anding the product portfolio of fungal fatty acid synthases · pdf filealbugo laibachii...

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

mailto:[email protected]

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



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.



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



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Phytophthora capsici 537721

Schizochytrium aggregatum 86957

Aurantiochytrium limacinum 83486

Aurantiochytrium mangrovei AKV56231.1 A0A0K1R5P4

Aplanochytrium kerguelense 103951

Meredithblackwellia eburnea 311302, 346975



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



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



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



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



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



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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



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



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


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

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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


519 ---KDGTGVRVIVAGTLDINPDDDYGFKQEIFDVT--SNGLKKNPNWLEEYHPKLIK-------NKSGKIFVETKFSKLIGRPPLLVPGMTPCT--VSPD

14









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










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










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










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










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








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



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


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


22


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


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



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


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


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



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