGTGGCATTTGCAAAATG	PCR control of ∆pdc1
JWP021-CC-Pdc5_Fw	ACGAAGTAACCTCACAATCGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGG	PDC5 target for pRCCK
JWP022-CC-Pdc5_Rv	CGATTGTGAGGTTACTTCGTGATCATTTATCTTTCACTGCGGAG	PDC5 target for pRCCK
DR_PDC5-Fw	ACTTATTTCACATAATCAATCTCAAAGAGAACAACACAATACAATAA CAAGAAGAACAAAGCTAATTAACATAAAACTCATGATTCAACGTTT GTGTATTTTTTTACTTTTGAAGGTTAT	Donor DNA for ∆pdc5
DR_PDC5-Rv	ATAACCTTCAAAAGTAAAAAAATACACAAACGTTGAATCATGAGTTT TATGTTAATTAGCTTTGTTCTTCTTGTTATTGTATTGTGTTGTTCTCTT TGAGATTGATTATGTGAAATAAGT	Donor DNA for ∆pdc5
A1-PDC5	CGTATACGAATTCCTTCAACAAAGGCC	PCR control of ∆pdc5
A4-PDC5	TAAGAAGGCATGTTGGCCTCTGTTTC	PCR control of ∆pdc5
JWP023-Mth1/Pdc1_Fw	TGCTTATAAAACTTTAACTAATAATTAGAGATTAAATCGCATGTTTG TTTCACCACCACCAG	Primer for synthesis of Donor DNA of Mth1 wt
JWP024-Mth1/Pdc1_Rv	TCATAACCTCACGCAAAATAACACAGTCAAATCAATCAAATCAGGA TACTGAATCCGGCTGC	Primer for synthesis of Donor DNA of Mth1 wt
JWP025-Mth1-∆T_Rv	CATTAGTTAGTTGCGTGTGCACAGTAGAGGGGGCAGAAAACATTG ATAGTGGCAAACTTTG	Primer for synthesis of Donor DNA of Mth1∆T
JWP026-Mth1-∆T_Fw	CAGTGATAATGCTTCTTTTCAAAGTTTGCCACTATCAATGTTTTCTGC CCCCTCTACTGTG	Primer for synthesis of Donor DNA of Mth1∆T
JWP043-CC-MTH1_Fw	GCAGTATGCATTCAGCGAGCGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGG	MTH1 target for pRCCK
JWP044-CC-MTH1_Rv	CGCTCGCTGAATGCATACTGCGATCATTTATCTTTCACTGCGGAG	MTH1 target for pRCCK
JWP045-DR-MTH1dT_Fw	CAGTGATAATGCTTCTTTTCAAAGTTTGCCACTATCAATGTTTTCTGCCCC CTCTACTGTGCACACGCAACTAACTAATG	Donor DNA for MTH1∆T modification
JWP046-DR-MTH1dT_Rv	CATTAGTTAGTTGCGTGTGCACAGTAGAGGGGGCAGAAAACATTGATAG TGGCAAACTTTGAAAAGAAGCATTATCACTG	Donor DNA for MTH1∆T modification
JWP047-MTH1_Fw	ATGTTTGTTTCACCACCACCAGC	PCR control of MTH1 modifications
JWP048-MTH1_Rv	TCAGGATACTGAATCCGGCTGC	PCR control of MTH1 modifications
JWP053-CC-Adh1_Fw	TAACTTGATGGCCGGTCACTGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGG	ADH1 target for pRCCK
JWP054-CC-Adh1_Rv	CAGTGACCGGCCATCAAGTTAGATCATTTATCTTTCACTGCGGAG	ADH1 target for pRCCK
JWP055-DR-Adh1_Fw	TCAAGCTATACCAAGCATACAATCAACTATCTCATATACAGCGAAT TTCTTATGATTTATGATTTTTATTATTAAATAAG	Donor DNA for ∆adh1
JWP056-DR-Adh1_Rv	CTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTGTATA TGAGATAGTTGATTGTATGCTTGGTATAGCTTGA	Donor DNA for ∆adh1
Sbp26-Chk_DelADH1_A1	GCAACCAAACCCATACATCG	PCR control of ∆adh1
Sbp27-Chk_DelADH1_A4	GGGCGGAGCGTTCTAATTG	PCR control of ∆adh1
vsp381_CC-GPD1_rev	GCCTAGACCTTCGACGAAACGATCATTTATCTTTCACTGCGGAG	GPD1 target for pRCCK
vsp382_CC-GPD1_fw	GTTTCGTCGAAGGTCTAGGCGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGG	GPD1 target for pRCCK
vsp383_CC-GPD1-Donor	CACGTAGACTGGCTTGGTATTGGCAGTTTCGTAGTTATATATTTAT TGGAGAAAGATAACATATCATACTTTCCCCCACT	Donor DNA for ∆gpd1
JWP058-DR-Gpd1_Rv	AGTGGGGGAAAGTATGATATGTTATCTTTCTCCAATAAATATATA ACTACGAAACTGCCAATACCAAGCCAGTCTACGTG	Donor DNA for ∆gpd1
vsp384_pGPD1_fw	GTACAGCTGATGGGACCTTGCCG	PCR control of ∆gpd1
vsp385_tGPD1_rev	GCTCCGTATTATCTTCGTCGTGGGG	PCR control of ∆gpd1
JWP059-CC-Gpd2_Fw	CCCTTACATGAGGGGCCACGGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGG	GPD2 target for pRCCK
JWP060-CC-Gpd2_Rv	CCGTGGCCCCTCATGTAAGGGGATCATTTATCTTTCACTGCGGAG	GPD2 target for pRCCK
JWP061-DR-Gpd2_Fw	CTCTTTCCCTTTCCTTTTCCTTCGCTCCCCTTCCTTATCAACACTCTCC CCCCCCCTCCCCCTCTGATCTTTCCTGTTGC	Donor DNA for ∆gpd2
JWP062-DR-Gpd2_Rv	GCAACAGGAAAGATCAGAGGGGGAGGGGGGGGGAGAGTGTTGA TAAGGAAGGGGAGCGAAGGAAAAGGAAAGGGAAAGAG	Donor DNA for ∆gpd2
vsp269_pGPD2_fw	GGAACATCCGAGCACCCGCGCC	PCR control of ∆gpd2
vsp270_tGPD2_rev	GGCGGCATCGAAATCTTCTTCTTGCCC	PCR control of ∆gpd2
Vsp388_CC-ALD6_fw	AAAACTTTGGCCTTAGCCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG	ALD6 target for pRCCK
vsp389_CC-ALD6_rev	CGGGCTAAGGCCAAAGTTTTGATCATTTATCTTTCACTGCGGAG	ALD6 target for pRCCK
vsp390_CC-ALD6-Donor	AACATCTTTAACATACACAAACACATACTATCAGAATACATGTACCAACCTGCATTTCTTTCCGTCATATACACAAAATA	Donor DNA for ∆ald6
JWY092_DR_Ald6_c	TATTTTGTGTATATGACGGAAAGAAATGCAGGTTGGTACATGTATTCTGATAGTATGTGTTTGTGTATGTTAAAGATGTT	Donor DNA for ∆ald6
vsp393_ALD6_rev	TACCGGCCTTCAACATCTTGGCC	PCR control of ∆ald6
vsp394_ALD6_fw	TCCACGACACTGAATGGGCTACCC	PCR control of ∆ald6

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