Supporting Information

Subtle control of carbon chain length in polyunsaturated fatty acid

synthases

Mai Naka1, Kenshin Ikeuchi1, Shohei Hayashi1, Yasuharu Satoh2, Yasushi Ogasawara2, and Tohru

Dairi2*

1
Graduate School of Chemical Sciences and Engineering, Hokkaido University, N13-W8, Kita-ku,

Sapporo 060-8628, Japan

2
Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo 060-8628,

Japan

*Corresponding author: Tel: +81-11-706-7815, E-mail: dairi@eng.hokudai.ac.jp

1
Table of Contents

Methods

Supporting Information Figures

Figure S1. PUFA biosynthetic pathway of PUFA synthases.
Figure S2. Sequence alignments of Epa-C and Dha-C in PUFA synthases.
Figure S3. Modelled structure of the KSC domain in Dha-C.
Figure S4. Modelled structure of the CLF-like domain in Dha-C.
Figure S5. GC-MS analysis (traced at m/z 79) of products produced by E. coli expressing
Dha-ABD and KSC domain mutants of Dha-C.
Figure S6. Preparation of truncated recombinant KSC domain, CLF-like domain, and
KSC/CLF-like domain of EPA synthase.

Supporting Information Tables

Table S1. Primers used in this study

References

2
Methods
Plasmid constructions.
PCR primers used for construction of plasmids were shown in Table S1.

Plasmid construction of pCDF-dha-C_cassette.

For easy constructions of the mutated dha-C domain gene, we first constructed a plasmid
carrying three artificial restriction enzyme sites without amino acid changes in the translated
enzymes. NcoI and SpeI sites were introduced at 5ʹ and 3ʹ terminal sites of KSC and CLF-like
domains. A SacII site was created at a boundary site between KSC and CLF-like domains. First,
DNA fragments carrying KSC gene (fragment A), CLF-like gene (fragment B), and downstream
region of CLF-like gene (fragment C) were amplified with pCDF-dha-C [1] as template and primers,
FE1/FE2, FE3/FE4, and FE5/FE6. Then, DNA fragments carrying KSC and CLF-like genes
(fragment D) were assembled by overlap extension PCR with fragment A/B as templates and
primers, FE01/FE04. Finally, DNA fragments carrying KSC gene, CLF-like gene, and downstream
region of CLF-like gene were amplified by overlap extension PCR with fragments C/D as templates
and primers, FE01/FE06. After digestion with NcoI and EcoRI, the fragments were inserted into
the same sites of pCDF-dha-C to construct pCDF-dha-C_cassette.

Plasmid construction of pCDF_dha-C_KSC_mutants

Plasmids carrying the mutated KSC genes were constructed as follows. DNA fragments were
amplified with pCDF-dha-C_cassette as template and primers, FE1 and six kinds of FM_Rvs, each
of which was designed to introduce each amino acid substitution (Table S1). DNA fragments
carrying remaining KSC genes were amplified with pCDF-dha-C_cassette as template and primers,
FE2 and six kinds of FM_Fws. The thus obtained two fragments were used as templates for
overlap extension PCR with primers, FE1 and FE2. After digestion with NcoI and SacII, the
amplified fragments were inserted into the same sites of pCDF-dha-C_cassette to construct
pCDF-dha-C_KSC_mutants.

Plasmid construction of pCDF-dha-C_CLF_mutants

Plasmids carrying the mutated CLF-like genes were constructed by the same strategy as
those of pCDF-dha-C_KSC_mutants. DNA fragments carrying the mutations and the parental
sequences were amplified with FE3/six kinds of FM_Rvs and six kinds of FM_Fws/FE4 primers.

3
The amplified two fragments were used as templates for overlap extension PCR with primers, FE3
and FE4. After digestion with SacII and SpeI, the amplified fragments were inserted into the same
sites of pCDF-dha-C_cassette to construct pCDF-dha-C_CLF_mutants.

Plasmid construction of pET-epa-C_KSC and pET-epa-C_CLF

Truncated KSC and CLF-like genes of epa-C from Photobacterium profundum were
amplified by PCR using pCDF-epa-C [2] as template and gene-specific primers (Table S1). The
DNA fragments digested with NdeI and BamHI were inserted into the same sites of the pET28
vector (Merck KGaA, Darmstadt, Germany) to construct pET-epa-C_KSC and pET-epa-C_CLF.

Plasmid construction of pCDF-epa-C_KSC and pCDF-epa-C_CLF

Truncated KSC and CLF-like genes of epa-C from P. profundum were amplified by PCR
using pCDF-epa-C [2] as template and gene-specific primers (Table S1). The DNA fragments
digested with NdeI and BamHI were inserted into the same sites of the pCDF-orfB (a derivative of
pCDF1b vector (Merck), CloDF13 ori, T7 promoter, streptomycin resistance gene) [3] to construct
pCDF-epa-C_KSC and pCDF-epa-C_CLF.

4
 MAT
O O HOOC
SH
HO S
ACP DHA C22:6
KS ACP
HOOC

O O
EPA C20:5
R S CO 2
O O O
R
ACP R S R S S

ACP ACP ACP

NADPH
NADP +
KR
ER
NADP + NADPH

O
OH O
R S
R S

ACP
ACP

H 2O

DHFabA or DHPKS

Figure S1. PUFA biosynthetic pathway of PUFA synthases.

5
Sone MPKIAIVGLAVQYPDADTPEQFWQNLLDKKDSRSQIDAAKLNANPADYQGIQGQADRFYC 87
Swoo QSKIAIVGLATLYPDAKTPGEFWQNLLDKRDSRTTLTNKKLGANSCDYQGVQGQSDRFYC 70
Spie QSKIAIVGLATLYPDANSPQQFWQNLLEKRDSRSTLTDKKLGANSADYQGVQGESDRFYC 71
Vtap VGKIAIVGLANQYPDADTPNDFWQNLINKKDSRSTLTNEKLGANTEAYQGTQGQSDRFYC 78
Pss9 SNKIAIVGLANQYPDADTPKDFWQNLLAKKDSRTTLSPDKLGANPDAYQGIQGESDRFYC 118
Vtas CNKIAIVGIANQYPEADTPKDFWQNLLDKKDSRTTLSAEKLGAKPENYQGVQGESDRFYC 77
Cpsy MENIAVVGIANLFPGSSAPEEFWQQLLKKQDCRSKATKEQMGVDPEKYTGKKGDTDKFYC 60
C_sp MENIAVVGIANLFPGSSAPEEFWQKLLKKQDCRSQATKEQMGVDPAKYTGKKGDTDKFYC 60
Mmar MENIAVVGIANLFPGSQAPDQFWQQLLEQQDCRSKATAVQMGVDPAKYTANKGDTDKFYC 60
Mvis MENIAVVGIANLFPGSQAPDQFWQQLLEQQDCRSKATAVQMGVDPAKYTANKGDTDKFYC 60
Ping MEDIAVVGIANLFPGSDTPETFWQQLLKKQDNRSQITKRELGVEPQKYYGRKGDMDKFYC 60
P_sp MENIAIVGIANLFPGSSQPDAFWEQLLNKQDLRSKITEQEMGVDPLEYLGKKGDVDKFYC 60
.**:**:* :* :. * **::*: ::* *: ::... * . :*: *:***

Sone DKGGYIRNFRFDPQGY---------QLLPATFAGLDESFLWALDCSKKALLNAGVDLTA- 137

Swoo DKGGYIENFHFDATGY---------QLPSESLTGLDDSFLWALDTSRKALQDAGINLGSA 121
Spie DKGGYIENFSFDPSGY---------KLAADSLESLDNSFLWALDTSRKALDDAGIALNND 122
Vtap DQGGYIKDFNFDANGY---------KVPAEQFSGLDDSFLWALDTSRKALLDAGINLDA- 128
Pss9 DKGGYIQNFSFDSNGY---------RLPAETFEGLDESFLWALDTSRKALADAGIPLDD- 168
Vtas DKGGYIENFNFDSNGY---------RLTAESFNGVDQSFLWALDTSRKALVDAGIDLNA- 127
Cpsy VHGGYIRDFNFDATSFIQNTAGLTAPLSEEYLNQLDDLNKWALYVTQQALTDAGYWGS-- 118
C_sp VQGGYIRGFDFDATSFVQAKNGLT----ADYLNQLDDLNQWALYVTQQALTDAGYWRS-- 114
Mmar VHGGYISDFNFDASGY---------QLDNDYLAGLDDLNQWGLYVTKQALTDAGYWGS-- 109
Mvis VHGGYISDFNFDAAGY---------QLDSSYLTELDDLNKWGLYVTKQALADAGYWGC-- 109
Ping MYGGYIRDFEFDSGPFISK------GLNETYLNQLDSLHQWGLYVTYQALIDAGYWGS-- 112
P_sp MYGGYIRNFNFDVSSFVDH------GFDADYLMGLDELNKWGLYVTEQALKNAGYWGS-- 112
**** .* ** : : :*. *.* : :** :**

Sone PLLERTGIVMGTLSFPTARSNELFLPIYHQAVEKALKTKLNQPQFALAPFANASI----A 193

Swoo -SLARTGIIMGALSFPTTRSNDLFLPIYHSAVEKALQDKLGASAFKLNPTNAHTSRAQGE 180
Spie DLLARTGVIMGALSFPTERSNDLFLPIYHSAVEKALQDKLGSQNFKLSATNAASPRATNE 182
Vtap DILARTGVIMGALSFPTTASNDLFLPIYHSVVEKALQTKLGNSSFQLNPSNGN------V 182
Pss9 AVLERTGVIMGALSFPTKRSNDLFLPIYHSAVEKALQTKLGNEHFTLTPSNAN------I 222
Vtas DVLERTGVIMGALSFPTTRSNDLFLPIYHSVVEKAFKDKLANDQFSLLPTNET------A 181
Cpsy DKLEQCGVILGNLSFPTKSSNHLFMPLYHQVVDNALKAGID-KDFQLSHFSDTDI---ST 174
C_sp DKLAQCGVILGNLSFPTKSSNHLFMPLYHQVVDSALKNILH-KDFQLSHFSPLQQ-ANLA 172
Mmar TALENCGVILGNLSFPTKSSNQLFMPLYHQVVDNALKAVLH-PDFQLTHYTAP------- 161
Mvis DVLDNCGVILGNLSFPTKSSNQLFMPLYHQVVDNALKSVLH-PDFQLTHYTAP------- 161
Ping EKLQDCGLILGNLSFPTKTSNHLFLPLYGHVVEVALKQLTS-SSFQLSNFSAG------- 164
P_sp ASLQQCGLILGNLSFPTRSSNHLFLPFYHHVVEQALQSITH-RDFKLKNFTAP------- 164
* *:::* ***** **.**:*:* .*: *:: * *

Sone GSQLAANGVIAHTASKLLSDALGLGGAQLSLDAACASSVYALKLACDYLTTGKADMMLAG 253

Swoo ATLDNANGAIAHNASKVVADALGLGGAQLSLDAACASSVYSLKLACDYLSAGKADVMLAG 240
Spie STLNTANGAIAHNTSKVVADALGLGSAQLSLDAACASSVYSLKLACDYLSTGKADVMLAG 242
Vtap GPLNPSNGSAAHNASKVVAEALGLGNAQLSLDAACASSVYSLKLACDYLNTGKADMMLAG 242
Pss9 TSLNPANGSAAHNASRLVADALGLGSVQLSLDAACASSVYSLKLACDYLNTGKADMMLAG 282
Vtas QDLNPINGAAAHNASKLVADALGLGNVQLSLDAACASSVYSLKLACDYLNTGKADMMLAG 241
Cpsy NNIHADNALVAGYPAALLAKAAGLGGTHFALDAACASSCYSVKLACDYLHTGKADMMLAG 234
C_sp ENLHADNALVAGYPSALLAKAAGLGGTHFSLDAACASSCYSVKLACDYLHTGKADMMLAG 232
Mmar KKTHADNALVAGYPAALIAQAAGLGGSHFALDAACASSCYSVKLACDYLHTGKANMMLAG 221
Mvis KKTHADNALVAGYPAALIAQAAGLGGSHFALDAACASSCYSVKLACDYLHTGKADMMLAG 221
Ping QTVNPDNALIAGFPSALLSQAAGLGGSVLSLDAACASSCYSVKLACDYLHTGKADMMLAG 224
P_sp RKIEADNALIAGYPSALLAKVAGLGGAHFSLDAACASSCYSLKLACDMLHTGEADMMLAG 224
*. * : :::.. ***. ::******** *::***** * :*:*::****

6
Sone AVSGADPFFINMGFSIFHAYPDHGISAPFDSNSKGLFAGEGAGVLVLKRLEDAERDGDNI 313
Swoo AVSGADPFFINMGFSIFHAYPDHGISVPFDGNSKGLFAGEGAGVLVLKRLDDAERDDDNI 300
Spie AVSGADPFFINMGFSIFHAYPDHGVSVPFDGNSKGLFAGEGAGVLVLKRLEDAERDNDKI 302
Vtap AVSGADPFFINMGFSIFHAYPDHGVSAPFDSNSKGLFAGEGAGVLVLKRLDDAERDGDQI 302
Pss9 AVSGADPFFINMGFSIFHAYPDHGVSVPFDTNSKGLFAGEGAGVLILKRLEDAERDGDNI 342
Vtas AVSGADPFFINMGFSIFHAYPDHGVSVPFDSNSKGLFAGEGAGVLVLKRLADAERDGDNI 301
Cpsy AVSGSDPMFVNMGFSIFQAYPANNIHAPFDKNSQGLFAGEGAGMMVLKRHSDAVRDGDKI 294
C_sp AVSGADPMFVNMGFSIFQAYPANNIHAPFDKNSQGLFAGEGAGMMVLKRHSDAVRDGDKI 292
Mmar AVSAADPMFVNMGFSIFQAYPANNVHAPFDQNSQGLFAGEGAGMMVLKRQSDAVRDGDHI 281
Mvis AVSAADPMFVNMGFSIFQAYPGNNVHAPFDKNSQGLFAGEGAGMMVLKRHSDAVRDGDNI 281
Ping AVSGADPLFVNMGFSIFQAFPEDNNHAPLDKNSQGLFAAEGAGMMVLKRHADALRDGDKI 284
P_sp AVSGADPMFVNMGFSIFQAFPDNNKHAPFDSASKGLFAGEGAGMMVLKRHSDALRDGDTI 284
***.:**:*:*******:*:* .. .*:* *:****.****:::*** ** **.* *

Sone YAVVSGIGLSNDGKGQFVLSPNSKGQVQAFERAYAAANTHPSNIEVIECHATGTPLGDKV 373

Swoo YAVVSGVGLSNDGKGQFVLSPNPKGQVKAFERAYAASDIEPKDIEVIECHATGTPLGDKI 360
Spie YAVVSGVGLSNDGKGQFVLSPNPKGQVKAFERAYAASDIEPKDIEVIECHATGTPLGDKI 362
Vtap YAVVSGIGLSNDGRGQFVLSPNSKGQVQAFERAYQASDLTPDSIEVIECHATGTPLGDKV 362
Pss9 YAVVSGIGLSNDGRGQFVLSPNSKGQVQAFERAYEATDLSPESIEVIECHATGTPLGDKV 402
Vtas YAVVSGIGLSNDGRGQFVLSPNSKGQVQAFERAYEASNLSPDSIEVIECHATGTPLGDKV 361
Cpsy HAIIKGGALSNDGKGEFVLSPNTKGQVLVYERAYEDAAVDPRDVDYIECHATGTPKGDNV 354
C_sp HAIIKGGALSNDGKGEFVLSPNTKGQVLVYERAYDNAGVDPRDVDYLECHATGTPKGDNV 352
Mmar YAIIKGGALSNDGKGEFVLSPNTKGQVLVYERAYADADVDPSTVDYIECHATGTPKGDNV 341
Mvis YAIIKGGALSNDGKGEFVLSPNTKGQVLVYERAYADAGVDPAAVDYIECHATGTPKGDNV 341
Ping HAIIKGGALSNDGKGEFVLSPNSKGQILAYQRAYQNAKVNPADVDYIECHATGTPKGDKV 344
P_sp HAIIKGGALSNDGKGEFILSPSSKGQVLAYERAYADANVSPSDVGYIECHTTGTPKGDNV 344
:*::.* .*****:*:*:***. ***: .::*** : * : :***:**** **::

Sone ELTSMERFFEDKL-DGTKAPLIGSAKSNLGHLLTAAGMPGIMKMIFAMRSGHLPPSINLT 432

Swoo ELTSMETFFEGKL-AGNQAPLIGSAKSNLGHLLTAAGMPGIMKMIFAMKEGALPPSINIS 419
Spie ELTSMETFFEDKL-QDSSAPLIGSAKSNLGHLLTAAGMPGIMKMIFAMKEGLLPPSINIS 421
Vtap ELTSMEQFFQHKL-NDSQVPLIGSAKSNLGHLLTAAGMPGIMKMIFAMKQGVLPPSINLE 421
Pss9 EMTSMERFFADKL-NGSQAPLIGSAKSNLGHLLTAAGMPGIMKMIFAMKEGVLPPSINLS 461
Vtas ELTSMERFFADKL-NGSNPPLIGSAKSNLGHLLTAAGMPGIMKMIFAMKEGVLPPSINLD 420
Cpsy ELGSMDTFFSRFPRENGNKPLLGSVKSNLGHLLTAAGMPGMTKAIWALNEAKIPATINLN 414
C_sp ELGSMDTFFSRFPRENGNKPLLGSVKSNLGHLLTAAGMPGMTKAIWALKEGKIPATINLN 412
Mmar ELRSMETFFSRV----NNKPLLGSVKSNLGHLLTAAGMPGMTKAMLALGKGLIPATINLK 397
Mvis ELRSMETFFSRV----NNKPLLGSVKSNLGHLLTAAGMPGMTKAMLALGKGLIPATINLK 397
Ping ELSSMETFFSQY----HNKPLLGSAKSNLGHLLTAAGMPGMTKAIWALNEGKIPATISLK 400
P_sp ELNSMEVFFSKY----NTKPLLGSAKSNLGHLLTAAGMPGMTKAIYALNKGKIPATISLK 400
*: **: ** **:**.***************: * : *: .. :* :*.:

Sone APISSPKGLFSVNNLPTQRQAWPDKAG-----NDRRHAGVSVFGFGGCNAHLLLESYQPT 487

Swoo DAISSPNHLFGSSTLPNQVQPWPAKTG-----NAQRHAGVSVFGFGGCNAHLLLESYLPP 474
Spie DAISSPNQLFGQATMPNLVQGWPDKPSNNHFGVKTRHAGVSVFGFGGCNAHLLLESYSQK 481
Vtap TPLSSPNNMFGAHTLPTQVQAWPDKAG-----NEQRRAGVSVFGFGGCNAHLLLEAYSAP 476
Pss9 TPLSSPEGLFGSHTLPTQVQAWPDKAG-----NTERCAGVSVFGFGGCNAHLLLEAHSAN 516
Vtas KPLSSPEGLFGSQTLPTQVQSWPSKAG-----NSERCAGVSVFGFGGCNAHLLLEAYSDT 475
Cpsy EPLSSKKGYLGGAQMPTDTIDWPVPAN-S--ANKPRTAGVSVFGFGGSNAHLVLQQPTQQ 471
C_sp EPLTSKNGYLGAEQMPTDTIDWPIKAN-----NKPRTAGVSVFGFGGSNAHLVLQEPTQT 467
Mmar QPLQSKNGYFTGEQMPTTTVSWPTTPGAK--ADKPRTAGVSVFGFGGSNAHLVLQQPTQT 455
Mvis EPLQSKNGYFTGEQMPTSTVSWPTSHGSK--TTGLRTAGVSVFGFGGSNAHLVLQQPTQA 455
Ping NPMSSKNGYFGAAQMPVDMVNWPDTK------DKTRSAGVSVFGFGGSNAHLVFQPGNTA 454
P_sp NAIKTKLGYLTQAQIPTDTVNWESKN------GKPLVAGVSVFGFGGSNAHLVLEGADAP 454
: : : :* * **********.****:::

7
Sone AHSAEKQA------NKPVYQQQALTVIGMASHFGPLASINALDKALIAQTDAFIPLPPKR 541
Swoo KDAAGAQATGAVKQTQPTVTPQPMKITGLASHFGPLNSINQLNSAITSNSNGFITLPKKR 534
Spie SAPSQAQS-----SVVTAATPAPLKIVGLSSHFGPLSTINQLDNAIASNSDAFIELPEKR 536
Vtap SNQQPANK------TLHSQANRSLDVVGIASHFGSLSSINALSDAIKTNTTAFGALPEKR 530
Pss9 SARNSPAA----NSPAKPAVSAPLKVTGLASHFGSLKTINALHNAITTGADAFVALPKKR 572
Vtas SHVNP-TQ----ESASPSLKPSNLSITGLASHFGSLQSINALSTAIETNNDAFIALPKKR 530
Cpsy LEPI----------TVKAKPREPLAIIGMDAHFGGAEDLASFKTLIETNDNTFRELPTNR 521
C_sp LTPV----------TNTVKARQPLAIIGMDAHFGGAKDLASFNTLIQTNGNTFRELPTKR 517
Mmar LETN----------FSVAKPREPLAIIGMDSHFGSASNLAQFKTLLNNNQNTFRELPEQR 505
Mvis LESN----------FGVAKPREPLAIIGMDSHFGSASNLAKFKTLLDNNQNTFRELPEQR 505
Ping YAPS----------SAAVKPREPLAIVGMDCHFGSATCLAEFDTLLTTGQTTFRELPPKR 504
P_sp FIKR----------FNQPKPRALMSIVGMDCLLSGVDSIVAFDALLKSNKNTFIELPPKR 504
: : *: . :. : : : * ** :*

Sone WKGLDKHPDILQQFGLNRAPKGAYIEQFDFDFLRFKVPPNEDDRLISQQLLLIKVADEAI 601

Swoo WKGLEQHSELLAEFGLSAAPKGAYVDSFELDFLRFKLPPNEDDRLISQQLMLMKVTDEAI 594
Spie WKGLEKHPELLAQFGLNAAPKGAYVDNFELDFLRFKLPPNEDDRLISQQLMLMRVTDEAI 596
Vtap WKGLDKHPELLANFGLDTVPHGAYIDQFELDFLRFKVPPNEDDRLISQQLLLMKVADEAI 590
Pss9 WKGLDQHPELLSQFGLDAIPHGAYIDQFELDFLRFKVPPNEDDRLISQQLLLMKVADEAI 632
Vtas WKGLDQHPELLNQFGLHGIPNGAYIDQFDLDFLRFKVPPNEDDRLISQQLLLMKVADEAI 590
Cpsy WKGIDNDTDVMNALQLSKAPQGGYVENFDIDFLRFKVPPNEQDCLIPQQLMMMKVADNAA 581
C_sp WKGIEKNSDVMNALSLTKAPMGGYVEDFDIDFLRFKVPPNEQDCLIPQQLMMMKVADNAA 577
Mmar WKGMESNANVMQSLQLRKAPKGSYVEQLDIDFLRFKVPPNEKDCLIPQQLMMMQVADNAA 565
Mvis WKGMESNANVMQSLQLTKAPKGGYVEQLDIDFLRFKVPPNEKDCLIPQQLMMMQVADNAA 565
Ping WKGMEQHPSIKQTLLLNKAPKGGYIENFDIDFLRFKVPPNKQDCLIPQQLLMMKVADNAA 564
P_sp WKGIQNDAQVMRTLGLKKAPKGGYIEKFDIDFLRFKVPPNAQDCLIPQQLMMMKVADNAA 564
***::.. .: : * * *.*::.:::******:*** .* ** ***::::*:*:*

Sone RDAKLTAGSKVAVLVAMETELELHQFRGRVNLHTQLADSLKKQGVHLSNDEYLALEAIAM 661

Swoo RDAKLEPGQKVAVLVAMETELELHQFRGRVNLHTQLKESLAAVGVTLDEDEYQALEAITM 654
Spie RDAKLQAGQKVAVLVAMETELELHQFRGRVNLHTQLQQSLAALGVNLSIDEYQALEAIAM 656
Vtap RDAKLEAGQKVAVLVAMETELEMHQFRGRVNLHTQLADSLNNMGIALNQEEYQALETIAM 650
Pss9 RDAKLEVGQKVAVLVAMETELEMHQFRGRVNLHTQLADSLENMGVQLTDSEYQALEAIAM 692
Vtas KDAKLVAGQKVAVLVAMETELEMHQFRGRVNLHSQLADSFANMGIELTQDEYQALETIAM 650
Cpsy KDAGLKEGSNVAVLVAMGIELELHQYRGRVNLSTQIEESLLQQGVTLNSEQRETLTNIAK 641
C_sp KDAGLKEGSNVAVLVAMGIELELHQYRGRVNLTTQIEESLLQQGISLSDEQRETLTNIAK 637
Mmar KDGGLVEGRNVAVLVAMGMELELHQYRGRVNLTTQIEDSLLQQGINLTVEQREELTNIAK 625
Mvis KDAGLVESSNVAVLVAMGMELELHQYRGRVNLTTQIEDSLLQQGINLTVEQCEELTNIAK 625
Ping KDAQLTPGSNVAVLVAMGVELDLHQYRGRVNLSTQIEQSLKEQGIELSVDQRLTLTGIAK 624
P_sp KDGALTEGSNVAVLVAMGMELDIHQYRGRVNLSTQIEQSLKEQGITLTELQRAELINIAK 624
:*. * . :******* **::**:****** :*: :*: *: * : * *:

Sone DSVLDAAKLNQYTSFIGNIMASRIASLWDFNGPAFTISAAEQSVARCIDVAQNLLSKEAL 721

Swoo DSVLDAAKLNQYTSFIGNIMASRIASLWDFHGPAFTISAAEQSVSRCIDVAQNMMVSEAL 714
Spie DSVLDAAKLNQYTSFIGNIMASRVSSLWDFNGPAFTISAAEQSVSRCIDVAQNLIAEDNL 716
Vtap DSVLDAAKLNQYTSFIGNIMASRISSLWDFNGPAFTISAAEQSVARCIDVAENLMSQESL 710
Pss9 DSVLDAAKLNQYTSFIGNIMASRIASLWDFNGPAFTISAAEQSVARCIDVAQNLMSQESL 752
Vtas DSVMDAAKLNQYTSFIGNIMASRISSLWDFNGPAFTISAAEQSVARCIDVAQNLMSQESM 710
Cpsy NGVAHAAQLNQYTSFIGNIMASRISALWDFTGPAITLSAEENSVYRCVELAENLFQTSDI 701
C_sp DGVAHAAQLNQYTSFIGNIMASRISALWDFSGPAITLSAEENSVYRCIELAENLFQTSDV 697
Mmar DGVASAAQLNQYTSFIGNIMASRISALWDFSGPAITVSAEENSVYRCVELAENLFQTSDV 685
Mvis DGLASAAQLNQYTSFIGNIMASRISALWDFSGPAITVSAEENSVYRCVELAENLFQTSDV 685
Ping EGIASAAQLNQYTSFIGNIMASRIAALWDFSGPAFTVSSEENSVYRCLELAENLFQTSEV 684
P_sp ESIASPAQLNQYTSFIGNLMASRISALWDFSGPAFTVSSEENSVYRCLQIAQSLFATSDV 684
:.: *:**********:****:::**** ***:*:*: *:** **:::*:.:: . :

8
 Sone DGVVIAAVDLSGSVEQVILKNAQVAVDLDAN----------S-------ANPQWKVGEGA 764
Swoo DAVVIAAVDLSGSFEQVILKNSVEPVSIDAT----------AAQHSTAQESGSWNVGEGA 764
Spie DAVVIAAVDLSGSFEQVILKNSIAPVAMTPK----------G----NTAAQASWNVGEGA 762
Vtap DAVVIAAVDLSGSAEQVILKNSVTPVANTA-------------------QDAGWHVGEGA 751
Pss9 DAVVIAAVDLSGSVEQIILKNSVTPVALHP-------------------QDSGWNVGEGA 793
Vtas DAVVIAAVDLSGSAEHVILKNSVSPVTLAPK----------FGQ----LQDGSWNVGEGA 756
Cpsy DAVIIASVDLAGSVENITLRQHFGPVEKGQV--ETGSVSTNSATSANVLEQNTWRVGEGA 759
C_sp DAVIIASVDLAGSIENITLRQHFGKVSV------------DAESATNTLDQEQWLVGEGA 745
Mmar EAVIIAAVDLSGSIENITLRQHYGPVNEKGSVSECGPVNESSSVTNNILDQQQWLVGEGA 745
Mvis DAVIIAAVDLAGSIENITLRQHFGPVS------ECGPVNKKG-TSNNIFDEQKWLVGEGA 738
Ping EAVLIASVDLAGSLENISLRQRYGPVSENAT------------NRPNLLSSQQWLVGEGA 732
P_sp EAVLIASVDLAGSFENVSLRQRYGPVSENAS------------SHADPLRNKEWLLGEGA 732
:.*:**:***:** *:: *:: * . * :****

Sone GAIVLTNQQAS----NSQQAGYGQIRGQAFGTNHQLPKL---LDSLITETAIANPSMPTA 817

Swoo GAIVLVANDAK------PEKSYGTINSLAFGNATQANLV---TDNLLTQAGADIN----T 811
Spie GAVVLVKDEST------SGRAYGQIDALAFGHAQQLPQV---TDKLLTQTATNST----S 809
Vtap GAMVFVDSQLVN-----NNQSYGQLKSLAFGNVNGSHIT---TDTLLTSAGLNAN----D 799
Pss9 GAIVLVDTDNAS-----TKNSYGEITALDFGSVAQSNIT---SDRLLTTAGITAN----N 841
Vtas GAIVLVEENQVA---SNQDAAYGSINALAFGSSENNNAV---VDELLTQVGMSSN----D 806
Cpsy GAFVVKPLSKVI--QVAEQSIYATIDGISFANGKDAAAITKAASASLNIAGLNSA----D 813
C_sp AAFVIIPESKVPQSKVGERSVYATIDSISFANGCDEQAIVKAANKSLALAGIQAA----D 801
Mmar AAIVVKPSSQ-----VTAEQVYARIDAVSFAPGSNAKAITIAADKALTLAGISAA----D 796
Mvis AAIVVKLVSQ-----VASQQVYASIDAVSFASGCDAQAIASAADKALTLAGISAA----D 789
Ping GAFVVKPNSK-----ANSNSVYATVDGLSFAAGSDAQAIEKAALKVCQIANLSCA----Q 783
P_sp GAIVLRRSEL-------NDARYAQLDAISFAPGSDAKAIKKASAKACQLASISAN----E 781
.*.*. . *. : . *. .

Sone IHMIEQCIAPE-EQLPAEHLLAQLNLLGTSCNRVANTLGHNFAAAGMASLLSALLSLKNR 876

Swoo VKVVETSIAPG-SHR-N--TPVTDLFPNAIITSADERVGHSFAAAGMASLLHGLLNLSTQ 867
Spie INVVETNIAPG-SLTTN--VTLNDSFVNAVATSADDRIGHCFAAAGMASLLHGLLSVSRN 866
Vtap IQLIERNQAPE-SANLT----VNIPLPQSRATQANLRLGHSFAASGMASILHGLIELSQV 854
Pss9 VSLLELNQAPE-SVETV-----QFPLPSATHIQANQRLGHCYAASGMASILHGLLSLNAI 895
Vtas VSLLELNHAPE-SSSHQ---FSSLSLGNTKTTQASQRVGHCSAASGMASLLHGLLNLNLA 862
Cpsy ITSVEAHASGFSAENIAEAQALPALYAGKVISSVKSNIGHTFNASGVASIIKTALLLDDK 873
C_sp ISQVEAHASGFIAENSAEKQALPKLYSGKTISSVKSNIGHTFNASGVASIVKSALLLDER 861
Mmar VASVEAHASGFSAENNAEKTALPTLYPSASISSVKANIGHTFNASGMASIIKTALLLDQN 856
Mvis VVCVEAHASGFSAENSAEKTALSQLYPNVNISSVKSNIGHTFNASGIASIIKTALILDQQ 849
Ping INHVEAFASGFSLDNQAEKSAFNKLYPSIKIDSVKRQVGHLFNASGMASIIKTALLLDKQ 843
P_sp IDHVEAFASGFEADNAAETEAFTQLYPHVKPDSVKRQIGHLFNASGMFSLIKSALLVNSK 841
: :* : . :** *:*: *:: : :.

Figure S2.Sone
Sequence alignments of Epa-C and Dha-C in PUFA synthases. KSC and
SANSDK---------NAEKQALVSTQSQGVSSLLLLSQTATQAAQLELRLAQDLTLSEQK 927 CLF-like
Swoo NQSQKQ----------QAGSLLVANSSEAQISQLLISQTASENSALTNRLSHELKSDAKH 917
domains are indicated
Spie by blue and red lines, respectively. Light blue boxes show catalytic
AAA--------------TNKALITNVSENQASQLLISQSQCEQQALAARLANELKSDAKH 912 Cys/His
Vtap SAQANG----------TDKHAVIANISENQCSQLLFGQSESQTRALQDRLAVPLASDTKR 904
residues ofPss9
KSC domains. Green dots show the mutated amino acid residues. Sone,955
PKQTTIPSLNTSVTAAVTKAAIVANVSENQCSQLLLTQTSTETQSLTARLNSELANDSKR Shewanella
Vtas PLNPNV----------SSKSAIVANISEGQCSQLLLSQSSVESQSLSVRLSNELASDAKR 912
oneidensisCpsy
(EPA producer); Swoo, S. woodyi (EPA producer); Spie, S. piezotolerans
VLNEERLTS-----HVASHIAVNGLGKDESCAHLILSSSKLAHQAA-PPP----TGKQRP 923 (EPA
C_sp SAKTNTATD-----IARSHIAVNGLGKDGSCAHLILSSSKYAHQSA-PVSTLTLAGKPKP 915
producer); Mmar
Vtap, Vibrio tapetis (EPA producer); Pss9, P. profundum (EPA producer),
T-----SQD-----QKSKHIAINGLGRDNSCAHLILSSSAQAHQVA-PAPVSG-MAKQRP 904 Vtas, V.
Mvis NTL---SSD-----QKNKHIAINGLGRDNSCAHLILSGSEQVHQAA-PAPV----GKRRP 896
tasmaniensis
Ping
(EPA producer); Cpsy, Colwellia psychrerythraea (DHA producer); C_sp,
--------------KAGVNTAINGLGKDFCCAHLILSATDSARQSA-PKNSLG--RVKTP 886
Colwellia
sp. MT41 P_sp --------------Q-AKQVAINGLGHDMSCAHLLVSNAEPMRKLA-H--VVS--KKSAF
(DHA producer); 881
Mmar, Moritella marina (DHA producer); Mvis, M. viscosa (DHA
: : : *:. :
producer); Ping, Psychromonas ingrahamii (DHA producer); P_sp, Psychromonas sp. CNPT3
(DHA producer).

9
 (A) (B)

Figure S3. Modelled structure of the KSC domain in Dha-C.

(A) A homology model constructed using Phyre2 software
(http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index; template, 2VZ8 (mammalian fatty
acid synthase); amino acid sequence identity, 25%) and the catalytic residue (C196) and the
mutated amino acid residues (M229, Q239, N244, N245, H247, and K337) are shown in green
and red, respectively. (B) The cavity is highlighted in yellow.

10
(A) (B)

Figure S4. Modelled structure of the CLF-like domain in Dha-C.

(A) A homology model constructed using Phyre2 software
(http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index; template, 2VZ8 (mammalian fatty
acid synthase); amino acid sequence identity, 22%) and the mutated amino acid residues (E665,
Y670, E674, F680, N701, and R705) are shown in red. (B) The cavity is highlighted in yellow.

11
Figure S5. GC-MS analysis (traced at m/z 79) of products produced by E. coli expressing Dha-
ABD and KSC domain mutants of Dha-C. The traces show wild-type (WT), M229F, Q239H, N244H,
N245G, H247S, and K337L mutants (from top to bottom).

12
 (A) (B)
M I S kDa M I S P
kDa
250 250
150 150
100 100
75
75

50
50
37
37

25

20 25

(C) (D)
kDa M I S
250
150
100
75

50

37

25

20

Figure S6. Preparation of truncated recombinant KSC domain, CLF-like domain, and KSC/CLF-
like domain of EPA synthase.
SDS-PAGE analysis of (A) His-tagged KSC domain (58.6 kDa), (B) His-tagged CLF-like domain
(55.7 kDa), (C) nontagged CLF-like domain (53.6 kDa), and (D) KSC/CLF-like domain (110.4 kDa).
M, marker; I, insoluble fraction; S, soluble fraction; P, purified protein; arrows, target proteins.

13
Table S1. Primers used in this study
Primers used for
pCDF_dhaC_cassette
FE1
FE2
FE3
FE4
FE5
FE6
Primers used for KSC
domain mutations
FM_Fw _M229F_dhaC
FM_Rv _M229F_dhaC
FM_Fw _Q239H_dhaC
FM_Rv _Q239H_dhaC
FM_Fw _N244H_dhaC
FM_Rv _N244H_dhaC
FM_Fw _N245G_dhaC
FM_Rv _N245G_dhaC
FM_Fw _H247S_dhaC
FM_Rv _H247S_dhaC
FM_Fw _K337L_dhaC
FM_Rv _K337L_dhaC
Primers used for CLF-like
domain mutations
FM_Fw _E665A_dhaC
FM_Rv _E665A_dhaC
FM_Fw _Y670A_dhaC
Sequences
tttaaaccatggaaaatattgcagtagtaggtattgctaatttgttc
ctgcggtccgcggtttatctgccttgg
gataaaccgcggaccgcaggtgtgagcgtatttg
cttttctgatcttgactagtattctgatctaacagcagcgcc
cagaatactagtcaagatcagaaaagcaaacatattgc
aaagtgacaagggaaataccaatgctcagggtctaaatctttctg
Sequences
gcagatcctttcttcgtaaatatgggtttctcgatattccaagc
catatttacgaagaaaggatctgctgcagataccgcacc
cgatattccacgcttacccagctaacaatgtacatgcc
ctgggtaagcgtggaatatcgagaaacccatatttacgaac
cccagctcataatgtacatgccccgtttgaccaaaattc
catgtacattatgagctgggtaagcttggaatatcgagaaac
cagctaacggtgtacatgccccgtttgaccaaaattc
gggcatgtacaccgttagctgggtaagcttggaatatcgagaaac
aacaatgtatcggccccgtttgaccaaaattcacaag
caaacggggccgatacattgttagctgggtaagcttggaatatcg
gggcacacctttgggtgacaatgttgaattgcgttcg
gtcacccaaaggtgtgcccgttgcatgacattcaatatag
Sequences
ccgtatcggctgcggaaaactctgtttatcgttgtgttgaattagctg
cagagttttccgcagccgatacggtaatagcaggaccag
ctctgttgcgcgttgtgttgaattagctgaaaatctatttcaaacc
14
FM_Rv _Y670A_dhaC caacacaacgcgcaacagagttttcttcagccgatacggtaatag
FM_Fw _E674D_dhaC gtttatcgttgtgttgacttagctgaaaatctatttcaaaccagtgatgttg
FM_Rv _E674D_dhaC gattttcagctaagtcaacacaacgataaacagagttttcttcag
FM_Fw _F680M_dhaC gctgaaaatctaatgcaaaccagtgatgttgaagccgttattattgc
FM_Rv _F680M_dhaC cactggtttgcattagattttcagctaattcaacacaacgataaacag
ctgctgttgatttgtctggttcaattgaacagattactttacgtcagcactacggtcc
FM_Fw _N701Q_dhaC
ag
FM_Rv _N701Q_dhaC caattgaaccagacaaatcaacagcagcaataataacg
ctgctgttgatttgtctggttcaattgaaaacattactttaaagcagcactacggtcc
FM_Fw _R705K_dhaC
agttaatgaaaagg
FM_Rv _R705K_dhaC taaagtaatgttttcaattgaaccagacaaatc

Primers used for KSC

Sequences
domain expression
Fw_epaC_KSC_NdeI_ ttccttcatatgcattgcccagttaattacgcacc
Rv_epaC_KSC_BamHI gccggatccttatgcaggctttgcaggactattcg

Primers used for CLF-like

Sequences
domain expression
Fw_epaC_CLF_NdeI aaagcccatatgtcagctaactctgcaagaaatagccctg
Rv_epaC_CLF_BamHI ttcggatccttaagcaacgggtttagcaatagcttgaatg

15
References
[1] Hayashi, S., Naka, M., Ikeuchi, K., Ohtsuka, M., Kobayashi, K., Satoh, Y., Ogasawara, Y.,
Maruyama, C., Hamano, Y., Ujihara, T., Dairi, T. (2019) Control mechanism for carbon-chain
length in polyunsaturated fatty-acid synthases. Angew. Chem., Int. Ed. 58, 6605–6610.
[2] Hayashi, S., Satoh, Y., Ogasawara, Y., Maruyama, C., Hamano, Y., Ujihara, T., Dairi, T. (2019)
Control mechanism for cis double-bond formation by polyunsaturated fatty-acid synthases.
Angew. Chem., Int. Ed. 58, 2326–2330.
[3] Hayashi, S., Satoh, Y., Ujihara, T., Takata, Y., Dairi, T. (2016) Enhanced production of
polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins. Sci. Rep.
6, 35441.

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