Supplementary Information
Expanding the product portfolio of fungal type I fatty acid synthases
Zhiwei Zhu1-3, Yongjin J Zhou1,3, Anastasia Krivoruchko1,3,4, Martin Grininger5, Zongbao K Zhao2,6* and Jens
Nielsen1,3,4,7,8*
1. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
2. Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, China
3. Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg,
Sweden
4. Biopetrolia AB, Systems and Synthetic Biology, Chalmers University of Technology, Gothenburg, Sweden
5. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster
of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany
6. Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian, China
7. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark
8. Science for Life Laboratory, Royal Institute of Technology, Stockholm, Sweden
*Correspondence to: Zongbao K Zhao Email: [email protected]
Jens Nielsen E-mail: [email protected]
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
1
Supplementary Table 1. Determination of the molecular weight of ACPs by MOLDI-TOF mass spectrometry. The molecular
weight of ACPs increases 340 Da after the phosphopantetheinylation.
Protein Theoretical MW (Da) Measured MW (Da)
ΔMW (Da) Untreated by PPT Treated by PPT
GST-mRtACPI 49515 49508 49510 2
GST-wRtACPI 49531 49519 49851 332
GST-mRtACPII 49417 49465 49419 46
GST-wRtACPII 49433 49476 49798 322
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
2
Supplementary Table 2. Intracellular total fatty acids produced by S. cerevisiae PWY12 expressing RtFAS and it mutants.
C6 C8 C10 C12 C14 C16 C16:1 C18 C18:1 Total
RtFAS01 0.009 ± 0.0008 0.003 ± 0.0002 0.036 ± 0.005 0.051 ± 0.005 0.094 ± 0.008 5.02 ± 0.27 25.19 ± 1.77 1.35 ± 0.18 37.81 ± 1.15 69.6
RtFAS02 0.009 ± 0.0006 0.005 ± 0.0016 0.037 ± 0.003 0.043 ± 0.006 0.077 ± 0.007 4.13 ± 0.35 21.59 ± 1.72 1.88 ± 0.32 39.69 ± 3.74 67.5
RtFAS03 0.010 ± 0.0017 0.007 ± 0.0003 0.034 ± 0.006 0.050 ± 0.002 0.079 ± 0.002 4.08 ± 0.08 21.34 ± 0.39 1.73 ± 0.12 40.41 ± 0.67 67.7
RtFAS04 0.012 ± 0.0002 0.092 ± 0.009 0.140 ± 0.006 0.304 ± 0.006 0.192 ± 0.0003 4.15 ± 0.03 22.83 ± 0.06 1.47 ± 0.01 29.95 ± 0.47 59.1
RtFAS05 0.012 ± 0.0007 0.145 ± 0.008 0.196 ± 0.007 0.507 ± 0.017 0.271 ± 0.003 4.52 ± 0.10 26.35 ± 0.04 0.84 ± 0.02 22.71 ± 0.59 55.6
Unit. mg/g DCW. Total fatty acids (in the form of FAMEs) were extracted from dry cell mass. The cell samples were the same
as those in Fig. 2b. Data from three independent cultures, and mean ± sd was presented.
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
3
Supplementary Table 3. Rare FAS variants containing duplicated ACPs found in fungal species.
Source organism
Accession number
NCBI/EBI/DDBJ UniProt JGI
Rhodosporidium toruloides XP_016272280.1, XP_016272387.1 M7WSW5, M7XM89
Sporidiobolus salmonicolor CEQ41577.1, CEQ41181.1 A0A0D6ENG2, A0A0D6EMT7
Lichtheimia corymbifera CDH58408.1 A0A068S7Q9
Absidia idahoensis var. thermophila CDS12520.1 A0A077WZ31
Mucor ambiguus GAN07093.1 A0A0C9MY03
Mucor circinelloides f. circinelloides EPB92410.1 S2KJ19
Parasitella parasitica CEP14499.1 A0A0B7NAM6
Amylomyces rouxii ADN94479.1 E2DDL2
Capsaspora owczarzaki XP_004364985.2 A0A0D2WGC9
Aphanomyces invadans XP_008874394.1 A0A024TSY3
Saprolegnia diclina XP_008613180.1 T0QIJ7
Aphanomyces astaci XP_009832890.1 W4GDM9
Phytophthora parasitica INRA-310 XP_008899478.1, XP_008891777.1 W2QNR7,W2RBA8
Saprolegnia parasitica CBS 223.65 XP_012208825.1 A0A067BPP7
Phytophthora infestans T30-4 XP_002997955.1, XP_002901724.1 D0NXJ3, D0NFR3
Phytophthora sojae XP_009519258.1 G4Z031
Phytophthora ramorum H3GFG1
Schizochytrium sp. ATCC 20888 ABJ98780.1 A0FJY8
Mortierella verticillata KFH67626.1
Pichia kudriavzevii KGK40757.1, KGK36891.1 A0A099P8Y8, A0A099NYH5
Albugo laibachii CCA25392.1 F0WVF1
Albugo candida CCI41838.1 A0A024G5L5
Hyaloperonospora arabidopsidis M4BNV4
Pythium ultimum K3WEQ6
Lichtheimia hyalospora 203878
Mortierella elongata 606355
Backusella circina 315358
Coemansia reversa 10860
86596
Piromyces sp. E2 65122, 38102
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
4
Phytophthora capsici 537721
Schizochytrium aggregatum 86957
Aurantiochytrium limacinum 83486
Aurantiochytrium mangrovei AKV56231.1 A0A0K1R5P4
Aplanochytrium kerguelense 103951
Meredithblackwellia eburnea 311302, 346975
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
5
Supplementary Table 4. Plasmids used in the study.
Plasmids Description Sources
pMD19T TA cloning vector Takara Bio
pET-22b(+) pBR322 origin, ampR, T7 lac, His∙Tag (C-terminal), Signal sequence Novagen
pET-41a(+) pBR322 origin, kanR, T7 lac, His∙Tag (Internal and C-terminal), S∙Tag (Internal), GST∙Tag (N-terminal), Thrombin and Enterokinase
cleavage sites
Novagen
pET-24b(+) pBR322 origin, kanR, T7 lac, His∙Tag (C-terminal), T7∙Tag (N-terminal) Novagen
pMD19T-RtFAS1 cDNA of RtFAS1 This study
pMD19T-RtFAS2(69-3021) ~3 kb cDNA fragment of RtFAS2 This study
pMD19T-RtFAS2(2660-4529) ~1.9 kb cDNA fragment of RtFAS2 This study
pMD19T-RtFAS2(2660-4529)-S1062A ~1.9 kb cDNA (S1062A mutant) fragment of RtFAS2 This study
pMD19T-RtFAS2(2660-4529)-S1253A ~1.9 kb cDNA (S1253A mutant) fragment of RtFAS2 This study
pMD19T-RtFAS2(4304-8881) ~4.5 kb cDNA fragment of RtFAS2 This study
pET-41(a)-wACPI Overexpression of GST-wRtACPI (1022-1184 aa of RtFas2, wild type) This study
pET-41(a)-mACPI Overexpression of GST-mRtACPI (1022-1184 aa of RtFas2, S1062A mutant) This study
pET-41(a)-wACPII Overexpression of GST-wRtACPII (1213-1375 aa of RtFas2, wild type) This study
pET-41(a)-mACPII Overexpression of GST-mRtACPII (1022-1184 aa of RtFas2, S1253A mutant) This study
pET-41(a)-PPT Overexpression of GST-RtPPT (2809-2928 aa of RtFas2) This study
pET22b-RtFAS1 Overexpression of RtFas1 Ref 4 and 5
pET24b-RtFAS2 Overexpression of RtFas2 (wild type) Ref 4 and 5
pET24b-RtFAS2 (S1062A) Overexpression of RtFas2 (S1062A mutant) This study
pET24b-RtFAS2 (S1253A) Overexpression of RtFas2 (S11253A mutant) This study
pYX212 2 μ, AmpR, ScURA3, TPI1p, pYX212t Ref 19
pUG6 Template for loxP-kanMX-loxP cassette Ref 24
pWJ1042 Template for KlURA3 cassette Ref 23
pRtFAS01 2 μ, AmpR, ScURA3, TPI1p-RtFAS1-FBA1t, TEF1p-RtFAS2-ADH1t This study
pRtFAS02 2 μ, AmpR, ScURA3, TPI1p-RtFAS1-FBA1t, TEF1p-RtFAS2(S1062A)-ADH1t This study
pRtFAS03 2 μ, AmpR, ScURA3, TPI1p-RtFAS1-FBA1t, TEF1p-RtFAS2(S1253A)-ADH1t This study
pRtFAS04 2 μ, AmpR, ScURA3, TPI1p-RtFAS1-FBA1t, TEF1p-RtFAS2(‘AcTesA-RtACPII)-ADH1t This study
pRtFAS05 2 μ, AmpR, ScURA3, TPI1p-RtFAS1-FBA1t, TEF1p-RtFAS2(RtACPI-‘AcTesA)-ADH1t This study
pAkFAS01 2 μ, AmpR, KlURA3, TEF1p-AkFAS-ADH1t This study
pAkFAS02 2 μ, AmpR, KlURA3, TEF1p-AkFAS(S2150A)-ADH1t This study
pAkFAS03 2 μ, AmpR, KlURA3, TEF1p-AkFAS(S2340A)-ADH1t This study
pAkFAS04 2 μ, AmpR, KlURA3, TEF1p-AkFAS(‘AcTesA-AkACPII)-ADH1t This study
pAkFAS05 2 μ, AmpR, KlURA3, TEF1p-AkFAS(AkACPI-‘AcTesA)-ADH1t This study
pScFAS01 2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2-FBA1t This study
pScFAS09 This study
pScFAS10 This study
pScFAS11 This study
pScFAS14 This study
pScFAS15 This study
pScFAS16 This study
pScFAS17 This study
pScFAS27
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(wRtACPI-wRtACPII)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(mRtACPI-wRtACPII)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(wRtACPI-mRtACPII)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(‘AcTesA-ScACP)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(ScACP-‘AcTesA)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(ScPPT-‘AcTesA)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2-FBA1t, TDH3p-'AcTesA-CYC1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(G1250S, M1251W)-FBA1t This study
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
6
pScFAS28 This study
pScFAS23 This study
pScFAS25 This study
pScFAS26 This study
pZWM1 This study
pZWM1-‘AcTesA
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(G1250S, M1251W, ScACP-‘AcTesA)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(ScACP-ShMks2)-FBA1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2(ScACP-ShMks2)-FBA1t, TDH3p-ShMKS1-CYC1t
2 μ, AmpR, ScURA3, TEF1p-ScFAS1-ADH1t, TPI1p-ScFAS2-FBA1t, TDH3p-ShMKS1-CYC1t
2 μ, AmpR, KlURA3
2 μ, AmpR, KlURA3, TEF1p-’AcTesA-ADH1t This study
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
7
Supplementary Table S5. Strains used in the study.
Strains Description Sources
E. coli
DH5α F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK- mK
+) supE44 λ- thi-1 gyrA96 relA1 Takara Bio
BL21 (DE3) F- ompT hsdSB(rB- mB
-) gal dcm(DE3) Novagen
S. cerevisiae
BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ATCC
YJZ02 MATa MAL2-8c SUC2 his3Δ1 ura3-52 pox1Δ Ref 15
PWY12 MATα ura3 leu2 his3 trp1 can1 Δfas1::HIS3 Δfas2::LEU2 Ref 20
ZW201 YJZ02 ura3Δ::loxP-kanMX-loxP+TEF1p-ScFAS1-ADH1t+TPI1p-ScFAS2-FBA1t This study
ZW202 YJZ02 ura3Δ::loxP-kanMX-loxP+TEF1p-ScFAS1-ADH1t+TPI1p-ScFAS2(G1250S, M1251W)-FBA1t This study
ZW206 YJZ02 ura3Δ::loxP-kanMX-loxP+TEF1p-ScFAS1-ADH1t+TPI1p-ScFAS2(ACP-‘AcTesA)-FBA1t This study
ZW207 YJZ02 ura3Δ::loxP-kanMX-loxP+TEF1p-ScFAS1-ADH1t+TPI1p-ScFAS2(G1250S, M1251W, ScACP-‘AcTesA)-FBA1t This study
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
8
Supplementary Table S6. Primers used in this study.
Primers Sequence (5’- 3’)
FAS1-L1 CCTAGACTCCCGCAACTCCTC
FAS1-R1 GGCTAAGAATGACCACGGAAG
FAS2-69F GACTCGTCAAACACCGCACAGGATG
FAS2-3021R CTGGTAGTAGATCTCCTTCTGGTGCTTG
FAS2-2660F TCTCGAAGGAGTACGCCGAACGCAT
FAS2-4529R CGGACCCAGTTCCACGACGAGTTG
FAS2-4304F AGGACAACTTCGTCTCGCAGCAGGT
FAS2-8881R GGATCCGACTGAGCATCTACTTCTGG
S1062A-F CGTCGGCGGCAAGGCGACCCTC
S1062A-R GAGGGTCGCCTTGCCGCCGACG
S1253A-F TGTCGGCGGCAAGGCGACCCTG
S1253A-R CAGGGTCGCCTTGCCGCCGACA
41-GST-ACPI-F TGGTGGCTCCGGTGATGACGACGACAAGGTCGCCGACGAGCCGCTCAAGG
41-GST-ACPI-R ATTAGTGGTGGTGGTGGTGGTGGTGGTGGAGCGAGATGCCAGCCATCGAGG
41-GST-ACPII-F TGGTGGCTCCGGTGATGACGACGACAAGGTCCCCGACGAGCCGCTCAAGG
41-GST-ACPII-R ATTAGTGGTGGTGGTGGTGGTGGTGGTGGAGGGTGATGCCAGCCTGCGAG
41-GST-PPT-F GGTGGCTCCGGTGATGACGACGACAAGGGTGCTTTCGGCGTCGGCACG
41-GST-PPT-R ATTAGTGGTGGTGGTGGTGGTGGTGGTGCTTCTGGGCGATGACGACGGCG
FAS1-5-NdeI GGCATTCCATATGgcaagctggagccacccgcagttcgaaaagggtgcaATGAACGGCCGAGCGACGCG
FAS1-3-EcoRI GGAATTCTCAGAGCCCGCCGAAGACG
FAS2-5-HindIII GCCCAAGCTTATGGTCGCGGCGCAGGACTTG
FAS2-3-NotI CCGCATTGCGGCCGCCTTCTGGGCGATGACGACGGC
pFAS-TPI1p-F GAATTGGGGATCTACGTATGGTC
RtFAS1-TPI1p-R CGACGTCCCAGTCACGCTCCGCGTCGCTCGGCCGTTCATTTTTAGTTTATGTATGTGTTT
RtFAS1-F ATGAACGGCCGAGCGACGCGGAG
RtFAS1-R1 AGACGGACGCAAAGTGGTTGAAGG
RtFAS1-R TCAGAGCCCGCCGAAGACGTCGAGC
RtFAS1-F1 ATGTCTACTCGATCAACGGTGTCCTCC
RtFAS1-FBA1t-F CTCAAGCTCGACGTCTTCGGCGGGCTCTGAGTTAATTCAAATTAATTG
RtFAS2-ADH1t-R GTCGCTTGCGCCGTCGTCATCGCCCAGAAGTAGGCGAATTTCTTATGATTTATG
RtFAS2-R CTACTTCTGGGCGATGACGACGG
RtFAS2-F1 CAACGCCGAAGTCGCCAACAAGC
RtFAS2-F ATGGTCGCGGCGCAGGACTTG
RtFAS2-ACPI2AcTesA-F1 GCGGTGGAGCTGCTGCTGCTGCC
RtFAS2-ACPII2AcTesA-R1 CGGGCGCTCCACCGCCAGCTGCG
RtFAS2-4594-R CGACAAGATGCAGGAGAAGCAGGACA
RtFAS2-4264-F GACAAGGCAGCGAGCGGTGATG
RtFAS2-S1253A-F ACCCTGCAGAACGAGATCCTCGGCGAC
RtFAS2-S1253A-R GTCGCCGAGGATCTCGTTCTGCAGGGTAGCCTTGCCGCCGACAAGGTCCTTGATG
RtFAS2-ACPII2AcTesA-F CTCCGGCCGCAGCTGGCGGTGGAGCGCCCGCTAAGACTATATTGATATTGGGTGAC
RtFAS2-ACPII2AcTesA-R GACAGCCGCGCCGCCTCCGCCTCCACCAGCTAATGCACCCTTGATGTAAGGGTAAGC
RtFAS2-S1062A-F ACCCTCCAGAACGAGATTCTCGGCGAC
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
9
RtFAS2-S1062A-R GTCGCCGAGAATCTCGTTCTGGAGGGTAGCCTTGCCGCCGACGAGCTCCTTGATC
RtFAS2-6628-R GGTCGTCAACTACAACGTCGAGGGA
RtFAS2-6583-F GGGATTGGAGGGCGACGAGGTGA
RtFAS2-ACPI2AcTesA-F CCCGCTCCTGTCGCTGCCGCGCCTGCCGCCAAGACTATATTGATATTGGGTGAC
RtFAS2-ACPI2AcTesA-R TCGCGGGGGCAGCAGCAGCAGCTCCACCGCCGGCTAATGCACCCTTGATGTAAGGGTAAGC
RtFAS2-ACPI2AcTesA-R1 GGCGGCAGGCGCGGCAGCGACAGGA
RtFAS2-ACPII2AcTesA-F1 GCTGGTGGAGGCGGAGGCGGC
RtFAS2-R1 ATGCCCTGGTCTGCCTTGTTGCC
RtFAS2-TEF1p-R GCGCGAGCGGCAAGTCCTGCGCCGCGACCATTTTGTAATTAAAACTTAGATTAG
TEF1p-F ATAGCTTCAAAATGTTTCTACTC
TEF1p-HA-F GAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGATCCCTAGCTCCAATTCGCCCTATA
pFAS-pYX212t-R GCCGTAAACCACTAAATCGGAACC
TPI1p-ScFAS2-R GCAAAATATGAGCTAATTCTTGCTCAACTTCCGGCTTCATTTTTAGTTTATGTATGTGTTT
ScFAS2-F AAGCCGGAAGTTGAGCAAGAATTAG
ScFAS2-R1 ACAGGTTCATCGGCAATCTCA
ScFAS2-R CTATTTCTTAGTAGAAACGGCGACC
ScFAS2-F1 TCAGAGCCGTGTCCATCACTT
FBA1t-ScFAS2-F GTCGCGGTCGCCGTTTCTACTAAGAAATAGGTTAATTCAAATTAATTG
ADH1t-ScFAS1-R CGACAACTGGGAAAAGTATGAACAATCCTAAGCGAATTTCTTATGATTTATG
ScFAS1-R GGATTGTTCATACTTTTCCCAG
ScFAS1-F1 CTGCTGTCAAGCCTCGCCCACT
ScFAS1-R1 TGTGGTAGGCTCGTCATCTGC
ScFAS1-F GACGCTTACTCCACAAGACCA
TEF1p-ScFAS1-R GGGTTAATGGTCTTGTGGAGTAAGCGTCCATTTTGTAATTAAAACTTAGATTAG
ScFAS2-ACPI2TE-R1 GAGTCACCCAATATCAATATAGTCTTAATCTCAGCAGCTGCTGCAGCTGG
AcTesA-F AAGACTATATTGATATTGG
AcTesA_ScACP_F CCGCAGCTGGCGGTGGAGCGCCCGCTGCCGATGAACCTGTCAAGGC
ScFAS2-R2 AAGATTGTTTAGCCCAGTTCC
ScFAS2-F2 CGGTGTTGCTACTTCTTTCTC
ADH1t-F GCGAATTTCTTATGATTTATGA
pFAS-FBA1t-F ATAGTTTTTTAATGAGTATTGAATC
AcTesA_ScACP-R GGCAGCAGCAGCAGCTCCACCGCCGGCAGCTGATGATAAGTCAACACC
AcTesA-R ACCAGCTAATGCACCCTTGATG
ScFAS2-ACPII2TE-F1 CCTTACATCAAGGGTGCATTAGCTGGTGCTAGCGCTAGTGGTGCTGCCGGT
AcTesA-ScFAS2-R TTTCTTAGTAGAAACGGCGACC
ScFAS2-linker-AcTesA-F GGTCGCCGTTTCTACTAAGAAAGGAGGTGGCGGTGGGTCAGGTGGCGGCGGAGGATCTGGAGGTGGTGGAGGCAGTAAGACTATATTGATATTGG
FBA1t-AcTesA-R CTATATCAATTAATTTGAATTAACTTATAATGCACCCTTGATGT
FBA1t-F GTTAATTCAAATTAATTGATA
AcTesA-TDH3p-R CACCCAATATCAATATAGTCTTCATTTTGTTTGTTTATGTGTG
CYC1t-AcTesA-R CGACAAAGGAAAAGGGGCCTGTTTATAATGCACCCTTGATGTA
CYC1t-F ACAGGCCCCTTTTCCTTTGTC
CYC1t-R GCAAATTAAAGCCTTCGAGCG
CYC1t-HA-F GGGACGCTCGAAGGCTTTAATTTGCCTAGCTCCAATTCGCCCTATAG
ScFAS2-ACP-R1 GTCGACAGCCTTGAGCGGCTCGTCGGCAATCTCAGCAGCTGCTGCAGCTGG
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
10
Rt2ACP-F GCCGACGAGCCGCTCAAGGCTG
Rt2ACP-R ACCAGCGCCGAGGGTGATGCCA
ScFAS2-ACPII-F1 CAGGCTGGCATCACCCTCGGCGCTGGTGCTAGCGCTAGTGGTGCTGCCGGT
mFAS2-GM1250SW-F CATGTTTCTGAGGTTGGTAACTGTTCTGGTTCTTCTTGGGGTGGTGTTTCTGCCTTACG
mFAS2-GM1250SW-R AGAACCAGAACAGTTACCAACCTC
ScACP-tShMKS2-F CGCAGCTGGCGGTGGAGCGCCCGCTATGTCTGATCAAGTTTACCA
ShMKS2-R CAAATGATGTTGAACACCAC
ShMKS2-ScFAS2-F CTTGTGGTGTTCAACATCATTTGGCTAGCGCTAGTGGTGCTGC
TDH3p-ShMKS1-F CGAATAAACACACATAAACAAACAAAATGGAAAAATCTATGTCACC
CYC1t-ShMKS1-R CGACAAAGGAAAAGGGGCCTGTTCATTTGTACTTATTAGCAATAG
pZWM-2μ-F AAGGGCCATGACCACCTGATGCACCAATTAGGTAGGTCTGGCTATGTCTATACCTCTGGCCGTCGCATCCCCGGTTCATTTTC
pZWM-AmpR-R TTGGCAATTTTTTGCTCTTCTATATAACAGTTGAAATTTGAATAAGAACATCTTCTCAAAGAGAGGCGGTTTGCGTATTGG
pZWM-KlURA3-F TTTGAGAAGATGTTCTTATTCAAATTTCAACTGTTATATAGAAGAGCAAAAAATTGCCAATTCGATGATGTAGTTTCTGGTT
pZWM-KlURA3-R CACCTTTCGAGAGGACGATGCCCGTGTCTAAATGATTCGACCAGCCTAAGAATGTTCAACGTGATTCTGGGTAGAAGATCG
pZWM-TEF1p-F GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGCATCGTCCTCTCGAAAGGTGATAGCTTCAAAATGTTTCTACTCC
TEF1p-R ATTAAAACTTAGATTAGATTGC
TEF1p-AkFAS-F GAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAATGGAACAACATAAAACTGAGAAAC
AkFAS-499-R TTCTGTCGTTGTCAGGCGATGTA
AkFAS-149-F TGTATGAAACTGGCACAGATGAG
AkFAS-5039-R GTAAATGACGTGGTATTACTATGGA
AkFAS-4919-F AGTTGACTGTTCGTTTCGGTGGA
AkFAS-10868-R ATCGGTAGCAGTGTTTGACAGAGCA
AkFAS-10610-F TAGCGGGCGGGTATGATGACTTT
ADH1t-AkFAS-R TTAATAATAAAAATCATAAATCATAAGAAATTCGCTTAATTTGTTATTGCTTGGGCTACT
pZWM-ADH1t-R GCCAGAGGTATAGACATAGCCAGACCTACCTAATTGGTGCATCAGGTGGTCATGGCCCTTCCGGTAGAGGTGTGGTCAATAAG
pZWM-R GCCAGAGGTATAGACATAGCCA
pZWM-F GTTGAACATTCTTAGGCTGG
AkFAS-S2150A-R CTCCAATTAGCTCATTTTGAATCGCTGCTTTTCCACCAGAAACACCCTTG
AkFAS-S1250A-F GCGATTCAAAATGAGCTAATTG
AkFAS-6900-R AATTTGTACAAAGTTGTTTTGA
AkFAS-6800-F AAAAGTCCAAATTCGAACGAA
AkFAS-6300-R CTTTTGAAGTTCATTCCCCAC
AkFAS-ACPI2TE-F GGAGAAAGTGGGGAATGAACTTCAAAAGAAGACTATATTGATATTGGGTGAC
AkFAS-ACPI2TE-R CACACTTTCGTTCGAATTTGGACTTTTTAATGCACCCTTGATGTAAGGGTAAGC
AkFAS-S2340A-R CTCCAAGAATTTCGTTTTGAACTGCTGCCTTCCCATTCACCAAGCTC
AkFAS-S2340A-F GCAGTTCAAAACGAAATTCTTG
AkFAS-8000-R AGCACTATCAACTGTCACCCT
AkFAS-7900-F TGTAATCGTGCCACTCCAGAAC
AkFAS-ACPII2TE-F GACTGTTCAAAACAACTTTGTACAAATTAAGACTATATTGATATTGGGTGAC
AkFAS-ACPII2TE-R GGACTACTATCACCTGAAGAGAGAGTACGTAATGCACCCTTGATGTAAGGGTAAGC
AkFAS-7400-F CGTACTCTCTCTTCAGGTGATAG
dURA3-Up-F AAACGACGTTGAAATTGAGGCTACTGCG
dURA3-UP-R GAAGAAGAATGACCATACGTAGATCCCCAATTCGGACTAGGATGAGTAGCAGCACGTTCC
TPI1p-R TTAGTTTATGTATGTGTTTTTTGTA
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
11
pFAS-FBA1t-R GTAAGCTACTATGAAAGACTTTACA
TEF1p-KanMX-F GAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATAAGCTTCGTACGCTGCAGGTCG
dURA3-KanMX-R CTGGCCGCATCTTCTCAAATATGCTTCCCCGACTCACTATAGGGAGACCG
URA3-Down-F GGGAAGCATATTTGAGAAGATGCGGC
URA3-Down-R GGAAACGCTGCCCTACACGTTCGC
ID-dURA3-UP AGGGAAGACAAGCAACGAAA
ID-dURA3-DOWN CGTCAAGGTCTGTTGAGTGC
TEF1p-AcTesA-F TGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAATGAAGACTATATTGATATTGG
TEF1p-AcTesA-R GAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCCACACAGGAAACAGCTATGAC
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Results
12
Supplementary Table S7. Codon optimized genes used in this study.
Genes Sequence
‘AcTesA AAGACTATATTGATATTGGGTGACTCATTGTCCGCTGGTTATGGTATTAATCCTGAACAAGGTTGGGTCGCCTTATTGCAAAAGAGATTGGATCAACAATTCCCAAAGCAACATAA
AGTAATCAATGCATCTGTTTCAGGTGAAACTACATCTGGTGCTTTGGCAAGATTACCAAAGTTGTTAACCACTTACAGACCTAACGTTGTCGTAATTGAATTGGGTGGTAACGAC
GCCTTAAGAGGTCAACCACCTCAAATGATCCAATCAAATTTGGAAAAGTTAATACAACACTCCCAAAAAGCTAAGAGTAAGGTTGTCGTATTCGGTATGAAGATCCCACCTAAC
TATGGTACAGCATACTCTCAAGCCTTCGAAAATAACTATAAGGTTGTCTCACAAACCTACCAAGTCAAATTGTTACCATTTTTCTTGGATGGTGTTGCTGGTCATAAGTCCTTAATG
CAAAATGACCAAATCCACCCAAACGCCAAAGCTCAAAGTATATTGTTGAACAACGCTTACCCTTACATCAAGGGTGCATTATAA
ShMKS1 ATGGAAAAATCTATGTCACCATTCGTTAAGAAACATTTCGTTTTGGTTCATACTGCATTTCATGGTGCTTGGTGTTGGTACAAGATCGTTGCATTGATGAGATCATCTGGTCATAAT
GTTACAGCTTTGGATTTGGGTGCTTCTGGTATTAATCCAAAGCAAGCATTGCAAATCCCAAACTTCTCTGATTACTTATCACCATTGATGGAGTTTATGGCATCTTTGCCAGCTAAC
GAAAAGATTATCTTGGTTGGTCATGCTTTAGGTGGTTTGGCAATTTCAAAAGCTATGGAAACTTTTCCAGAAAAGATTTCTGTTGCAGTTTTCTTGTCAGGTTTGATGCCAGGTCC
AAACATCGATGCTACTACAGTTTGTACAAAAGCAGGTTCTGCTGTTTTGGGTCAATTGGATAACTGTGTTACTTATGAAAACGGTCCAACAAATCCACCAACTACATTGATCGCA
GGTCCAAAGTTCTTGGCTACTAACGTTTACCATTTGTCACCAATTGAAGATTTGGCTTTAGCAACAGCTTTGGTTAGACCATTGTATTTGTACTTGGCAGAAGATATCTCTAAGGA
AGTTGTTTTATCTTCAAAGAGATACGGTTCAGTTAAGAGAGTTTTTATTGTTGCAACTGAAAACGATGCTTTGAAGAAAGAATTTTTGAAATTGATGATTGAAAAGAATCCACCA
GATGAAGTTAAGGAAATCGAAGGTTCTGATCATGTTACAATGATGTCAAAGCCACAACAATTGTTTACTACATTGTTATCTATTGCTAATAAGTACAAA
ShMKS2 ATGTCTGATCAAGTTTACCATCATGATGTTGAATTAACAGTTAGAGATTACGAATTGGATCAATTCGGTGTTGTTAACAACGCTACTTATGCATCATACTGTCAACATTGTAGACAT
GCATTTTTGGAAAAGATTGGTGTTTCTGTTGATGAAGTTACTAGAAATGGTGACGCTTTGGCAGTTACAGAATTATCATTGAAGTTCTTGGCACCATTGAGATCAGGTGACAGAT
TCGTTGTTAGAGCTAGATTGTCTCATTTCACAGTTGCAAGATTGTTTTTCGAACATTTCATTTTTAAATTGCCAGATCAAGAACCAATTTTGGAAGCTAGAGGTATTGCAGTTTGG
TTGAACAGATCATACAGACCAATCAGAATACCATCAGAGTTTAATTCTAAGTTCGTTAAGTTCTTGCATCAAAAATCTTGTGGTGTTCAACATCATTTG
Nature Chemical Biology: doi:10.1038/nchembio.2301
1 MDAYSTR------------PLTLSHGSLEHVLLVPTA---SFFIASQLQEQFNKILPEPTEG------------------FAADD
1 MAALFSSAAP----------MRPLVLHDAATRVSVHVPASPLSAWVTSQVVAQDFQDSIVGQHTP----------------APVADED
1 MSGIKTTPSDDSYVELSATSSQSSNNSNGLLLRALTIASATGIEVNIPVPSQPFDAWIVADVLREEFQRSLASSEAN--------------DVLEVQEEQ
1 MKPLITR----------PHQITHQPSGARLSLPLPSS-EEIISHAESTRDDFTAWLDHQPDSPLLQLSNGQQGILRSQEEGEQEQ
1 MNGRATRSVTGTSTPVHTATTR----------PLVLLHPSTQTRISLHVPST-SQEWIAAEVARDTFQDWLHAAEKS----------GNLVGFEAAELDD
1 MPVSQSLLVSSTQLVDKFVSNSQPIYDDARLN----------------------------
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
53 EPTTP------------AELVGKFLGYVSSLVEPSKVGQFDQVLNLCLTEFENCYLEG--NDIHALAAKLLQENDTTLVKTKELIKNYIT-ARIMAKRPF
63 EEPSAPSVEP------QVKLLAQFLSFVADRAVQDASHELSEVLLAAYNRFNELFLSA--TNVHSLVQSFDPET--RAEILKAYFKAFAT-AREQLGDKV
87 EQAGAADKAAG---EAKINLAAKFLGFLASRATLKTSSHELNLLQHAWAHFHDEFLATR-GSVHDLVAAIDSD--LRTPVLRSYFEAYVQ--LESAGQAK1 MEQHKT---EKLSAAD75 EEQEEGKEEKQNERHHQEASLILLAHYLHFLSIHPNRPHHKQLIQISLTYFHSEILNNRAIDLHSAAFQLTTSDLARRLVIKAYYLARN--AIPELLPNC
80 EQAGEGDDEK--ELVLTAYFLKHVAGLLPFPSTATS-PATAAVLLAAFNHFASVYLSG--TDVHTLTASLAAP--VRALVISSFFLAKTKLEVEGLGKVL
32 ----------------KVVLVAEFLDFCLQHN--------KELAVEVFAYFVASFCKKDVTSIYQVVEHLNKTD--AKAVLKTFYAGWTD----------
138 DKKSNSALFRAVGEGNAQLVAIFGGQGNT------------------------------------------------------DDYFEELRDLYQTYHVL
152 QIAHPSALLEAAKDGSTELYALFGGQGVN------------------------------------------------------EHYFGELQLLYDTYTPF
179 PSLITPKLFSLSAQNKAEVHAVFGGQGNNEEARRDPAASFVIVGESGRQRRVFAILLRRPDAFVTVVSRGLTLISLLPQFSNSQAYFKELRTLFDTYRPL14 EKLSESTIHWLAATDVPSNFVLFSGQGYQ--------------------------------------------------------YFDELRELYETGTDE173 PSPPVGRLWKDDQP-NKKLAGVFGGQGVN------------------------------------------------------ETYWQELVNLYSLYPTI
173 PKQSESALLQKAATGQAEVFALFGGQGMN------------------------------------------------------EVYFDELQTLHDLYTPL
96 -SNKSVPLECAQFPDDVKVIGIFGGTLGCDGG---------------------------------------------------AQCLEAIRELLEIYRPL
184 VGDLIKFSAETLSELIR--TTLDAEKVFTQGLNILEWLENPSNTPDKDYLLSIPISCPLIGVIQLAHYVVTAKLLGFTPGELRSYLKG-ATGHSQGLVTA
198 VAPIISEITDLLISLGHKADNDGYTYYS-QGLDLLSWLDGSSPQPSVEYFASIPLSLPLIGVAQLAQYVISCRVTDISPAEMRERFKG-ATGHSQGIVSA
279 VQPLLESIQPVLDKLVQDAVKQGFASYYIHGMDVLAWLTGAAPEPAVLYLASVPLSLPLIGLTQITQFLVSIRASGLTPAEFRSQIKS-ATGHSQGIISA58 VKGFLLLASKTLQEEICSEKAKTVAFKFVNELDFTKWIEEDIDVDQT-YIFSAPVSFPLIFVAQVARYLQTLNLLRTDHKQFIQTIKG-SSGHSQGIVAA218 LHPFLELADRHLHSLCSSPHAQASSLYKPHGIQILKWLNEPGSKPPTTYLASCALSLPLIGLVQLAHYIVLGEAQGLTPNEISSQLKGGVAGHSQGVVVA
219 LTPFLARASEHLVSLAA---AEQHTLLYDHSLDALAWLQDPSTRPEVPYLATCAVSLPLIGLTQLCQYVVYGKGSSLGPAELGAKFKG-ATGHSQGVVSA
145 VAEYLVAIVEFLERECQ---EPDIVHLFEHGFDLMGWLENADQIPATEYLDSAPIATPLLGLLQLLRVLVTSKTSGFSVNELVKRFEA-VVGFSHGLIVA
281 VAIAETDS---------WESFFVSVRKAITVLFFIGVRCYEAYP-------NTSLPPSILEDSLENNEGVPSPMLSISNLTQEQVQDYVNKTNSHLPAGK
296 VAIASSNS---------WSSLYENILKAVKQLFYIGLRGQEGFP-------LLAIDPKIVSDSVENNEGVPTPMLSINGLGLKALEGHIKKVNSHLASNS
378 VVLSASDS---------LESFYVNVAKALELLFHIGKRGQEFFP-------TLAIDPALVKDTVEGGEGEPTPMLSVSGLEQTVLEKHIQKTNSHLAESQ156 VLISTSPD---------NDRIVENAVKLCRYMLWQGLRCHESSVNRFSGKNHKSKKSKVYSFDESTRMVKYSPMLAVNGLTESLLNDFMKKTESIIEARR318 ALVAGELPGPENN----WAEFHCGAIHAITVLFHIGLHASLRFP-------QTSLPPKLIGTTAEH-EGLPTPMLAVTGLALDQLQKAIQAIQPYFAPN-
315 LVIAHEYPPASKDGSDAWEPFYEQALRGLTVLFQIGLQGTLAFP-------SIAISPALESSSVENGEGVPTAMLAVTGLDLKSLEKKIAEVNGHVKSEG
241 AAVSASSDN--------PDAFMHESMSALGHLLLFGTLSQIVDP--------QPALHALAIAEHIEHEGTPTSLAIVQGADRDIVEIMLKRYNVFVGSDE
364 ---------------------------QVEISLVNGAKNLVVSGPPQSLYGLNLTLRKAKAPSGLDQSRIPFSERKLKFSNRFLP-VASPFHSHLLVPAS
379 ---------------------------QIGISLHNGPTNFVVTAPAKALYGLVTALRKVMAPAGLDQSKVPFSKRKAVFTMRFLP-VNVPYHSHYLEGAT
461 ---------------------------AVSISLFNGPRSFIVTGYPKALVGLVGSLRAIRADTGADQGKIPFSKRKAVFRMNFLP-VGVPYHSSYLIGAT247 TIQQKYNRRESFFETPTEKDEKSKRLNMFQIALANGPKSFVVSGAPKTLIELEQRIKESTTSSPNSQTRIPFSKRKPVVKMYFLK-VGAAFHTEICNDAS404 ---------------------------DANVSLFNGPKAFVVSGHPRTLVGLVAALRNSKAEPGLDQSKIPFSKRRPVFSMRFLP-IGVPYHSAHLEGCT
408 RDE------------------------TVSISLYNGARAFVVTGAPKDLVGLADGLRKNRAPAGKDQSKIPHSKRLPVFSMRFLP-INVPYHSHLLQGAT
325 TER-----------------------HVHLAATLVKDSVFAISGNTRSLSQVVLNVRRKAAAKGEDQSAVPFMERKPQVTVTYVKSAVAPLHSPHMRIVY
437 DLINKDLVKN------NVSFNA--KDIQIPVYDTFDGSDLR-VLSGSISERIVDCIIRLPVKWE------TTTQFKATHILDFGPGGASGLGVLTHRN--
452 QKVEKDLG--------EELWDA--KALGMAIYHTEDGSDLR-SSEISLTTSLCDQIFTRPIHWV----KACNFPSTATHAVDFGPGGNSGIGPLTGRA--
534 EKMLQEDYAG-----RTIVWDA--SELKTTVKNTEDSSDLASYSSEDILKSIADQIFVKHIYWS----KVSDLPKSATHLIDFGTGGLSGIGGLSARI--346 SKLKADVVQM------KLSISP--KEILIPVYHTKDGSNLSEISADVNVVDLLIDMQTSQVNDFRKTLKSISSQNGVSNVIDFGPG--DGTAKLCLKR--477 ARLMGPVEEGGVGEEERAWWAAHKARLSCPVFNTENGVDMR-VEHKDLLSSLADLIFTSPIHWT----KACAFPDDTTHIIDFGLGTLSGIGSLVARN--
483 EKALATFS-----AEEAAHWAP--SSFTCAVYNTEDGSDMRQLSASSVLESVFQQIFTSPIHWVS---HATNFPSSATHAIDFGTGGASGIGSLCARN--
402 NRHLAMARAKG--------WVLG-APQVPILGATAAKVPGSIADDNDVAAALVEHVYLQAANWP----RALDATDGVTHIVDFAPKSGSPSNEGLLRMST
13
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
534 ---IEGRGVRIVVVGE-------KGKAAAEFYDA----TKIRREPVWAKEWSPKLVK-------TLDGKVHIDTPFSRLLGKPPIMVAGMTPST--VGAN
620 ---LEGRGVRILTPSG-------THARSTELYNA----STVKFEERWEDVWSPRLIK-------TKNG-IKIDTPFSRTTGRGILMVGGMTPTT--VSAN433 ---KSGTGILVVAATGPLRNRRCAYGRNLVGLNSVLLEKNPILGMNWGEEFKPRLSSR------KNDNQIIVGTRFSDLTGKPPVILPGMTPTTSFHGID569 ---IEGKGHRLVFVGLP--ASGQGHKSMNEVYDS----RDIIREQKWAEKYKIRLVK-------TKDGRLQIDTPFSRLLSKPPLMVAGMTPCT--VPTD
570 ---WEGRGIRTIMLGN----RGEGVGAGKEAWG-----KKVPTEEKWNERFHPRLVR-------TSDGKIHLDTPFSRLLSKPPLMVGGMTPTT--VKAG
489 RTVIEGRGIALICAESATSVEFASDTPNHEISGLHS--LNTCRVRSWQQDFGAVIVEDGTIVNEGQQHPQRIRSRLSEVIGMPPIIIGGMSPTT--THVE
606 FVAATTNAGYTIELAGGGYFSAAGMTAAIDSVVSQI-EKGSTFGIN--LIYVNPFMLQWGIPLIKELRSKG-YPIQFLTIGAG--VPSLEVASEYIETL-
612 LVAATLDAGYHIELAGGGHYNPKALRAKVAEIQRRV-KPGVGITLN--ALYINQRQFSFQFPLWQEMRKEG-LPIEGFCVAAG--IPSSEKATEIITALK
697 INAAVLNAGYVIELAGGGLHSEAGLRKRVNEILAQL-EPGNGLELN--GLFIDQRRFGWQYPLWGQMRREG-FPCKGFTVAAG--IPSTEKAKEIIDGLK525 LVAACSNGGYHGELAGGGLPLPEYFKAKIDELVSKQ-NSGVGININ--MLYLNSYLWGFQFSTAIQMAKDG-YPIESITCAAG--VPTTEKAKEIMHQLK652 FNAAVMNAGYHIELAGGGHYNAKALRSKISAIQAKLQKPGLGFTLN--ALYINQKQWAFQFPLWLEMRKEG-LPMEGFVVAAG--IPSTEKAKEIIEGLR
650 FVSAVLRAGYHIELAGGGHYNEKAVRAKVAEIQKLVNKPGMGITLN--SLYINQRQWTFQFPLWAKMKQEG-EPVEGLCVAAG--IPSTEKAKEIIDTLR
585 FVAAASRAGYLAELEIGHLETATALEKLVVAACETAVQAGHGLALSSTTQAVSDGPQAWLVDSIVHIRQELRLPILGISLGVAPSTATSSTVAADILRLH
698 --GLKYLGLKPGSIDAISQVINIAKAHPNFPIALQWTGGRGGGHHSFEDAHTPMLQMYSKIRRHPNIMLIFGSGFGSADDTYPYLTGEWSTK---FDYPP
706 EAGIKHIAFKPGSVEGIRQVVNIAAANPDYPIIMQWTGGRAGGHHSCEDFHQPIIATYPAIRQNPNISLIAGSGFGGAEDVWPYLSGEWSVKM--FGLQP
791 AAGIEYVGFKPGTADGLRAVAAIAAANPDFKIICQWTGGRAGGHHSTEDFHQPILQTYASLRKQKNLILVGGSGFGDAEGVWPYLTGEWSVK---LGYAP619 EAGIKYIAFKPGSRAAIMDVLSIARENPNSSIVLQWTGGRGGGHHSFEDFHAPLLDTYQEIREHPNVVLVVGSGFGDAEKSYPYLDGSWSTLPPHNCESR747 EAGIKHVSFKPGSVDGIRQVINIAAANPDFPVICQWTGGRAGGHHSCEDFHQPILATYASIRSQSNLILVVGSGFGSAEDVYPYLTGHWSRDR--FGVEV
745 EAGIKHVSFKPGSVDGIRQVVNIASANPDFPIILQWTGGRAGGHHSCEDFHAPILATYASIRQHPNIKLVAGSGFGSAEGCYPYLSGEWSEKQ--YGVAR
685 GAGLQYVGFTASTTDEVHRVLAIAEQSAPALIMLQWTGGRGQGRHSLDEFHMVLAATYSDVRRHSNVLLLGAAGFGTSEGALPYLSGEWATWF---DRPV
794 MPFDGFLFGSRVMIAKEVKTSPDAKKCIAACTGVPDDK-WEQTYKKPTGGIVTVRSEMGEPIHKIATRGVMLWKEFDETIFNLPK-NKLVPTLEAKRDYI
804 MPFDGVLYASRVMVAKEADTSASVKQLIVDAPGVDDAQ-WEGTYDRPTGGILTVRSELGEPIHKIATRGVKLWREFDDTVFAQPR-EKRAAWLENKRDYV
888 MPFSAFLFASWMLVAKEAATADAVKQLLVDTPGCGDDK-WQDTYGKGAGGTITCNSELGESISYVHNRAAALWRQLDDQLFSLPR-EKQRVWLSEKKSWL719 MPCDAVLFGSRCMVAKEAATSPEVKQLIVNAKGVRENESWEMSYENDAGGILTVTSELGEPIHKINNRGMKLWREFDRKYFSLPSGQEREKAIVKDKKEI845 MPFDGVLFASRMMVAKEAATSLSVKELIVQAKGVDDQE-WEGTYERETGGIITVTSELGEPIHKIATRGIKLWKEFDQTVFALPR-EKRAAWLKTHKEYV
843 MPFDGFMFASWVMVAKEAHTSESVKQLIVDAPGVEDGQ-WEQTYDKPTGGILTVNSELGEPIHKVATRGVKLWAEFDKKVFSLSK-EKQLAWLADNKKYV
782 MPLDGIVVHSRVIAARESALAGPAKALVAAAAGLDALDLPALFRSSNGSGVISIRDLDDSPTHVAANRASQLCQDIGQWVLDRPA-DQHPKLLRSHGSQI
892 ISRLNADFQKPWFATVNGQAR-------DLATMTYEEVAKRLVELMFIRS----------TNSWFDVTWRTFTGDFLRRVEERFT----KSKTLSLIQSY
902 IDRLNKDFNKPWFGEKSDGTVV-----ADIGKMTYEEITKRMVRLMYIAK----------QDRWIDVSLRNLVGDWLRRVEERFAGVDGIRTKESLLQSF
986 IERLNKDFQKPWFAAKLDGTVL-----MDVADMTYEEVTRRMLELLFVGH----------QSRWIDPSLKKLFGDWCRRVEERFAGIETAGKKESLLQSY819 IRRINADFQKVYFGRKADGTV------VDIAYMTYGEILRRMVDLMYVTGGGDGKQHRFAPNRWIDVTYQTRTFKFLVRTERRFLR----DKKMAFATDM943 IKRLNADFQKPWFAEK-DGHP------AELGEMTYQETVSRLVRLLFVKH----------QARWIDPTLRNLVGDWLRRIEERLSVVNGP-PKVSEIQSY
941 IDRLNADFQKPWFPAKADGSP------CDLADMTYAEVNARLVRLMYVAH----------EKRWIDPSLRNLVGDWIRRVEERLSNVNDSGIKISALQSY
881 MKRLSSDFMRPWFAVRRSDSDDGSAEPIELTEMSYIETIDRLVELMYVRS----------QHRWLAPSYRNFVAKFAERAAMRLLG----SARPFEQPAL
971 SLLD-KPDEAIEKVFNAYPAAREQFLNAQDIDHFLSMCQNPMQ-KPVPFVPVLDRRFEIFFKKDSLWQSEHLEAVV-------DQDVQRTCILHGPVAAQ
987 SDLD-KPLPTIESFFGTYPRAKSQLVAAEDKAFFLAICQRPGQ-KPVPFIPILDNNFEVWFKKDSLWAAEDVEAVF-------DQDPQRVCILQGPMAVK
1071 SVLDKEPSKFVDAFFAEYTGAKEIRLAAEDVGYFLTICQRRGQ-KPVPFIPELGDNFQTLFKKDSLWQSEDLDAVV-------GKDAQKVIILQGSVAVQ909 RKLESFPIKCIDEFVKCYPQVDKVLVSDEDAGYFIELCRNLRNGKPVNFIPQIDGSLAYWFKKDSLWCSEQIDAVP-------DNDPGRVCILHGPVAAQ1025 SELD-EPFSKLETFFTRYPEASTQILASEDIAYFLALCQRPGQ-KPVPFIPVLDAQFGIWFKKDSLWQSEDIDAVV-------DQDPQRVAILQGPVAVR
1025 SELN-EPEAFLKQFLAQYPQAEDQILASADVSYFLAISQRPGQ-KPVPFIPVLDANFSIWFKKDSLWQAEDIEAVF-------DQDPQRVCILQGPVAAK
967 SAHYDAVLDEIETLRRSCAHVLNQPLASADVQFFLQLCTRRGQ-KPVPFVPVLDANLRRYLMADAWWMAEDLDAVASAAACSSEDAAQRVLLRLGPVSAA
1062 FTKVIDEPIKSIMDGIHDGHIKKLLHQYYGDDESKIPAVEYFGGES-------------PVDVQSQVDSSSVSEDSAVFKATSSTDEES-WFKALAGSEI
1078 HATVVDEPIKDMLGNIEALLVKKILKEFYNNDESKVPEIDFIGAKP--------------GKPKAGLVSESVSGDVRTLKVGKNVPALDDWLEVVAGANV
1163 HCTKANVPAGEMLGDIEKQLVAKVLAKYYDNDESKVPVADYVSNDP--------LAIDEADVYARYGVEASIEGNLKTLKIGSQLPDTTEWLEALATRRT1002 YSVVSDEPICEILGNIHKGYVDKLKEEKY--EVAKVETIGKLP-----------------------EYKGSNKSFRKFLRYG------------------1116 HSKTTEETAGQILGGIEEGLVSRLLRDEYGGDESLVPEQDYLCREEGGMEAEERTAMLAAARIKYRKVTSSDR-VLHTYDIHGILPPPSQWLACLVGSSV
1116 HCTSTQTPIAEMLGNIEHQLVKNVLDDYYGGDESQIPTIDYLAPPP---KPVDAGAILAENNIAHSVEELADGGKKHVYSINGVLPPTGDWHAALAGPKL
1066 HVTAVDEDLADILDNIHQGLLAALP-PALDAGAAEISALEPLPESL------------------LLTVENEMRQRTLSVDDNCVVPKGLAWATALSGNSS
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
519 ---KDGTGVRVIVAGTLDINPDDDYGFKQEIFDVT--SNGLKKNPNWLEEYHPKLIK-------NKSGKIFVETKFSKLIGRPPLLVPGMTPCT--VSPD
14
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
1164 S--WLRAALTSVNVVQGAGYISNPFRRIFS--PRAGQTVVIKSE-NGKAVSVTVYGAARSFGPHAAD---FKAVELTFNSNTNAMSLIMYE---------
1255 S--WLRALLRNVSVVQGKNYADNPMLRMLA--PRKNQTAKIAYAADGEPISLTLFGAARSYGGHDDG---FKALEITKSSDSAKIQVTLFE---------1058 ---WFNALFQSTVMVKEKKWVRNIIPGIIQT-ENENIVFSERTAMNDMCLIEVLLNDSQR-------------TLAFIEFEGKHISVSLQD---------1215 S--WISALFNSISFLQGNAIVDNHLDTILLK-PRLHQRVQIVTGINGKPLNVKVFAAHLLS1 MAEIDYDSLPQTVDISFEPDSNIIDVKLFDYLVPSGSGP1213 D--WLQAFLSNVSIQAGEQSIPNPVKKVLA--PRHGQRVELTLNKDGQPLKLDVFGGL1 MVAAQDLPLALSISFAPESSTISMTLFN-------QP1147 NGGWLAAALSSAVIVQGVHHVPNYIARLLRARPGYTFIVSFSDENMSEPTRIEVREISSS----------QVTLVVTWISEEQKLAMRIPH---------
1228 TMDGKPVSLPLLYNFNPDNGFAPISEVMEDRN---QRIKEMYWKLWID----EPFNLDFDPRD-------VIKGKDFEITAKEVYDFTHAVGNNCEDFVS
1247 ERRGVSVPLHFAFAYHPEQGYAPIHEVVEGRN---KSIKDFYWRLWFG---DDEKLPSLKLDT-------TFTCDESKVDALAVQRFCDVVGNQGEAFKS
1339 ERLGESIPLHFYFTYRPDQPFASIHEVMNGRN---DRIKQFYWQLWFH--ADMPAAAQLAAAT-------QFESPVKSLDETAIRTFCRIVENRGEVFT-1132 -SEPVNSTLTLYFEYHPETPHSPLFQVTNGSKSIARSIKQYYRNIWDC--------QDVVSIH-------DTFSESFTVTREDIERFNGAIQYAVNTSEG
40 NAEPKVTQLDLKYKYSPETPWAPIHEVVEDRI---DRIKRYYWNVWDLGTEEEFENLPTAPSA-------IFHGPKVDILAEDIVSFSTIVGNDSDAYR-
31 EASKPALPLELKYKYDPSTPYAPIHEITEDRN---QRIKQHYWDLWGLGNKADQGISQLKITD-------EFQGDLVTISADEIEAFCRVVGIEGEAYKR1228 QAADFESAIELEFIFSPQTPLAPIHELMIGRD---ARIREFFIDTWLRSSAEEDIETSSEVNSEEANTNLEFITRAIRVDAERVGAFCRAAGLRLSAFPP
1314 RPDRTMLAPMDFAIVVGWRAIIKAIFPNTVDGDLLKLVHLSNGYKMIPGAKP---LQVGDVVSTTAVIESVVNQP-TGKIVDVVG--TLSRNGK------
1334 ARNEKIMAPMDFAIVLGWQSVMKAIFPDDINGDLLKLVHLSNGFRMMDGVAP---LRAGDACSAEARVVSVINSD-SGKTVKVKG--YVLRGGE------
1425 ----KGQAPMDFAIVAGWEAIMQALMAS-CDADLLSLVHLSNSFKVIDDARP---LRAGDVCTATAHASSIKISD-TGKSVSVTGTVLRKDASG------1216 --------TLDFAIVAGWKSLIKALFSKEIDGSLLDLVHISHSYKLLVEEKERTLVQAGEKICSESRVISVQILPGVGKSITVKG--VLSRNTV------
129 SSGPNSEVPMDFGIKLGWKAIMKPLFPKSIPGDLLALVHLSNRFDMRDRAPR---LKVGDTVTSEAKIASITNSE-TGKTVAVKGTVFLLKDGE------
121 NHKAGMQVPLDFAIKLGWKAIMKPIFPSTIDGDLLKLVHLSNGFRVLPDTPT---LQVGDVVTTTSRIESITNSD-TGKTVSVRGVISLVSSADSKGKDA1325 NSDIAAQVPMDYLPVLALQSAFKALTSRAVHSGLLHMVQTGCEIRREGGSAT--QLRLNDLVDATARVEELANCA-QGKRVVLASQILRDGL--------
1401 ------PVMEVTSSFFYR-GNYTDFENTFQKTVEPVYQMHIKTSKDIAVLRSKEWFQLDDEDFDLLN-KTLTFETETEVTFKNANI-FSSVKCFG-----
1421 ------PVIEISSSFLYR-GKFTDYENTFETIDEDDYVVELSKPTSVGILQAKPWFEWDDDSVPLDVGTTLTFKTKSELRYKDKST-FSSVKVSG-----
1510 ---DFVPVISVVSSFFFR-GRFTDFDTCFETSQN-EFTLEIKTKADATVLLAKEWFDWVAP-QPLAPGTVLHFNIQSLLKYKDSTS-YSDVEVTG-----1300 ------KWVEVQSEFLIR-GNFFDFQKTFK-EYSYKAEVLCKDDTVPQILNSQEWLFLDSGVRPLSKHDKVVFQIDRVVEKRKSVTNISDILVAGNVVRI
218 ----KTPVMDVISSFFYR-GRFDDFDATFMSEDDPEYKVTMNSTTDISVLKSKDWFDWKDENVKLAPGQTLTFQTSSSYRYKEKGV-YASVEVEG-----
217 STEDRIPLIEVTSSFFYR-GKFSDYAQTFSRVAHPTYSVPITTPEAVAVLQSKEWFQWDDDSKPLEVGTKLQFKVESNYVYADKSS-YAMATVTG-----1413 ------PIADIRSSFLFLGAALDDSNSGFRRTSHPLLAIELQSAADLAVLLSKEWFVCHDD-TGLAEGARLEIELKSDHRLDARGA-YATLHTYG-----
1487 ------PIKVE---LPTKETVEIGIVDYEAGAS---HGNPVVDFLKRNGSTLEQKVNLENPIPIAVLDS-----YTPSTNEPYARVSGDLNPIHVSRHFA
1508 ------AAFIR---DSTKALIQVATIDYEAHNL---QGNPVIEYLKRHGTAVGKPTPLENG-YSLIEDPTAAVFTTPATNEPYSKISGDFNPIHVNPYFS
1598 ------GAYVR---DYKDDLIKVGEIEYTADAIS--YGNPVIEYIKRNGGASTGGVAPLETGYTLGASGVPTTFVAPATNEPYSNASGDFNPIHINPYFS1393 NTPEEIDGFIELKDQGSPKGIIIGKVDLSLENAN-LSENPIITYLDSIKESKLHGSVFESGGYTVMPRPELVTAPSIVESIVYASASRDMNPIHRNRTFA
307 ------SAYLTGIGSDPNKLVQVAIISYTSATS--SKGNPVLEYLKRSGKPVGQHILFPTGGYLIKDENNISEIKTPTNNLPYSQASADWNPIHCNPYFA
309 ------GAYV--ITPELKLAVKVATVDYTSEGEGVIQGDPVIEYLKRHGSALDQPIMLENGGYSLTKAG-QCTFTTPASNLDYSLTSGDTNPIHTNPYFA1500 ------RVYLCG---LLHDRRHIADIDYQEGAG--AQDDAALAFLQARAEPTEPAPSNFDRAYSLAAPLS---TTAPASALAYAHASADHNPHNTNVFAI
1571 SYANLPG--TITHGMFSSASVRALIENWAADSVSSRVRGYTCQFVDMVLPNTALKTSIQHVGMI-NGRKLIKFETRNEDDVV--------VLTGEAEIEQ
1596 DLASLPG--TITHGMWSSAATRKYLESVVADNHPERVVSYQVGFVGMVLPGDEIQVKLTHTAMR-DGKKVVKVEAFNQRGEK--------VIDGSAEVLQ
1688 SFASLPG--TITHGLFSSAASRKFVEVVAAEGYPDRCLSFEANFVGMVLPGDELTVKLRHIAMR-DGNKVIKVETFNQNGDK--------VIDGQAEVKQ1492 KLAGLPGGSTIVHGMWTAAMSRRILELHTVLGDHRRISTYSVKFTGMVFPEDKLAVMVKHVGVT-EGRLILDVDVSKVETQER-------VLHGRAEVEG
400 NLASLPG--TITHGMWSSAATRSVVERVAAEGHGARVKSYDVSFTGMLLPNTTLKIELKHIGQTSKGYKLISVTTYALPGESSSSAEPTKVLVGTAEVAQ
401 SLAYLPG--TITHGMHSSARTRKFVEQVAADNVGARVRKYEVGFTAMCLPSRKMEVRLKHVGMTADGNRLIKVETVDVEGGN-------VVLSGTAEVAQ1587 DLAGLDAP--LMHGLWTSAQARALVELHAANGDPTRMRRFSVEFVAAVRPGAKLTTRLSHVAMR-AGRMVVRATTTSNATDEV-------VLRATAEVAA
1660 PVTTFVFTGQGSQEQGMGMDLYKTSKAAQDVWNRADNHFKDTYGFSILDIVINNPVNLTIHFGGEKGKRIRENYSAMIFETI-VDGKLKTEKIFKEINEH
1685 PPTTYVFTGQGSQEVGMGMELYNNSEVARAVWDVADAHLTSTYGFSIVDIVKNNPKELTIHFGGIKGQAIRQRYMDLTYDIIDESGRVKTLPLFGDIDLY
1777 PATVYVFTGQGSQEQGMGMDLYNSSPTAKALWDEADGHLRSTMGFSILEIINQNPLTKTIHFGGVRGHTIRERYMSMTYESTDADGNTVTLPLFPEISAS1584 PSTSFLFTGQGSAKVGMGMDSYEEKQSARAVWQTADKFLRKKFGFSIVDIVKNNPKELTVRFGGSQGKVIRENYRAIKREDG--------SQLIREIHSN
498 ASTGYVFTGQGSQEPGMGMALYNESAVARAVWDEADRHLGEVYGFSILEIVRNNPKEKIVHFGGIKGHGIRQRYMEMSYQTTDKDGNVKTLPLFGDIDLR
492 APTAYVFTGQGSQEPGMGMELYANSPVARAVWDEADRHLGEVYGFSILEIVRTNPKEKTVHFGGLKGQATRQKYMDMSYTTTDHEGNVKTLPLFGDIDLR1677 PRTVYVFTGQGSQEPGMAMELYERSAAARAVCDKVDHHMRERFGFSLLHIIRSNPHELTVHFGGKVGAQIRRNYMAFTRRVAGGK---QMVPLFPEITLN
1148 N--WRHASFLCSFITQDKMFVSNPIRKVFK--PSQGMVVEISNGNTSSKTVVTLSEPVQG---------ELKPTVILKLLKENIIQMEMIEN-------R
15
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
1877 SQSYTFQSPKGLLFATQFAQIALVLVELSAFRDMKQRGLITPDAAFAGHSLGEYASLAAVASVLEVKSLCDVVFYRGLSMQNCVQRDPVTGASNYAMMAC1676 TTSFTFRSPNGLLFATQFSQPALVLVQKAAFEEMLQGGFVPSNSMFAGHSLGEYAALASYANVLSIEDLVETVFLRGMVMQNAVARD-DEGTSDYGMVAA
598 TSRYTFSSPTGLLYATQFAQIALVVTEKAAFEDLREKGLVQEGAPFAGHSLGEYSALASIAGVLPISSLVDVVFFRGITMQRAVERD-EQNRSKYAMAAI
592 TSRYTFSSPTGLLYATQFAQIALVVTEKAAFEDMRAKGLVQKDCVFAGHSLGEYSALASIADILPISALVDVVFYRGITMQRAVERD-HLNRSSYGMVAV1774 SRQYTFRSPTGLLHATQFAQPAIMLFDVAVTADMRARGVFVDDALFAGHSLGEYGALAAFR-MMALEDIVDITFIRGMTMQATVERD-HNHRSAFAMVAA
1858 NPGRVA----ASFSQEALQYVVERVGKRT--GWLVEIVNYNVENQQYVAAGDLRALDTVTNVLNFIKLQKIDIIELQKSLSLEEVEGHLFEIIDEASKKS
1884 NPSRVG----KTFNEMALREIVDTISQQK--SVLLQIVNLNVANQQYVCAGELRALATLTNVLNMLKIQKIDLEKLSTMMSIEEVKEKLGEIIEGCWDMM
1977 NPLRIVGEGNPAFAGQALAEIVDSISKES--GRLLQIVNHNVAGQQYVTAGDLVALTTLGNVLNFIKIQKIDLKKLNEIMPIEEVREKLAEIVREVLSAA1775 NPSRVS----KTFSAQLLMEIVQKIDDESDDSHLLQVVNFNIRDAQYVIAGNLELLDSLSNVLNVISNSHDKTFDRSIIQAAMIKAENRRRSCKEN----
697 NPSRIG----KSFSDAALREVVDTISKRC--QVLLEIVNFNVEGQQYVTAGELVALQTLTNVLNFLKVQKIDIAQLQQTMSLEKVKEHLIEIVDECHKES
691 NPSRIG----KSFGDAALREVVDTIARRG--NILIEVVNYNVEGQQYVVAGHLVALQSLTNVLNFLKIQKIDLAKLTETMSIEQVKEHLCEIVDECVQKA1872 NPSRVH----RSFDEAALAFVIAEISAAG-TSELLEIVNYNVRRMQYVVAGTRHQLALLGGVLDAVHYREINVSGDSGRAVVKQLVAQLVS---------
1951 ---AVKPRPLKLERGFACIPLVGISVPFHSTYLMNGVKPFKSFLKKNIIKENVKVAR--LAGKYIPNLTAKPFQVTKEYFQDVYDLTGSEPIKEIIDNWE
1978 KEKEEKDGSVILDRGFATIPLPGIDVPFHSRYLWPGVLSFRNYLVKKIDPSQLNPDR--LIGKYIPNLIAEPFEVSKAYVQKIFDQTASPRMETVLNNWE
2075 LEEEMK-GRIELKRGFATIPLPGIDVPFHSRYLTGGVAPFRSYLSKRINVDAIRPQL--LVGRYIPNLVAAPFELSKSYAELIFAQTDSPRLDKVLKNWE1866 ------NQPFRLKRGIATIPLHGIDVPFHSRQLLNGVPAFRSLLEPRFTEEMIQKYLPRLIGKYIPNVHAEPFSISKEYIEKVASVTKSPALYKLLSTYS
791 LLKEEKQGFIVLERGFASIPLPGIDVPFHSRYLWAGVMPFRAYLSKKLNPAHMNPEL--LIDKYIPNLTAEPFQISKAYAERIYQQTNSPRLEKTLKNWV
785 RDLQAKTGFITLERGFATIPLPGIDVPFHSRYLWAGVMPFRTYLSKKVNPAHFNADL--LVGRYIPNLTAVHYEVSKEYAERIHTQTSSPRLNKILKAWD1957 ----NGGCGDEVTRTRATIPIPGLDVPFHSSHLLQGADQFRTCINMMIRETRTDCAG--YEARYIPNLTAVPFAISREYFQQVYDQTGSPVIRAELDCWP
2047 KYEQS1 MKPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNT--ERVVEIGPSPTLAGMAQRTLKNKYESYDAALSLHREILCYSKDAKEIYY---2076 KEAWES-PSQRQRLAYNILTECLAYQFASPVRWIETQDVLFTYAKF--ERLIEVGPSPVLSGMATRTLKAKYEAQDGAISLQRQILCHAKNQKEVYY---
2172 KDEWES-PDRVQRLTATLVVELLAYQFASPVRWIETQDIFFTDYNF--ERLVEFGPSPTLVGMAKRTQALKYSAVDLAQGRKRVMYCSSKDKEALYY---1961 TMCDSS-------KARILLIELLAHQFAMPVQWIDTQDYILSRHTQ---RIIEMGPAPTLVGMAKKTLQSRIYGDEED--YKPSILWWNQNQEEIFYNLD
889 EDGWDL-PENRSKLGYVIIVELLAYQFASPVLWIQTQDQLFSHQKYNIERLIEFGPSPTLTGMASRTLKLKYDQQDTAHNMSREILCISKNIKEIYY---
883 EERWGA-PENRNKLGYAILIELLAYQFASPVRWIETQDILFRDFKF--ERLVELGPSPTLTGMATRTQKLKYDAHDSSVGIKRSIYCIAKHQKEIYY---2052 EPPTAR---DRTRLSRLMIIELLSYQFASPVRWIETMDRLLHEFDV--EQIIEIGPQATLCRMAEGALMLAGKE------KQVAVRHIFRHEDEIFF---
90 ------TPDPSELAAKEEPAKEEAPAPTPAASAPAPAAAAPAP-VAAAA----PAAAAAEIA--------------------------------------2169 --------AFEDEAADEAPAAAAASTSAAPAPVAAPVAAAPAP-VAASA----GPAAAVE----------------------------------------
2265 --------AFADDESEVEAAPAETSAAAPVAAAPTPVA-AIAAPAASGG----AAAAAVP----------------------------------------2049 NQGISIQQFLQELKGTEDDESQSDDMSSYTQSDETIQRTAKNRVMPEANANDKEKVGNELQKKEVTNRPLHVLRVLISTKLKKKMLEVKATDTIKGVSGG
984 --------QYEDEVEAEAAPAAVSEPTAASPAAVAVAVAVAAPVAASG-----PVAAVA-----------------------------------------
976 --------QFDDVAGEEAPAPAAVAPS-APAPKAAPVAAAPPPPAPVAA----APAAAVA---DEPLKAVDTLRIIIAQKLKKPVGEVPLTKSIKELVGG2137 -TAARAAAEEREAQEAKAAAATLEAQAPAAAPEIQVSTIPEVQAPVTAPEIVPPVAATSSVP--------------------------------------
141 ----------------------------------------------------------------------------------------------------2216 ----------------------------------------------------------------------------------------------------
2312 ----------------------------------------------------------------------------------------------------2149 KSAIQNELIGEINAEYSSFRDIEDIS-EVSLAELARRAPSRQETVLGKVTTKMVNKMFSSKLPGKFGPNDTKEYLQSKLSDA-STVDSVSLHALTLQPVS
1030 ----------------------------------------------------------------------------------------------------
1061 KSTLQNEILGDLQGEFSS---APEKGEEMPLQELGAALQQGYSGKLGKYTTGVISRMIGAKMPGGFGLSAVQGHLGKTYGLGAGRIDGVLLFAVTQEPAK2198 ----------------------------------------------------------------------------------------------------
141 ---------------------------------------------------------DEPVKASLLLHVLVAHKLKKSLDSIPMSKTIKDLVGGKSTVQN2216 ---------------------------------------------------------DVPPKAVDTVRIIVAQKLKKQVSEVPLSKSLKELSGGKSTLQN
2312 ---------------------------------------------------------DEPLKAVDTVRAILAQKLKKNISEVPLSKSIKDSVGGKSVLSN2247 RLATPGEAQAWLDSVCRDFENDQRISILKNKSPNSNESVMISEQRTVQNNFVQIS--EEPVNSKHVLLSLISMKLQKEISSINETSTIKSLVNGKSAVQN
1030 ---------------------------------------------------------DEPLKAVETLRVLVAQGLKKPLDDVPLSKAIKDLVGGKSTLQN
1158 RLANEGEAKAWVDSVAQGYASMAGISLAAGGGAAAAAPAMAFAAPAAAGGGAPAAVPDEPLKATDTLRAIIAQKLKKQIPDVPLTKSIKDLVGGKSTLQN2198 ---------------------------------------------------------DIPLQAVDVIRAVIAQKIKRTVNEVAADFSVKQLTGGKSTLQN
1759 STSYTFRSEKGLLSATQFTQPALTLMEKAAFEDLKSKGLIPADATFAGHSLGEYAALASLADVMSIESLVEVVFYRGMTMQVAVPRD-ELGRSNYGMIAI
1785 TTSYTFSHPQGLLFATQYTQIALVVTEKAAFDDMKAKGLIDQNAAFAGHSLGEYSALAAIADVLPISSLADVVFFRGITMQRAVQRD-AEGKSQYAMMAA
16
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
2356 ELVSDLLAEFGN---LPERGEELPLEELGSALQAGYTGTLGKTTSNLVTRTVAGKLPGGFNLSAVKSHFQKAWGLG-AGRTDGALLVAVTMEPAKRLGSE2345 EILGEIASEFRTD--ESEGAADISLSELGEKLGIGYAGPGSVVSR-HINKMLNSKLPGSFNSSSAKKYLS-MQGFG-KGRSDSVLVHALTMQPKSRLQGV
1074 ELLGSAQAEFGS---APDKAEEMPLGELGSALQSSYSGTMGKHMSALVSRLIGSKMPGGFGMSGVKSHLSKSWGLG-PGRTEGVLLMALTMEPPKRLGSE
1258 EILGDLQGEFSS---APEKGEEMPLQELGAALNQGYSGTLGKHTSGLVARMMGAKMPGGFGLSAAKAHLSKAHGLG-PGRTDGALLVALTKEPEKRLGSE2242 EILGDLLKEFGGSGQIPDRPDEISLQELATGMGASFTGALGKHTQAQVSRLFSAKMPGGFSQSAARTHLATQLGVGRTHQQDAVLLLALTMEPSARLSSE
281 ADAKAFLDSMAQKYASIVGVDLSSAASASGAAGAGAAAGA--AMIDAGALEEITKDHKVLARQQLQVLARYLKMDLDNGERKFLKEKD-TVAELQAQLDY2355 AEAKAFLDQLAQSYASYSGISLSSGA--AAGGAGGAAGGG--AVMNSEEFDKFVLKQEEHAQREIELLSRYLGKDPREGEKKADEEKA-TAEELQAKLDA
2452 AEAKTWLDSVAQTYAQLAGISLAQAS--VGG--GGGGSGG--VAISSEELDKLKAQQDEHVRRQIQVLERYLNMDGRAGGRLAESTKA-ELEAAQAQLDA2440 EDAKSWLDSVCSSYGKWANIDLTVSRTLSSGDSSPAHSTK--DILSSTAMKKLKK----LYLDQVDVFNTFLGEDRRAKLQKVLTEETSAKDRIEAQLLE
1170 GEAKSYLDSVVQAYGQHSGLTLSQGG--PAGAAGGSAGG---AMINSEEFEKFQQSQDAFVSQQLEVLLRYLKRDSRDGYRLHDLKHA-DYMRVQDELDS
1354 ADAKAWLDGVAQAYASQAGITLGAGG--GGGGAAVGGAG---FMINTEQLDKMQEKQDNFVSQQVELFLRYLGKDSREGHRLADMQKA-EVANLQEKLDS2341 -EAPTWLDSVATAYATTHGITLAEPSKTAGSANDGSNSTANAPMVSSKALDDALRSQHALAMRQLETYAQHLGIDLRSDARRGEVAVN-ASAAQQQQLDQ
378 LNAELGEFFVNGVATSFSRKKARTFDSSWNWAKQSLLSLYFEIIHGVLKN----------------------------VDREVVSEAIN---IMNRSNDA2450 IKLEHGDAYIDGITPVFSALKARTFDSSWNWVRQSSIQLFYDIIHGDLDPSTVFNDPPRYRSNA1 MAAEGMDKESIVRRELTSRCIA---IMNRADPA2545 IAAEHGDTYTNGIMPKFTEKKVRHFSSYWNWARQDAMTLLYDIIYGRLTT----------------------------VDREITARCIT---LMNRADPS2534 LKEELGDPFCDGIQPQFNEARIRIYDSYWNWVVQDALELHYHTFSCVLNSKGKNVNIPNSSNSYFRAMSDWITSSNEKLEENKPPQAWFRNYLCNRATPE
1264 IQKEHGENYLKGIQPVFDPLKARHLDSAWNWVRQTALEMFFDIIYGRLKT----------------------------VDRDITAKCLV---IMNRANPA
1448 IAREHGDAYVQGIQPVFDPLKARHFNSSWNWVRQDALMMWMDILFGRLTT----------------------------VDRDITARCLV---IMNRADPS2440 LSEELGDEFIAGITGTFEPRKARHLASYWNWAREDAMAWIYGTLSGAEEKR------------------------PEAKELRLLQLANRADLRLLGLLDG
447 LIKFMEYHISNTDETKGENYQLVKTLGEQLIENCKQVLDVDPVYKDVAKPTGPKTAIDKNG--NITYSEEPR--EKVRKLSQYVQEMALGGPITKE-SQP
31 LLDYMKYHIDNADPSKGPTFQKIKEFGQILLDNCKEVVDKPPVYRDVALPTAPHTEISAKG--DIVYSEVSR--QNVRKLESYVKEMASGGEVE---PAV2614 LLRYMQYHIDQIDANQGPTYALAKEYGQQLIENCREACSEDAVYKEVHAPTAPHTEIDARG--NIAYSEVKR--VGVRKLEAYVKEMAAGSRLLGAGSQV2634 LLTVVQYFAARMNEQGHSEYAQAVSL---LAEQVSQWISRPPVHIALFASLEPRVTVDSANNFGLKYEEKPRKFVAVGENKAFSCDNASLYVKEMSKGLF
1333 LIDYMQYYLDHTDASKGERYRLAKEFGQMLLSNCREAVGTAPLYRDVTFPTAPKTTVTLKG--EIVYEEVNR--VGVSRLERYVAEMAAGSKIT---AEM
1517 LIDYMQYTIDNTPVERGEHYVLAKQFGQQLLDNCREMIGQAPLYKDVTFPTAPKTTVNAKG--DIITEEVNR--PGVSRLEKYVAEMAAGSKVTV--ASV2516 ILRMLDAQTNSSDSSVDLATVAARDLAQLIRSRCSSAVGSDPVYRELSQPTQPETLVSVGG--AVSYTETPR----------------------------
542 TIEEDLTRVYKAISAQADKQDISSSTRVEFEKLYSDLMKFLESSKEIDPSQTTQLAGMDVEDALDKDSTKEVASLPNKSTISKTVSSTIPRETIPFLHLR
124 NLEKVQSDIEKLWDLVNSQPSITAAQKSAIKSMYSEVVKSLGQSSGSALE-----DAESPAIARAKGPKQRRSSSQFLR-PNVEDRTEVEEAHLPFLHLK2710 NLDKAQENVAKLWTLIRNEPSVSKLSKATIKSLYSEVVRSLGPARQ-------------ARAPVRQAPRNRRLSSSTQR-PQVSDAAST-EDRTPFLHVK2731 YDHRVASKVEHPSQSVYLSPDNSFFPNEEESDQISSGMRLPKTQGEIQRDFSRLPAGQKLEVMRKSVNRSSDTDEEIANIVRQNYNSIHVAKNVPLVHLK
1426 NLDKVQESIQNLYKLVKSQPTIAKSQMAAIRSLYGEVVKGLSPHER-------------NRAAN--GPRSRRPSSQFLRPVQV-EPTALSGDKTPLLHLK
1611 NLDKVQEQVEKLYKLVKSQPQISKQHMTSIKSLYAEVVRGLGKDAG-------------PPPVHKAGTRARRPSSQFLRPAAVSEATFLPEDKVPLLHLK2585 ---INEVTMDEYVTLMSREPSKEESRGK--------------------------------------------------------------AAFAPLLHLR
642 K--KTPAGDWKYDRQLSSLFLDGLEKAAFN-GVTFKDKYVLITGAGKGSIGAEVLQGLLQGGAKVVVTTSR-----FSKQVTDYYQSIYAKYGAKGSTLI
218 R--KTGT-SFSYSAKLTNIYFDVLTEIATS-GVTFAKKAALLTGVGKGSIGVEILKGLLSGGCTCIVTTSR-----YSRAAVDYYKNIFHEIGSKGSKLI2795 R--KVAG-QWQLSRKLTSVYLDILGEMASQ-GVSFKGRNALLTGVGKASIGVEVVKGLLAGGARVVITTSS-----YSRSVVEYYQRIYQEVGSRGSTLT2831 SPSKFDKTVRVLDEPLTSMYLSCLHDIATS-GVSFAGQNALVTGAGFGSIGIELIKPLLEGGATVLVTVRLNRTDEQMQIVNERFQRLYEEFGSRGSKLV
1510 R--KVGS-NWVYSSKLTTLYLDVLTEIASA-GTTFEHKNALLTGVGKASIGVEILKGLLSGGAHVIVTTSR-----YSRATVEYYQAIYQEVGSRGSRLT
1698 R--KIGN-DWQYSSKLTSLYLDILKEIATS-GVTFEHKNALMTGVGKGSIGIEIVKGLLAGGARVVITTSR-----YSRSTVEYYQAIYQEVGSKGSSLT2621 E--KSASHHWEFSAEQSAVYYAALREMSSEKGTSFAGCTALITGCGRESIAAQVLQRLLLGGARVVATTSS----YAAPATLAFFEQSYRQFGARGAELI
734 VVPFNQGSKQDVEALIEFIYDTEKNGGLGWDLDAIIPFAAIPEQGIELEHIDSKSEFAHRIMLTNILRMMGCVKKQKSARGIETRPAQVILPMSPNHGTF
309 VVPFNGASRQDVEALVDYIYS-----TLQIDLDYIIPFAALPENGREIDSIDDKSELAHRLMLTNLLRLLGAVKQKKAARQFVTRPTQVVLPLSPNHGIF2886 VVPFNQASKQDVDNLVKYIYG-----SLDLDLDFVVPFAAISENGREIDGIDDKSELAHRLMLTNLVRLLGAIKTEKASRNFDTRPTQVLLPLSPNHGTF2930 LVPCNCASNQDVHSLIRHIYEK-----LKLDLDFIFPFAAIGEQGKDVSEIGSKSEVAHRLMLTNTIRLLGAVKKAKEDRCIETRPALVLLPCSPNHGDF
1601 VVPFNQGSKQDVEALVDYIYSTDKDKGLAMDLDYILPFAAIPENGREIDGLDDRSELAHRVMLTNLLRLMGEVKKKKHALKLVTRPTQVVLPLSPNHGNF
1789 VVPFNQGSKQDVEALVDFIYS--KDKGLGMDLDYILPFAALPENGREIDGIDDRSELAHRIMLTNLLRLLGAVKSKKAALKLTTRPTEVVLPLSPNHGLF2715 VVPFNQGSVADVQKLVAHIYR-----TLEWNLDYVVPFAAISEQGSDVTQLGSRSELAARIMLTNVLRLLGEIKSIKEELGHVTRPTLAVLPLSPNHGVF
185 EILGDLGKEFGT---TPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPGGFTITVARKYLQTRWGLP-SGRQDGVLLVALSNEPAARLGSE2260 EILGDLQVEFAS---APEKGEDLPLDELGAALSVGYA-ALGKHSMALTNRMVASKFPGGFNITAARAHLNKQWGLG-PLRTDSALFFGILNEPPKRLGSE
17
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
3025 GLDGLYAESKLGLESLVNKWKSEEWGNYLSICAAVIGWTR-SKLMWQNNVVAEGIEKLG--VRTFSTTETAFNLIGLLHPDIVSHAAEEPLWADLTGNWG
1701 GNDGLYSESKISLETLFNRWSSESWGEYLCLAGAVIGWTRGTGLMSANNLIAEGVETHG--VRTFSAKEMAFNILGLMHPLLFDVAQVEPVWADLNGGMD
1887 GNDGLYSESKISLETLFNRWSSESWGEYLCLAGAVIGWTRGTGLMSATNSVAEGIEAQG--CRTFSAKEMAFNILGLMHPLVFDVAQIEPVWADLNGGMD2810 GNDGLYGESKAALETVFNRWESERWGSLVSVAGAVIGWTRGTGLMAANNPIAQHLEQQPGAPRTFSAAEMAFNLVGLLHPTVADLAQHAPVWADLNGGLQ
932 FVPELKEFTAKLRKELVETSEVRKAVSIETALEHKVVNGNSADAAYAQVEIQPR-------------ANIQLDFPELKPYKQVKQIAPAELEGLLDLERV
502 RVAGLAEVMTSIRVDINRVAELRKAITIDNAADFRVINGGDAERLHQKVAITPR-------------ANFSFDFPKIDS-DDILN-ELKHLQGLIDLDKV3079 KIVDLAEQTGKFRAAIQEASSTKRALVADNSADFKVIKGSAAEALHKKVHINPR-------------SNFTFRFPEVGS-LQLNR-EIAKSYSQMDLEKV3122 AAPDLKEHSKRIRTSLLAQSKAAKAISLSSKSLVIEPSESKTKAQTEIVNTYLAGVNDKLRLPLANPEKFCNPFPKIPSQERMDS--LAYLKHSVDLKKV
1799 KLPDLAEISMKVRQELNEVANVRSKISLDNAIDFKVVHGVEAEAIHHPVKISPR-------------ANFTLPMPKLRPNFDDET-NMTLLRGMLDLDKV
1985 KLPDLANLTTEIRKKLNLTASTRRAIAKDNSFDYKVAHGPAMEQIHQRINVAPR-------------ANFSLPFPELKP-IDAKS-ELAKLRGLIDLEKV2910 RIPNVQHAVATARAQLVEEVGRRSAVVRGYGADLGVALGPRIARLHSDYALKPL-------------FNYKQHFAAARSYEELAH--LRHLQGMVNLEKV
1019 IVVTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYHNGNLK---GRPYTGWVDSKTKEPVDDKDVKAKYETSILEHSGIRLIEPELFNGYN
587 IVCTGFAEVGPWGSSRTRWEMEARGEFSIEGCIEMAWMMGFIKHLDGKLGN--GQTYVGWVDAKSGEPVDDKDVKTKYEKDIIKHAGIRLIEPDLFWGYN3164 VVITGFAEVGPWGSSRTRWEMEAFGHLTNEGVIELAWIMGLIKHHDGQLKS--GATYVGWVDVKSGEPVDDKDIRANYEEHIIKHAGIRLIEPELFRNYD3220 VVVVGYGEIGPWGNSRTRWEMESFGEFSLEGAIELAWLVGLIKPVTGPLKNDPRTQYFGWVDAESEEPVADHEIKTRYEKVLLQHSGIRLIEPELFEGYN
1885 IVVAGYAEVGPFGSSRTRWQMEAKGEFSIEGLLELATITGLIKFVDGKLKN--GKQYVGWVDAQTEEPVDDSQVKSKYEAQILAHTGVRFIEPELFRGYD
2070 VVMTGYAEVGPFGSSRTRWEMEANGTFSIQGTLELAYVMGLIKHFEGRLKD--GTLYVGWVDAKTNEPLDDKDVKAAYEKHILAHTGIRLIEPEIFNGYD2995 VVVTGYGEVGPYGHAETRWEMEAFGEFSLEGCVELAWVMGLIKHFNGSLPDT-GRHYTGWIDCASGEPVADTDVKARYEQQILAHSGIRLLEPELLEDQD
1116 PEKKEMIQEVIVEEDLEPFEAS-KETAEQFKHQHG-DKVDIFEIPE---TGEYSVKLLKGATLYIPKALRFDRLVAGQIPTGWNAKTYGISDDIISQVDP
685 PEKKGFIQEIELNHDLEPLEVA-AEEAARFKREHG-DKVDIWAQE----SGEWFVKFNKGARIFLPKAVKFDRVVAGQLPTGWDARRFGLPDDIIAQTDR3262 PNRKGFQQEIELNHDLEPFEVS-AEEASKYKLEHG-DKVDSWQDAN---TGGWFVVLKKGARIFVPKAVAFDRLVAGQLPTGWSGQRYGIPDDIVSQVDR3320 PKKKSILRQVAIAEDMKPIEVASLEEAQQYVNELGKEFIDVFNENVESNDGQWYIRLKAGAVVSIPGALSFNRFIAGQLPTGWDAKRLGIPDDIADSVDP
1983 PKRKGYTQEIELNHDLEAIETS-RADADKFKLQHG-DKVDVWLD-----GDKCFIRFKKNAKIMIPKAVRFDRLVAGQIPTGWDARVFGIPDDIIAQVDR
2168 PKRKGFTQEIEIQHDLEPIEAS-EEDAARFKREHG-ALVDVYTED----GSKFFVKFKKGAKLHIPKAVAFDRLVAGQIPTGWSHKAFGIPDDIASQVDR3094 PAVVPLMRELQLEADMDVFEAT-CDEAAAFKLRNG-DRVDVWENND----GSWSVRFLRGAVLMVPKALRFDRLVGGQLPTGWDPTRYGIPADVVDQVDM
1211 ITLFVLVSVVEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRGMFKDRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATSVE
779 TALWALVCTMEALIMSGVTDPYELYKYIHPSEVGTSLGSGMGGMHSMSAMFKDRREERDVQKDVLQETFINTVAGWVNLLLLSSSGPVKIPVGACATALQ3357 TTLWALVCASEALINSGVSDPYELYKHFHPSKVGSCIGSGMGGMTSLSKMFRDRRNDTDVQNDILQETFINSVAGWVNLLVMSAAGPIKTVVGACATALQ3420 VTLYALVSTVEALVCAGLTDPYELYQYVHVSQVGNTSGGGMGGMRSLKRMFLERKLDAEIPSDTLAESFINTMPAWVNMLLLSSSGPIKTPVGACATAAE
2076 TSLWALVCTAEALMMAGITDPYELYKYIHPSQVGTSLGSGMGGMSSLSKMFRDRREEKDVQKDILQETFINTVAGWVNLLLLSSSGPIKIPVGACATALQ
2262 TSLWALVSVAEALMMAGITDPYELYKWIHPSEVGSSLGSGMGGITSISKMFRDRREEKDVQKDILQETFINTVAGWVNLLLLSSSGPIKIPVGACATALQ3188 VTCYAIVATVEALVRAGITDPYELYQYFHVSQVGTALGSGAGGVHSIRDLYRRRLTDRAVQSDILQETFVNTTAAWINMLLLSSAGAIKPPTGGCGTSVL
1311 SVDIGVETILSGKARICIVGGYDDFQEEGSFEFGNMKATSNTLEEFEHGRTPAEMSRPATTTRNGFMEAQGAGIQIIMQADLALKMGVPIYGIVAMAATA
879 SVEIACDTILTGKAKVMIAGGFDDFSEEGSFEFANMKATSNAETEFAMGREPNEFSRPMTSTRSGFMESQGCGVHVMMSAKTAIEMGASIQGIVAYTSTH3457 SIDVAVETILSGKAELMLAGGFDDLSEEGSYEFANMKATSNGKEELAAGREPSEHSRPFSTSRSGFMESQGCGVQVLTTAANAIKLGMPIQSIVAYSNTH3520 SVDIGIETILAGKARVVIAGGYDDFCETGSNEFAMMGATSNSQTEAEKGRFPREASRPMTDTRAGFMESQGAGMQVLMDAELAIQMGLPVYGILALSNTA
2176 SIEIACDTILSGKAKVMIAGGFDDFDEEGSLEFANMQATSNTETELAAGREPNEMSRPTTSTRAGFMESQGCGVQVLMSAKTAIEIGASIYGIVAYTATA
2362 SVEIACDTILSGKAKIMVSGGYDDFSEEGSYEFANMKATSNSETEFAAGREPNEMSRPTTSTRAGFMESMGCGAQVLMSAKTAIEMGATIYGIVAYTATA3288 SVDVAADTIRAGKARVMLAGGFEGFVDQGSYEFAQMGATSSSVAELAAGRSPHSMSRPTTSTRAGFVEAQGAGVVVLMSASAAIECGAPIYAVLAYSGTA
1411 TDKIGRSVPAPGKGILTTAREHHSS--------VKYASPNLNMKYRKRQLVTREAQIKDWVENELEALKLEAEEIPSEDQ-------NEFLLERTREIHN
979 TDKAGRSIPAPGRGILSTAREVTP----------KEALPLLDIKYRSRQLAFRRKQISQWLENEHELLRMELETRKGGDN-------EEWFQNRVAFIDD3557 TDGAGRSLPAPGHGVIGSADG-------------------------------------------------------------------------------3620 TDRQGRSVPAPGRGILTTAREVRSKNSKSSHNGKPVENPLLSVKFRKKHLRQELDAIDLWASQEISNMSEEFSQNSRKME------------NRKGFVET
2276 TDKAGRSVPAPGRGVLSTAREAPG----------KVPAPILDIEWRKRQLAFRRMQISQWLDNEVDIFKSMVSNLEKAGQP----MPADEIAERYAAIEK
2462 TDKAGRSIPAPGRGVMGTAREITS----------KYPSPILDVTYRRRQLEFRRKQISQWLENETELLKFEVSSHGQATK-----LPDDYVSERLASIER3388 TDKQGLSLPAPGLGVLTSAAQRCTAN---------SVPCTLDISYRRRQINRAFDDAERWADDERALAQENADAMAQNDDTTDHTSVNAFLAEQHALIDT
834 GGDGMYSESKLSLETLFNRWHSESWANQLTVCGAIIGWTRGTGLMSANNIIAEGIEKMG--VRTFSQKEMAFNLLGLLTPEVVELCQKSPVMADLNGGLQ
404 GNDGLYSESKISLETLFNRWSAESWGEYLCIAGAVIGWTRGTGLMSATNFVAEGLEKLG--VRTFSAKEMAFNILGLMHPLLFDITQIEPIWADLNGGMD2981 GGDGLYGESKLGLETLLNRWESESWGQYLCITGAIIGWCRGTGLMSSSNTVAEEIEKLG--CRTFSTTEMAFNLLGLLAPTVSAVSQVEALLVDLNGGMD
18
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
2362 EAKRQEKEAQSTFG-MLSGDDPEVAPLRRALAVWGLNIDDIG--VASFHGTSTKANDKNESSVYNQQFQHLGRSRGNAVPVVAQKWLTGHPKGGAAAWMM
2547 EAKRQEAEALATYG-MLAGQDPTIAPLRRALAVWGLTIDDVGTVALQFHGTSTVANDKNESNAYNEQFRHLGRAKGNACPVIAQKWLTGHPKGGAAAWML3479 KLAERRASALDEWGSDFWQRLPEVAPLRGALAVWGLSADDIG--IASFHGTATVANDRNECRVLSSQLRHIGRSSGLAVPAVCQKWLTGHPKGPAAAWML
1594 NGALQILNSGIIPGNRNADNVDKILEQFEYVLYPSKTLKTDG--VRAVSITSFGFGQKGGQAIVVHPDYLYGAITEDRYNEYVAKVSAREKSAYKFFHNG
1159 NGMCQTINSALVPGNHNADNISEELRAFRHLFYPSKPIQHVR--LECGLLTSFGFGQVGGQVAIVHPRYLFAALQTHELEAYKKRRQDRELNTYSRMSSA3650 NGVMQALHTSIVCGNRNADDISPELQKFDMLLYNSTSIHRTPHHVNAAAVSSFGFGQVGGIILVLHAAHILGRLSDKAFEEYVARRKARQHQTYTRMHSA3806 NGLLQCMNSGRVPGNRNLDNVDGKLRTNGYLFYPNRTIEVPR--VEAAFLKSFGFGQAGAEVVIIHPDRLLAVLSEENLKSYILRRNEREKRAYRYHQGV
2459 CGVTQAIQDGIIPGNRNADNIGPELQEFEFLVYPSKSIQTDG--IKAGLLTSFGFGQVGGQVLVVHPDYLLAAIEPAEYESYKSKRFVRERASYRKFNDF
2646 NGLAQVIQSGLVPGNRNADNIGEELRAFEYLLYPSKSIQTDG--IKAGLLTSFGFGQVGGQALIVHPSLLIGALEPAQFEAYKKLNDQRKKWSYRRFNDF3577 NGAIQSLRTGIVPGNRNADDIDEELRQHDLIVFPSRSFQTPI--VKAVLLKSFGFGQVGAEMLVVHPDYLLATLDEQSLDKYNSRMKKRELSAYRYWQDT
1692 MI-YNKLFVSKEHAPYTDELEEDVYLDPLARVSKDKKSGSLTFNSKN------------------------------IQSKDSYINANTIETAKMIENMT
1257 LV-NNNMVQIKDGPPYTAELEGSVLLNPLARAGPSKN-SFAFQGKLP------------------------------TKVPLDIKNAETLKAMFDQAG--3750 LT-KEDLVRIKDNRPWPAELEDQVLMNLNARAVEIGD-SYGFKAPLPP-------------------------MPALATVEDTIVPAKPANVAAQQENLE3904 MSGHHTMVQVKEFAPYEDDILEEIYLNPRARASFDSSKSTWTFHKYRNDKDTVDDKDEMEQDSLNESVDLPKDITKVGSPSEIRIPVKTRLEVTVREGVE
2557 LT-KRSLVILKETPPYIPELEPHVLLNPLARASKDSTGSYAYPKKPD------------------------------HLPTKVNIATKTASLAATITQKY
2744 FT-NGKLVIIKDGTPFTPEQENTTLLNPLVRAVPDKTGSYSMPKEFP------------------------------ATVPRSNNAEVANKLVSAAVG--3675 FAENHPFVQVKSAPPYTSKQEVDVLLDPLARAQYDPATSQYHF
1760 ----KEKVSNGGVGVDVELITSINVE--NDTFIERNFTPQEIEYCSAQPSVQSSFAGTWSAKEAVFKSLGVKS--------LGGGAALKDIEIVRVNKNA
1322 --------ALSGVGVDTELISNVPT---SETFRERNFTADEISYCNSAADPTASFAGRWAAKEAVFKALSVPS--------KGAGAPLKEIEIVSTSS-G3822 ----KMMGHVAGVGIDCEKISTFPSD--NDNFVSRNFTDVEIEYCRAQPDPRASFCGRFCAAEAVVKSLGVRT--------QGAGASLKDVEIVPTPE-G4004 GLTKKDKFSSQGVGVDVEPVSTFAQHEEKTIFIQNNFTENEQLYCNHAASPAASYAGRWAAKEAVIKAISNSSLETRSLW-QGAEGKLIDIEIIQSNSGA
2625 ----ENEDSVFGVGTDVEMITAVPQ---SDVFLERNFTEQELAYCRQSSDFNASLAGKWTAKEAAFKAMKTLS--------KGAGAAMKEIEILSGPS-G
2810 --------GAFGVGTDVELISAVPT---SESFLERNFTQDEIAYCKAAPDFRASLAARWSAKEATFKALKTES--------KGAAASMQDIEVVSTSQ-G
1 MTSSGIGVDIELIQDINIEN--DTFIERNFTIREQEYCQSRPNPHASFAGKWCAKEAVIKAVSSLNLDCVKVWTKGDAAPLDEIEINIANSGA
1847 PAVELHGNAKKAAEEAGVTDVKVSISHDDLQAVAVAVSTKK-
1403 PTVKLSGDALAAAGG---KSVKVSLSHSDTSVVAFAVAQ---3908 PQVKLYGEALKASNG---SRFLVSISHGDDTAMAIVHRLPSA4103 PEVVLHGHAKEVFQTLGLTNVKVSISHTPEVAVAQAITN---
2710 PEIKLTGQASKVADEKGIKNFELSISHSDEVAMAFVVARH--
2891 PTIKLHGEVEKIAQAAGITAFEVSLSHSEDVACAVVIAQK--
92 PEVVLYGDAKEACAKAGVSSIKVSISHIDDFAIATAITS---
1496 EAESQLRAAQQQWGNDFYKRDPRIAPLRGALATYGLTIDDLG--VASFHGTSTKANDKNESATINEMMKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMM
1062 EARRQEKDALATFG-MLEGSHPNIAPLRRALAVWGLNADSVG--AISCHGTSTKANDKNESGVYNLQFEQLGRTPGNAVPVIAQKSLTGHPKGGAAAWMF3577 --------------------------MQRALARFNLTADDIG--VISAHMTATKANDKNESHVYQELFTRMGRTPGHAVPVMAQKWLCGHAKGGAAAWAL3708 MRKKKRSAAFETWGQGFYRNDDSIAPLRGALSVWGLTVDDLL--VGSFHGTGTNLNDTNESSLVNKQLKHLDRKEGNILLVVTQKYLTGHPKGAAAAWML
C9
Supplementary Figure 1. Sequence alignment of fungal type I FAS variants. Seven FAS representa�ves including FAS fromS. cerevisiae JGenbank accession number6 NP_yCR739hC and NP_yC5y93hCI6 C. neoformans JGenbank accession number6XP_57CCyyhC and XP_57Cy99hCI6 U. maydis JGenbank accession number6 XP_759CC8hCI6 A. kerguelense JJGI protein ID6Cy395CI6 P. graminis JGenbank accession number6 XP_yy33R4647hR and XP_yy3889657hCI6 R. toruloides JGenbank accessionnumber6 XP_yC6R7RR8yhC and XP_yC6R7R387hCI and C. reversa JJGI protein ID6 Cy86y and 86596I were shownh The domainborders were indicated by bold lines of different colors according to Fig. 1ah Cataly�c residues were indicated by trianglesh
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
ScFas1ScFas2CnFas1CnFas2UmFasAkFasPgFas1PgFas2RtFas1RtFas2CrFasCrPPT
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
ScACP Fas2 142CnACP Fas1 2217UmACP Fas 2313AkACPI Fas 2112AkACPII Fas 2302PgACP Fas2 1031RtACPI Fas2 1215RtACPII Fas2 1024CrACP Fas 2199
ScACP Fas2 239CnACP Fas1 2313UmACP Fas 2410AkACPI Fas 2211AkACPII Fas 2399PgACP Fas2 1128RtACPI Fas2 1121RtACPII Fas2 1312CrACP Fas 2299
DEPVKASLLLHVLVAHKLKKSLDSIPMSKTIKDLVGGKSTVQNEILGDLGKEFG---TTPEKPEETPLEELAETFQDTFSGALGKQSSSLLSRLISSKMPDVPPKAVDTVRIIVAQKLKKQVSEVPLSKSLKELSGGKSTLQNEILGDLQVEFA---SAPEKGEDLPLDELGAALSVGYA-ALGKHSMALTNRMVASKFPDEPLKAVDTVRAILAQKLKKNISEVPLSKSIKDSVGGKSVLSNELVSDLLAEFG---NLPERGEELPLEELGSALQAGYTGTLGKTTSNLVTRTVAGKLPEVTNRPLHVLRVLISTKLKKKMLEVKATDTIKGVSGGKSAIQNELIGEINAEYSSFRDIEDIS-EVSLAELARRAPSRQETVLGKVTTKMVNKMFSSKLPEEPVNSKHVLLSLISMKLQKEISSINETSTIKSLVNGKSAVQNEILGEIASEFRTD-ESEGAA-DISLSELGEKLGIG-YAGPGSVVSRHINKMLNSKLPDEPLKAVETLRVLVAQGLKKPLDDVPLSKAIKDLVGGKSTLQNELLGSAQAEFG---SAPDKAEEMPLGELGSALQSSYSGTMGKHMSALVSRLIGSKMPDEPLKAVDTLRIIIAQKLKKPVGEVPLTKSIKELVGGKSTLQNEILGDLQGEFS---SAPEKGEEMPLQELGAALQQGYSGKLGKYTTGVISRMIGAKMPDEPLKATDTLRAIIAQKLKKQIPDVPLTKSIKDLVGGKSTLQNEILGDLQGEFS---SAPEKGEEMPLQELGAALNQGYSGTLGKHTSGLVARMMGAKMPDIPLQAVDVIRAVIAQKIKRTVNEVAADFSVKQLTGGKSTLQNEILGDLLKEFGGSGQIPDRPDEISLQELATGMGASFTGALGKHTQAQVSRLFSAKMP
GGFTITVARKYLQTRWGLP-SGRQDGVLLVALSNEPAARLGSEADAKAFLDSMAQKYASIVGVDLSSAGGFNITAARAHLNKQWGLG-PLRTDSALFFGILNEPPKRLGSEAEAKAFLDQLAQSYASYSGISLSSGGGFNLSAVKSHFQKAWGLG-AGRTDGALLVAVTMEPAKRLGSEAEAKTWLDSVAQTYAQLAGISLAQAGKFGPNDTKEYLQSKLSDA--STVDSVSLHALTLQPVSRLATPGEAQAWLDSVCRDFENDQRISILKNGSFNSSSAKKYLSMQGFGK--GRSDSVLVHALTMQPKSRLQGVEDAKSWLDSVCSSYGKWANIDLTVSGGFGMSGVKSHLSKSWGLG-PGRTEGVLLMALTMEPPKRLGSEGEAKSYLDSVVQAYGQHSGLTLSQGGGFGLSAVQGHLGKTYGLG-AGRIDGVLLFAVTQEPAKRLANEGEAKAWVDSVAQGYASMAGISLAAGGGFGLSAAKAHLSKAHGLG-PGRTDGALLVALTKEPEKRLGSEADAKAWLDGVAQAYASQAGITLGAGGGFSQSAARTHLATQLGVGRTHQQDAVLLLALTMEPSARLSSE-EAPTWLDSVATAYATTHGITLAEP
R-
Supplementary Figure 2. Sequence alignment of ACP domains from fungal FASs. The sequences include ScACP from FasR ofS. cerevisiae JGenbank accession number6 NP_-S5-93vSp6 CnACP from FasS of C. neoformans JGenbank accession number6XP_57SS--vSp6 UmACP from Fas of U. maydis JGenbank accession number6 XP_759SS8vSp6 AkACPI and AkACPII from Fas of A.kerguelense JJGI protein ID6 S-395Sp6 PgACP from FasR of P. graminis JGenbank accession number6 XP_--3889657vSp6 RtACPIand RtACPII from FasR of R. toruloides JGenbank accession number6 XP_-S6R7R387vSp6 CrACP from Fas of C. reversa JJGIprotein ID6 S-86-pv Numbers le� to the sequences indicate the posi�on of amino acid residuesv And the black triangleindicates the conserved serine residues phosphopantetheinylated in hologACPsv
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
wRtACPI Fas2 1057-LVGGKSTLQNEILGDL-1072wRtACPII Fas2 1248-LVGGKSTLQNEILGDL-1263AkACPI Fas 2145-VSGGKSAIQNELIGEI-2160AkACPII Fas 2335-LVNGKSAVQNEILGEI-2350ScACP Fas2 175-LVGGKSTVQNEILGDL-190mRtACPI Fas2 1057-LVGGKATLQNEILGDL-1072mRtACPII Fas2 1248-LVGGKATLQNEILGDL-1263
GST-wRtACPII + + + +
GST-mRtACPII — — — —
CoA + — + —
GST-RtPPT + + — —
GST-ACP
GST moiety
ACP moiety
GST-ACP
GST moiety
ACP moiety
— — — —
+ + + +
+ + — —
+ — + —
GST-wRtACPI + + + +
GST-mRtACPI — — — —
CoA + + — —
GST-RtPPT + — + —
— — — —
+ + + +
+ + — —
+ — + —
b
c
CoA
HO
Apo-ACP
O
Holo-ACP
Phosphopantetheine moiety
3', 5' – ADP
+
+
PPT
a
S
S
Supplementary Figure 3. In vitro phosphopantetheinylation of ACP domains from R. toroloides FAS by cognate PPT domain.6a72Phosphopantetheinylation2reaction2catalyzed2by2PPT92The2phosphopantetheine2moiety2of2coenzyme2A26CoA72is2transferredto2the2hydroxyl2group2of2serine2residue2in2ACP926b72Phosphopantetheine2attachment2site2sequences2in2ACPs92The2sequencesare2extracted2from2Fasv2of2R. toruloides26Genbank2acession2numberN2XP_ID4vwvS%w9D7N2Fasv2of2S. cerevisae26Genbankaceesion2numberN2NP_IDEIRS9D72and2Fas2of2A. kerguelense26JGI2protein2IDN2DISRED792Numbers2indicate2the2position2incorresponding2protein2sequencesN2and2conserved2serine2residues2for2phosphopantetheinyl2attachment2are2highlight2in2bold9In2mRtACPI2and2mRtACPIIN2these2serine2residues2are2mutated2to2alanine2residues926c72SDS5PAGE2analysis2of2thephosphopantetheinylation2products92RtACPI26RtFasvN2DIvv5DD%-7N2RtACPII26RtFasvN2DvDS5DSwE72and2RtPPT26RtFasvN2v%IR5vRv%7were2expressed2and2purified2as2GST2fusion2proteins92The2phosphopantetheinylation2reaction2mixture2was2treated2withenterokinase2and2then2separated2by2D4_2Tricine5SDS5PAGE92The2phosphopantetheinylated2ACPs2with2slightly2higher2molecularweight2than2the2apo5ACPs2were2shown2by2arrows9
vD
Supplementary2Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary Figure 4. In vitro enzyme activity assays of the purified RtFAS complexes.3PaG3Recombinant3plasmids3forexpression3of3RtFAS43and3RtFAS2C3In3the3S4H62A3and3S4253A3mutantsv3the3corresponding3serine3residues3were3mutated3toalanine3residues3by3siteMdirected3mutationC3PbG3SDSMPAGE3analysis3of3the3purified3RtFAS3complexv3two3subunits3were3separatedas3indicatedC3PcG3Enzymatic3activity3assays3of3the3purified3RtFAS3and3its3mutantsC3MalonylMCoA3and3acetylMCoA3dependentNADPH3oxidation3was3monitored3by3decrease3of3absorption3at334H3nmC3Specific3activities3of3each3protein3complexes3areshownC
Time (S)
WT 129 mU/mg S1062A 106 mU/mg S1253A 124 mU/mg
Abs
orba
nce
(340
nm
)
a
c
b
pET24b-RtFAS2
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTT
AAGAAGGAGATATACATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCGAATTCGAGCTCC
GTCGACAAGCTTatg...tcg...tcg...aagGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGAnnnnnnnnnnnnatg...gcg...tcg...aag--S1062Annnnnnnnnnnnatg...tcg...gcg...aag--S1253ATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
RtFAS2 His6-tag
pET22b-RtFAS1TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTT
AAGAAGGAGATATACATATGgcaagctggagccacccgcagttcgaaaagggtgcaatg…tgaGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
RtFAS1Strep(II)-tag
T7npromoter lacnoperator
RBS
T7nterminator
Fas2
Fas1
10
Supplementary3Results
22
Nature Chemical Biology: doi:10.1038/nchembio.2301
RtFAS02
RtFAS01
RtFAS03
RtFAS04
RtFAS05
pYX212
SC-URA SC-URA+FA
a
b
A
kFA
S01
AkFA
S0
2
AkFAS03 A
kFAS
04 AkF
AS
05c
S
cF
AS
01
ScFAS09 ScFAS10
ScFA
S11
d
ScFA
S01
ScFA
S14
ScFAS15 S
cFAS
16 ScF
AS
17
S
cF
AS
01
S
cFAS23 ScFAS26 ScFA
S25
e
f0.0
0.2
0.4
0.6
0.8
1.0
0 10 20 30 40 50
OD
XRea
dXfro
mXB
ioS
cree
n
TimeX(h)
RtFAS05
RtFAS04
RtFAS03
RtFAS02
RtFAS01
ScFAS01
pYX212
Supplementary Figure 5. The complementation of ScFAS1 and ScFAS2 deletion in S. cerevisae PWY12 by different FASs andtheir mutants.vmauvGrowthvofvPWYM3vstrainvexpressingvindicatedvRtFASsvorvharboringvblankvvectorvpYX3M3vonvplatesvwithvfattyacidsvmSC/URA0FAvmediumuvorvwithoutvfattyvacidsvmSC/URAvmediumuLvmbuvGrowthvcurvesvofvthevPWYM3vstrainvexpressingindicatedvFASsvorvharboringvblankvvectorvpYX3M3vinv3BBvμlvDelftvmediavwithvMBBvmgTLvtryptophanLvThevcellsvgrownvinhoneycombvplatevwerevmonitoredvbyvBioscreenvCvMBRvinstrumentLvmcu/mfuvGrowthvofvPWYM3vexpressingvindicatedvFASsvonplatesvwithoutvfattyvacidsvmSC/URAuL
33
SupplementaryvResults
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary,Figure,6.,Schematic,presentation,of,ScFAS,with,embedded,(ScFAS14,and,ScFAS15),,peripheral,(ScFAS16),orfree,(ScFAS17),sTEs.
24
AT
ER DH
MPT
KSKR
PPTACP
ScFAS01
AT
ER DH
MPT
KSKR
PPTACP TE
ScFAS14ScFAS15
AT
ER DH
MPT
KSKR
PPTACP TE
ScFAS16
AT
ER DH
MPT
KSKR
PPTACP
TE
TE
TE
TE
ScFAS17
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
Supplementary2Figure27.2Production2of2S/MCFAs2in2YJZ022integrated2with2engineered2ScFASs.gkavgS8MCFAgproductionginZW7O,gstraingkYJZO7gwithgagchromosomegintegratedgScFASO,vgwithgagplasmidgexpressingg‘AcTesAgkpZWM,L‘AcTesAvgandZW7OKgstraingkYJZO7gwithgagchromosomegintegratedgScFAS,jvgwithgagblankgvectorgkpZWM,v4gCellsgweregculturedgingDelftmediumgsupplementedgwithg,OOgmg8LghistidinegforgH3ghours4gkbvgS8MCFAgproductiongandgopticalgdensitiesgkODvgofgstrainsZW7O,fgZW7OKfgZW7O7gkYJZO7gwithgagchromosomegintegratedgScFAS7±vgandgZW7O±gkYJZO7gwithgagchromosomegintegratedScFAS73v4gCellsgwereggrowngingDelftgmediumgsupplementedgwithg,OOgmg8Lghistidinegandg,OOgmg8LguracilgforgH3fg±7gandg–Khours4gkcvgpHgadjustedgculturegofgZW7O±gingDelftgmediumgsupplementedgwithg,OOgmg8Lghistidinegandg,OOgmg8Lguracil4gThearrowgindicatesgthegadditiongofgsterilegKOHgsolutiongk7gMvgtogchangegthegpHgtogK4O4gkdvgS8MCFAgproductiongofgZW7O±gingpHunadjustedgandgpHgadjustedgculturesgasgshowngingkcv4gMeang±gSDgofg9g–gHgindependentgculturesgaregpresented4
7j
4
5
6
7
8
9
10
11
12
0
1
2
3
4
5
6
7
8
0 24 48 72 96
OD, pH-UnadjustedOD, pH-AdjustedpHpH, pH-UnadjustedpH, pH-Adjusted
OD
pH
Time2WhM
C6 C8 C10 C12
0
5
10
15E
xtra
cellu
lar2
Fat
ty2A
cids
2Wm
gCLM
a
0
3
6
9
12
15
0
25
50
75
100
125C6 C8 C10 C12 OD
Ext
race
llula
r2F
atty
2Aci
dsWm
gCLM
OD
482h
722h
962h
482h
722h
962h
482h
722h
962h
482h
722h
962h
b
c
0
50
100
150
200
pHUnadjusted
pHAdjusted
Ext
race
llula
r2F
atty
2Aci
dsWm
gCLM
C6 C8 C10 C12
d
PlasmidFAS2intergrated
Host2strain
pZWM15‘AcTesAScFAS01ZW201
pZWM1ScFAS15ZW206
FAS2intergratedHost2strain
ScFAS01ZW201
ScFAS15ZW206
ScFAS27ZW202
ScFAS28ZW207
SupplementarygResults
Nature Chemical Biology: doi:10.1038/nchembio.2301
0
10
20
30
40
7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12
ScFAS01 ScFAS23
2-Undecanone 12-Tridecanone 2-Pentadecanone
H111111131111111C1111111 C1111111H111111131111111
O1111111
O1111111
O1111111
C1111111H111111131111111
H111111131111111C1111111
C1111111H111111131111111
O1111111
H111111131111111C1111111
O1111111
O1111111
C1111111H111111131111111
H111111131111111C1111111
C1111111H111111131111111
O1111111
H111111131111111C1111111
O1111111
O1111111
C1111111H111111131111111
H111111131111111C1111111
Decanoic1acid,1methyl1ester
Dodecanoic1acid,1methyl1esterTetradecanoic1acid,1methyl1ester
0
10
20
30
7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12
ScFAS25 ScFAS26
2-Undecanone
12-Tridecanone
2-Pentadecanone
Tetradecanoic1acid,1methyl1esterDodecanoic1acid,1methyl1ester
Decanoic1acid,1methyl1ester
Retention1time16min)
Retention1time16min)
Inte
nsity
16H
)1re
lativ
e1to
12-O
ctad
ecan
one1
6IS
)In
tens
ity16
H)1
rela
tive1
to12
-Oct
adec
anon
e16I
S)
SupplementaryYFigureY8.YGC-MSYanalysisYofYtotalYmethylYketonesYandYfattyYacidsYproducedYbyYPWY12YstrainYexpressingYtheScFASsYasYindicated.)Increased)production)of)2-undecanone,)2-tridecanone)and)2-pentadecanone)were)found)in)strainexpressing)ScFASs)containing)embedded)Mks2)lScFAS23)and)ScFAS25R.)Moreover,)the)formation)of)corresponding)fatty)acidsldodecanoic)acid)and)tetradecanoic)acidR)decreased)in)ScFAS23)and)ScFAS25)compared)with)ScFAS01)and)ScFAS26,)respectively.
26
Supplementary)Results
Nature Chemical Biology: doi:10.1038/nchembio.2301
TDH(p
m((l myxy muul g(ux
pRtFASrl
pRtFASrx
pRtFASr(
pRtFASrG
pRtFASr)
pAkFASrl
pAkFASrx
pAkFASr(
pAkFASrG
pAkFASr)
pScFASrl
pScFASru
pScFASlr
pScFASll
pScFASrl
pScFASlG
pScFASl)
pScFASlm
pScFASlg
pScFASrl
pScFASxg
pScFASl)
pScFASxy
pScFASrl
pScFASxm
pScFASx(
pScFASx)
RtFASx
RtFASx
RtFASx
RtFASx
RtFASx
RtFASl
RtFASl
RtFASl
RtFASl
RtFASl
AkFAS
AkFAS
AkFAS
AkFAS
AkFAS
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASx
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
ScFASl
(rml ())) (m(G Glxy
Gxl ul)
f
f
a
pZWMlv'AcTesA lwkb
ZWxrl
ZWxrx
ZWxrm
ZWxrg
ScFASx
ScFASx
ScFASx
ScFASx
ScFASl
ScFASl
ScFASl
ScFASl
KanMX
KanMX
KanMX
KanMX
DwDw
Dw
Dw
Dw
Up
Up
Up
Up
f
f
TEFlp
TPIlp
ADHlt
FBAlt
CYClt
ACPwofwR. toruloideswFAS
ACPwofwA. kerguelensewFAS
ACPwofwS. cerevisiaewFAS
InactivatedwACP
LinkerwbetweenwtwowACPswofwRtFAS
FlexiablewlinkerwYGGGGSo(
'AcTesA
ShMksx
ShMksl
Up
Dw
Homologywarmwupstreamwtowura3-52
Homologywarmwdownstreamwtowura3-52
LoxPwsite f Glx)rSiwSlx)lWwmutations
b
c
Supplementary Figure 9. Schematic illustration of the expression cassettes.2(a)2FAS2expression2cassettes2in2plasmids.2Thenumbers2indicate2position2in2the2gene2sequences.2(b)2Integrative2expression2cassettes2of2ScFASs.2(c)2Expression2cassette2of2‘AcTesA.
27
Supplementary2Results
Nature Chemical Biology: doi:10.1038/nchembio.2301