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Supporting Information
Functional dissection and module swapping of fungal cyclooligomer
depsipeptide synthetases
Dayu Yu,a,b,c Fuchao Xu,a,b,c David Gageb and Jixun Zhan*b
a
Department of Applied Chemistry and Biological Engineering, College of Chemical
Engineering, Northeast Dianli University, Jilin, Jilin 132012, China.
b
Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT
84322, USA.
c
These authors contributed equally to this work.
* Corresponding author: Fax: +1 435 797 1248; Tel: +1 435 797 8774; E-mail:
[email protected] (J. Zhan)
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1. Strains, plasmids, and culture conditions
Escherichia coli XL1-Blue (Agilent) was used for routine cloning and pJET1.2 (Fermentas) was
used as the cloning vector. E. coli cells were grown in Luria-Bertani (LB) medium at 250 rpm
and 37C. When necessary, 50 μg mL-1 ampicillin was added. S. cerevisiae BJ5464-NpgA
(MATα ura3-52 his3-Δ200 leu2- Δ1 trp1 pep4::HIS3 prb1 Δ1.6R can1 GAL) was obtained from
Dr. Nancy Da Silva at the University of California, Irvine. The strain was maintained on YPD
(yeast extract/peptone/dextrose) agar plates at 30C. The E. coli/S. cerevisiae shuttle vectors
pXW55 and pXW06 were gifts from Dr. Yi Tang at the University of California, Los Angeles.
pXW55 carries the ampicillin-resistance gene and URA3 as the selection markers, and was used
to express M1-M2 linker-containing or -lacking M23 of BbBEAS and BbBSLS. pXW06 carries
the ampicillin-resistance gene and TRP1 as the selection markers, and was used to express
M1-M2 linker-containing or -lacking M1 of BbBEAS and BbBSLS.
Beauveria bassiana ATCC 7159 was obtained from the American Type Culture Collection
and was grown in potato dextrose broth at 250 rpm and 28C. The genomic DNA was extracted
from the mycelia of the fungus and used as the template to amplify the bbBeas and bbBsls gene
fragments.
2. DNA manipulations
Isolation of plasmid DNA from E. coli was conducted with a GeneJET™ Plasmid Miniprep Kit
(Fermentas) by following the manufacturer’s instructions. Gene ligations were performed with
T4 DNA ligase (New England Biolabs).
3. Gene amplification and plasmid construction
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The gene fragments bbBeasM1 with M1-M2 linker, bbBeas-M23 with M1-M2 linker, bbBeas-M1
without M1-M2 linker, bbBeasM23 without M1-M2 linker, bbBeasM1 with partial M1-M2 linker,
and bbBeasM23a with partial M1-M2 linker were amplified by PCR from the genomic DNA of
B. bassiana ATCC 7159 with Phusion® High-Fidelity DNA Polymerase (New England Biolabs)
with specific primers (Table S1). Similarly, bbBslsM1 with M1-M2 linker, bbBslsM23 with
M1-M2 linker, bbBslsM1 without M1-M2 linker, bbBslsM23 without M1-M2 linker, bbBslsM1
with partial M1-M2 linker and bbBslsM23a with partial M1-M2 linker were amplified from the
template. These gene fragments were ligated into the cloning vector pJET1.2 (Fermentas) to
yield twelve plasmids including pDY38, pDY39, pDY60, pDY61, pDY62, pDY66, pDY69,
pDY72, pDY90, pDY91, pDY94 and pDY95. These plasmids were confirmed by digestion
checks and gene sequencing.
The bbBeasM1 with M1-M2 linker and bbBeasM1 without M1-M2 linker inserts were
excised from pDY39 and pDY94 with NdeI and PmeI and ligated into pXW06 between the same
sites to generate pDY45 and pDY99, respectively. The bbBeasM23 with M1-M2 linker and
bbBeas-M23 without M1-M2 linker inserts were excised from pDY38 and pDY91 with SpeI and
PmlI and ligated into pXW55 between the same sites to yield pDY58 and pDY98. The bbBslsM1
with M1-M2 linker and bbBslsM1 without M1-M2 linker inserts were excised from pDY69 and
pDY95 with NdeI and PmeI and ligated into pXW06 between the same sites to generate pDY73
and pDY102. The bbBslsM23 with M1-M2 linker and bbBslsM23 without M1-M2 linker inserts
were excised from pDY72 and pDY90 with SpeI and PmlI and ligated into pXW55 between the
same sites to generate pDY78 and pDY97.
The bbBslsM23a with partial M1-M2 linker insert was excised from pDY62 with SpeI and
ligated into pDY60 at the same site to generate pDY68. Then the bbBeasM1-bbBslsM23a insert
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was excised from pDY68 with NdeI and ligated into the pDY42 (harboring the entire bbBsls
gene) vector treated with NdeI to replace a fragment to afford pDY70. The bbBeasM23a with
partial M1-M2 linker insert was excised from pDY66 with SpeI and ligated into pDY61 at the
NheI site to generate pDY67. The bbBslsM1-bbBeasM23a insert was then excised from pDY67
with NdeI and ligated into the pDY37 (harboring the entire bbBeas gene) vector treated with
NdeI to replace a fragment to generate pDY71.
The plasmids constructed in this work are shown in Table S2.
4. Transformation of S. cerevisiae BJ5464-NpgA and fermentation of the engineered strains
Plasmids were introduced into S. cerevisiae BJ5464-NpgA using the lithium acetate method.
Correct transformants were selected by appropriate autotrophy. The engineered S. cerevisiae
strain carrying pDY37, pDY42, pDY70 or pDY71 was fermented in a SC-Ura dropout medium
(6.7 g L-1 yeast nitrogen base; 20 g L-1 glucose; 0.77 g L-1 -Ura DO supplement) at 30°C with
shaking at 250 rpm. When the OD600 reached 0.6, equal volume of YP medium (10 g L-1 yeast
extract; 20 g L-1 peptone) was added. The cultures were maintained under the same conditions
for an additional 72 h to produce the cyclooligomer depsipeptides (CODs). Strains carrying a
pXW55 derived plasmid and a pXW06 derived plasmid were fermented in a SC-Ura-Trp dropout
medium (6.7 g L-1 yeast nitrogen base; 20 g L-1 glucose; 0.72 g L-1 -Trp/-Ura DO supplement) at
30°C with shaking at 250 rpm until the OD600 reached 0.6, and then equal volume of YP medium
was added. The fermentation broths were cultured under the same conditions for an additional 72
h to produce the corresponding CODs.
5. Extraction and analysis of the products in the engineered S. cerevisiae strains
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A typical procedure used to analyze the products in the engineered S. cerevisiae BJ5464-NpgA
strains is given below. 100 mL of fermentation broth was centrifuged at 2,737 ×g for 5 min to
separate the supernatant and the cells. The supernatant was extracted three times with 100 mL of
ethyl acetate, and the cells were extracted twice with 50 mL of methanol. The extracts were then
combined and evaporated in vacuo to dryness. The residue was re-dissolved in 500 μL of
methanol and analyzed on an Agilent 6130 Single Quad LC-MS (C18, 5 μm, 4.6 mm × 250 mm
column). A linear gradient of acetonitrile-water (containing 0.1% formic acid) was programmed
from 80% to 100% over 20 min at a flow rate of 1 mL min-1 and the metabolites were monitored
at 210 nm. Standard curves based on the linear relationship between the amounts and the peak
areas were established for beauvericin, beauvericin A, beauvericin B, beauvericin C and
bassianolide for quantitative measurement of their production in different engineered strains.
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Table S1
Primers used in this work. The restriction sites are in bold.
Primer
BEAS-M1-5
BEAS-M1-short-3
BEAS-M23-short-5
BEAS-M23-3
BSLS-M1-5
BSLS-M1-short-3
BSLS-M23-short-5
BSLS-M23-3
BSLS-M1-long-3
BSLS-M23-long-5
BEAS-M1-long-3
BEAS-M23-long-5
20120725BEAS-M1-3
20120725BSLS-M23a-5
20120725BSLS-M23a-3
20120725BSLS-M1-3
20120725BEAS-M23a-5
20120725BEAS-M23a-3
Sequence
aaCATatggagccgctcaaaaatgt
aaGTTTAAACtcacatcacggcctgaagacgag
aaACTAGTtcttactctcaaggtcgatt
aaCACGTGtcacaaagccgagtttagac
aaCATatggagccacccaacaacgc
aaGTTTAAACtcaaatagctgcctggaggctgg
aaACTAGTtcatactcgcaaggtcgact
aaCACGTGtcataaagacgcattcaaagcc
aaGTTTAAACtcactgttccacaggaccatccaa
aaACTAGTggtggcagctcaatgaccgt
aaGTTTAAACtcactgctcaacgggtccgtccca
aaACTAGTagcggcgattctacgccctc
aaACTAGTgatggtcgagggcgtagaat
aaACTAGTatcccggcgctagctttgga
aaACTAGTgtgcatatgggatgcttgaaagt
atccaaagctagcgccgggat
aaACTAGTttgggacggacccgttgagca
aaACTAGTttgcatatgcgtggctggata
Restriction sites
NdeI
PmeI
SpeI
PmlI
NdeI
PmeI
SpeI
PmlI
PmeI
SpeI
PmeI
SpeI
SpeI
SpeI
NdeI, SpeI
NheI
SpeI
NdeI, SpeI
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Table S2
Plasmid
pDY38
pDY39
pDY45
pDY58
pDY60
pDY61
pDY62
pDY66
pDY67
pDY68
pDY69
pDY70
pDY71
pDY72
pDY73
pDY78
pDY90
pDY91
pDY94
pDY95
pDY97
pDY98
pDY99
pDY102
Plasmids constructed in this work.
Description
bbBeasM23 with M1-M2 linker in pJET1.2
bbBeasM1 with M1-M2 linker in pJET1.2
bbBeasM1 with M1-M2 linker in pXW06
bbBeasM23 with M1-M2 linker in pXW55
bbBeasM1 with partial M1-M2 linker in pJET1.2
bbBslsM1 with partial M1-M2 linker in pJET1.2
bbBslsM23a with partial M1-M2 linker in pJET1.2
bbBeasM23a with partial M1-M2 linker in pJET1.2
bbBslsM1-bbBeasM23a in pJET1.2
bbBeasM1-bbBslsM23a in pJET1.2
bbBslsM1 with M1-M2 linker in pJET1.2
bbBeasM1-bbBslsM23 in pXW55
bbBslsM1-bbBeasM23 in pXW55
bbBslsM23 with M1-M2 linker in pJET1.2
bbBslsM1 with M1-M2 linker in pXW06
bbBslsM23 with M1-M2 linker in pXW55
bbBslsM23 without M1-M2 linker in pJET1.2
bbBeasM23 without M1-M2 linker in pJET1.2
bbBeasM1 without M1-M2 linker in pJET1.2
bbBslsM1 without M1-M2 linker in pJET1.2
bbBslsM23 without M1-M2 linker in pXW55
bbBeasM23 without M1-M2 linker in pXW55
bbBeasM1 without M1-M2 linker in pXW06
bbBslsM1 without M1-M2 linker in pXW06
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BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1
1
1
1
1
MEPLK-NVNTGQPCSTVPFP-VSDETVEHLNGLYEEINRRFGLDRDAIETILPCTPFQYDVLDC
MEPPN-NANTGQLGPTLP-----NGTVDLPTDLSREITRHFGLEQDEIEEILPCTPFQRDVIEC
MASLNGMVGIGQHDPAITASSVPDDAGRHHDNLRKQVARRFGLDKAKIENILPCTPFQSDVMDC
MSLHTPSDGQQD--PALAS-----------KTLCEQISRALGLGQDKIENIFPGTPFQRDVIDC
MEYLTAVDGRQDLPPTPASF--CSHGDSPLNSSYEQLFHLYGLDSSRIEAIKPCTPFQLDMIDC
62
58
64
51
62
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
63
59
65
52
63
AANDARHAVGHAMYEISQHVHVQRFIAAWRETVRRTPALRACTFTSTTGESFQLVLRESFVLSR
ASDDKRRAVGHVVYEIPEDVDTERLAAAWKATVRYTPALRTCIFTSETGNAFQVVLRDYFIFAR
AVDDGRRAIGHVVYEIPRNIDTQRLATAWKEVVRQTPALRTCIFTSEAGDCFQVVLTESFFVWM
AADDKQRAVGHAVFEIPKDIDAARLAAAWKETVLHTPALRTCTFTSKSGDVLQVVLRDSFVFSW
NALDKQSAIGHAVYDVPTDIDISRFALAWKEIVNQTPALRAFAFTSDSGKTSQVILKDSFVFSW
126
122
128
115
126
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
127
123
129
116
127
IYWSSSSSLQAAVLKDETTAAIAGPRCNRLVLLEDPDTRKQLLIWVFHLALVDSTVQEPILRRV
MYCP-SAHLKSAIVKDEATAAVAGPRCNRYVLTGEPNSKRRVLVWTFSHSFVDSAFQGRILQQV
QLTS---FDLKEAVQDEAGAAMAGPRCNRYVILEDSSTKQRLLIWTFSHALVDNVLQERILRRV
MSGP-SVDLKEAVVQDEAAAALAGPRCNRFVLLEDPDTKERQLIWTFSHALVDSTFQERILRRV
MCWS-SSSSPDEVVRDEAAAAASGPRCNRFVLLEDMQTKKCQLVWTFSHALVDVTFQQRVLSRV
190
185
189
178
189
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
191
186
190
179
190
LAAYKSEDDQL---DSLPLTPDSSGGSDSD-SPS-TLKMPRAFDQEKATQFWQRQLSGLDASVF
LAAYKDGHGRV---FSLQPTTDLTESENGD-HLS-TPASERTVVIERATQFWQEKLHGLDASVF
IAAYDGGN-----------------VQYAN-GLE-ILANPQARDTEGAARFWRQHFDGLNASVF
LKAYKDANDEHPRQFETPDSSQATPEEDLQPNPSKMLKIPQAADMDRAVEFWKDHLSGLKCFCL
FAAYKHEKDTH--RPETPESSDAT-DTDSQ-SVS-VVSMSCEDNAVSATHFWQTHLNDLNASVF
249
244
234
242
248
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
250
245
235
243
249
PPLSSHLTTPKADAKIEHYISWPASAAQHRWSSTTVCQAALAVLLSRYSHSSEALFGVVTEQVC
PHLPSHKRVPAIDARADHYLPCPP-FIQHEWSSTTVCRTALAILLARYTHSSEALFGVVTEQSPPLPSHTVVPDPDTQSEHRISYPR-LAQQKWSNTAVCRAALAVLLARFTHAPEALFGVVTERPH
PAFVLSSVYAHPDAKAEHRISYSS-SAQQKMSSATICRTALAILLSRYTHSPEALFGIVTEQTP
PHLSDHLMVPNPTTTAEHRITFPL-S-QKALSNSAICRTALSILLSRYTHSDEALFGAVTEQSL
313
306
297
305
310
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
314
307
298
306
311
MFEGQRLLINGPTRSVVPFRVHCGPEQSVTDLLKSIASDNHDMRQFAHVGLCNISRIGDDQSAA
-HEEHPLLLDGPTSTVVPFRVLCALNQSVSKVMEAITTYDHDMRQFAHAGLCNISRIGDDASAA
AFKEQEHLMNSPTRTVVPIRVLCASDQSVSDILRAITTHDDAMHEFEQAGLRNIRNAGDDASAA
LLEEQ-LMLDGPTRTVVPIRVSCASEQSVSDIMSTIDSYDQTMRQFAHAGLRNIASAGDDESAA
PFDKH-YLADGTYQTVAPLRVHCQSNLRASDVMDAISSYDDRLGHLAPFGLRDIRNTGDNGSAA
377
369
361
368
373
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
378
370
362
369
374
CRFQTVLSVSNRRSSEDAASGEVLQILQESEGFAPCADRALLLRCETSRQGALLVARYDQGVIE
CGFQTVLMVTDSRT---AGDDEIHQVLEESEKFIPCTDRALLLSCQMTDEGVLLVARYDQSILE
CGFQTVLLVTDSDTP-QTPGSALHRIVEESDRFVPCTNRALLLNCQMADDSALLVARYDQSVID
CGFQTVLLVSDGDAQP-ASTWEILKKTEEPEGFIPCTNRALLLSCQMTSSGAHLTARYDQSIID
CDFQTVLLVTDGSHVN-NGINGFLQQITESSHFMPCNNRALLLHCQMESSGALLVAYYDHNVID
441
430
424
431
436
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
442
431
425
432
437
PPQMARFLRQLGWLMEQLQSAADDALSVKQLDIVTREDRAEIDSWNSDALEVQESLLHSAFVKR
PLQMARFLRQLGFLINKLQS-TDGSPCVGQLDVLAPEDRTEIEGWNSEPLQTQDCLIHSEVVRN
SRDVARFLRQLGGLIKQLQS-HAIDLPLGQLDVITQEDRAEIESWNSDRLQTSDVTIHDVIAKR
AEQMARLLRQLGHLIQNLQTSTDLP--VEKVDMMTQEDWLEIERWNSDSIDAQDTLIHSEMLKW
SLQTTRLLQQFGHLIKCLQSPLDLSS-MAEVNLMTEYDRAEIESWNSQPLEVQDTLIHHEMLKA
505
493
487
493
499
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
506
494
488
494
500
AAESPSDPAVLSWDGAWTYSELDNVSSRLAAHIRSLDLSHEQLIVPVYFEKSKWVVASILAVLK
AGDTPNKPAVCAWDGEWTYSELNNVSSRLASYISSLDLG-QQLIVPIYLEKSKWVMAAILAVLK
AADAPRKIAVSAWDGEWTYNELNNVSSRLAGHIQSLDLGQGQAVIPLCFEKSKWVVAGMLAVLK
TSQSPNKAAVAAWDGEWTYAELDNVSSRLAQHINSIDLGKEHAIVPIYFEKSKWVVASMLAVLK
VSHSPTKTAIQAWDGDWTYSELDNVSSRLAVHIKSLGLRAQQAIIPVYFEKSKWVIASMLAVLK
569
556
551
557
563
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
570
557
552
558
564
AGHAFTLIDPKDPPARTTRIVQQTSAKVALTSKLHQDTVQAIIGRCIVVDDDFVQSLGSASQCQ
AGHAFTLIDPNDPPARTAQIIKQASASIALTSALHQSKMQAVVGRCITVDDDLVQTLTTFEGSQ
TGRAFTLIDPSNPSARMSQVCRQTSARVALTSQLHYDTMRAVVGQCIVVDDNLLQSLPCDEDRL
AGHAFTLIDPSDPPARTAQVVQQTSATVALTSKLHRETVQSTVGRCIVVDEEFVKSLPQ-SSEL
SGNAFTLIDPNDPPARTAQVVTQTRATVALTSKLHRETVQKLVGRCVVVDDELLQSVSA-SDDF
633
620
615
620
626
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
634
621
616
621
627
EKSELTVKPHNLAYAIFTSGSTGDPKGIMIEHQAFASCVAKFGPAL-IPHNARALQFASHGFGA
VAS--AAKPGDLAYVIFTSGSTGDPKGIMIEHRAFYSSVVKFGKALGIRSSTRALQFATHGFGA
NPA--VK-PQDLAYVLFTSGSTGEPKGSMIEHRGFTSCALKFGPALGINSDTRALQFASYAFGA
SAS--VK-AHDLAYVIFTSGSTGIPKGIMIEHRSFSSCAIKFGPALGITSDTRALQFGSHAFGA
SSL--TK-SQDLAYVIFTSGSTGDPKGIMIEHRAFSSCALKFGASLGINSDTRALQFGTHAFGA
696
682
676
681
687
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
697
683
677
682
688
CLLEILPTLLRGGCVCIPSDLDRMHNIPDFIRRYNVNWMMATPSYMTTFKPEDVPGLQTLILVG
FLLEVLTTLIHGGCICVPSDHDRMHNIPGFIRQNQINWMMATPSYMTTMKPEDVPGLETLVLVG
CLLEIVTTLMHGGCVCIPSDDDRMNNVPDFIKRSGVNWALLTPSFIGTIQPESVPGLQTLVLVG
CILEIMTTLIHGGCVCIPSDDDRMNNVLEFINRTNVQLGHATPSYMGTFQPEVVPGLKTLVLVG
CLLEIMTTLINGGCVCIPSDDDRMNSIPSFINRYNVNWMMATPSYMGTFSPEDVPGLATLVLVG
760
746
740
745
751
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BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
761
747
741
746
752
EQMSASVNATWASRLGLFDGYGQSESCSICFIGKISPVS-EANNIGRAVGAHSWIVHPDDPDRL
EQMSSSINDVWLSELQLLDGYGQSESSSICFVGKIDDSSRDPNNLGWAIGAHSWIINPDNPDQL
EPMSATMRDTWAPRVRLLNGYGQSESATICSVTKINPFSSEPNSIGRAVGARFWITDPNEPNHL
EQMSASVNEVWAPRVQLLNGYGQSESSSICCVAKISPGSSEPNNIGHAVGAHSWIVDPEDPNRL
EQMSSSVNAIWAPKLQLLNGYGQSESSSICFASNMS---TEPNNMGRAVGAHSWVIDPNDINRL
823
810
804
809
812
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
824
811
805
810
813
APVGAVGELLIESPGIARGYIAAPATDRNPFLETAPAWYAPRQPPTGVKFYRTGDLARYAADGT
VPIGAIGELLIESPGIARGYLFSQSTET-PFLERAPAWYASKQPPYGVKFYRTGDLARYAPDGT
VPIGCIGELVIESPGIARGYIVSPPRDNSPFLAKAPAWYPTKQLPDGVKFYRTGDLVCYRSDGT
APIGAVGELVIESAGIARDYIVAPTQDKSPFIKTAPTWYPAKQLPDGFKIYRTGDLACYASDGS
VPIGAVGELVIESPGIARDYIVPPPPEKSPFFTDIPSWYPANTFPDGAKLYRTGDLARYASDGS
887
873
868
873
876
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
888
874
869
874
877
VVCLGRIDSQVKIRGQRVEMGAVETRLRQQVPSDITVVAEAVKRSGSSGSTVITAFLIDSSDKN
VICLGRMDSQVKIRGQRVELDAIENLLRRQFPSDVTVVAEAVKRSDLPSSVVITGFLISS--EY
VVYLGRRDSQVKIRGQRVETSEVEAGLRQQSSSHIMPVVEAVKRLDSSNSTVLVAFLIGLS--IVCLGRMDSQVKIRGQRVELGAVETHLRQQMPDDMTIVVEAVKFSDSSSTTVLTAFLIGA---IVCLGRIDSQVKIRGQRVELGAIETHLRQQMPDDLTIVVEATKRSQSANSTSLIAFLIGSS--Y
951
935
929
933
938
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
952
936
930
934
939
NSSAASAKDARILDQTATQEMNAKLCQVLPPHSVPSCYICMHALPRTATGKVDRKTLRSIGSKL
VVGAPSTEDTYILDQVVTQEINAKMRQILPAHSIPSFYICMKSLPRTATGKVDRRKLRSIGSSL
-KGGEEAADAYILETSVAGGINAKLQQVLPQHSIPSFYIRMKDLPCTATGKTDRRRLRSIASKL
---GEKNS--HILDQRATREINAKMEQVLPRHSIPAFYISMNNLPQTATGKVDRRKLRIMGSKI
--FGNRPSDAHILDHDATKAINIKLEQVLPRHSIPSFYICMLELPRTATGKIDRRRLRIMGKDI
1015
999
992
992
1000
BbBEAS 1016 LEQQAYKKSPETMQKSKSAETLETGPEARLKEVWLQSFNLEPASPKCGASFFELGGDSITAIKM 1079
BbBSLS 1000 LALQAQSTAP----RSSQAPDASAG-VTKLEEVWMDIFNLTPNSHNIGGNFFALGGDSITAIKM 1058
XsBSLS 993 LSELFQNVAS---QPSAKFGSSATSTEDKLREIWFRGLNLDLKSNSGRASFFELGGDSITAIKM 1053
FeESYN 993 LSQKTHSTPS---QQSQAAISSGTDTYTKLESIWITSLDLEPGSANMSATFFEMGGNSIIAIKM 1053
FvBEAS 1001 LDKQTQGAIV---QQAPAPIPVFADTAAKLHSIWVQSLGIDPATVNVGATFFELGGNSITAIKM 1061
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1080
1059
1054
1054
1062
VNMARAAGLELKVSDIFQNPTLARLQAVMSGDSTPSTITTPFATIPASTWDGPV-EQSYSQGRL
VNMARAAGIQLKVSDIFQNPTLASLQAAIGGSS--MTVTS----IPALALDGPV-EQSYSQGRL
VNMARSAGIVLKVSDIFHNPTLAGLIDVIGQGS--APYSP----IPTTTYSGPV-EQSYAQGRI
VNMARSNGIELKVSDIYQNPTLAGLKAIVIGTS--LPYSL----IPKVTRQGPVSEQSYAQNRM
VNMARSVGMDLKVSNIYQHPTLAGISAVVKGDP--LSYTL----IPKSTHEGPV-EQSYSQGRL
1142
1115
1110
1111
1118
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1143
1116
1111
1112
1119
WFLDQLDIGAVWYLIPYAVRMRGALNIDALRAALLALEQRHETLRTTFENQNGVGVQIVHQRLA
WFLDQLEIGANWYTIPYAVRLRGPLDVDALNRALLALEKRHETLRTTFEDQDGVGVQIIHETLL
WFLDQLKLGASWYLIPYGVRMRGPLRVDALTIALLALEQRHETLRTTFEERDGVGVQVVHASCI
WFLDQLSEGASWYLIPFAVRMRGPVDVDALTRALLALEQRHETLRTTFENQDGVGVQIIHDRLS
WFLDQLDVGSLWYLIPYAVRMRGPVNVDALRRALAALEQRHETLRTTFEDQDGVGVQIVHEKLS
1206
1179
1174
1175
1182
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1207
1180
1175
1176
1183
KELKIIDASS-HGDDGYLQPLEQEQTTPFDLTCEAGWRASLICVGEDHHVLSIVMHHIVSDGWS
DQLRIINAD--HAD--YVQLLKQEQTAPFNLASESGWRVSLIRLDDDDNILSIVMHHIISDGWS
KELRLIDVTDQKGD--YLQSLQQEQAAPFDVTSEAGWRVSLIRLGEDDHILSIVMHHIISDGWS
KELQVIDALD--GDEGGLKTLYKVETTTFDITSEAGWSSTLIRLGKDDHILSIVMHHIISDGWS
EEMKVIDLCG--SDLDPFEVLNQEQTTPFNLSSEAGWRATLLRLGEDDHILTIVMHHIISDGWS
1269
1239
1236
1237
1244
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1270
1240
1237
1238
1245
IDVLRQELGQLYAAVLHGDEDPLSAVSPLPIQYRDFSMWQRR-QQVAEHDRQLQYWRKQLADCS
IDVLRRELGQLYAAALHGADLFGSALSPLPIQYRDFSVWQKQDAQVAEHERQLQYWQKQLADCS
IDILRQELSKFYAAALRG-HDPLSGISPLPIHYRDFAVWQKQEEQVAEHQRQLEYWTHQLADST
IDVLRRELIQLYAAALQ-GKDPSSALTPLPIQYSDFAVWQKQEAQAAEHERQLQYWKKQLADSS
IDVLRRDLNQLYSAALKDSKDPLSALTPLPIQYSDFAKWQKD--QFIEQEKQLNYWKKQLKDSS
1332
1303
1299
1300
1306
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1333
1304
1300
1301
1307
PAKLPTDFPRPPLLSGDAGSVPVEISGELFQKLHRFCNVTSTTPFAVLLAAFRAAHYRLTGVDD
PAKLPTDFHRPALLSGKATTVPVTITSELYYRLQEFCSTFNTTSFVVLLATFRAAHYRLTGVDD
PAEFLTDLPRPTILSGQAGFVPVTIDGELYKKLREFCKAQQMTSFAVLLAAFRAAHYRLTGAED
PAKIPTDFPRPDLLSGDAGVVPVAIDGELYQKLRGFCNKHNSTAFSILLAAFRAAHYRLTAVDD
PAKIPTDFARPALLSGDAGCVHVTIDGELYQSLRAFCNEHNTTSFVVLLAAFRAAHYRLTAVED
1396
1367
1363
1364
1370
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1397
1368
1364
1365
1371
AVVGTPIANRNRPELERLIGFFVNTQCMRITVDDD-DTFEGLVRQVRRTTTEAFENEDVPFERV
AVIGTPIANRNRHELENLIGFFVNTQCMRITINEDEDTFESLVRQVRSTTTAAFEHEDVPFERV
ATIGTPIANRNRWEIENMIGFFVNTQCMRITIDGE-DTFESLVRQVRSTATAAFEHQDVPFERV
AVIGIPIANRNRWELENMIGFFVNTQCMRIAVDET-DTFESLVRQVRSTTTAAFAHEDVPFERV
AVIGTPIANRNRPELEDIIGCFVNTQCMRINIDHH-DTFGTLINQVKATTTAAFENEDIPFERV
1459
1431
1426
1427
1433
BbBEAS
BbBSLS
XsBSLS
FeESYN
1460
1432
1427
1428
VSAMLPAGGGSRDLSQTPLAQLIFAVHSQENLGKFELEGLESEPVANKAYTRFDAEFHLFQTRD
VSAMLP---GSRDLSQNPLAQLVFAIHSHKDLGKFELEALESEPLQNEVYTRFDAEFHFFQAPD
VSALLP---GSRDTSRNPLVQLIFAVHSQQDLGKFELEGLEGEPVSNAVVTRFDVEFHLFQEVD
VSALQP---GHRDLSRTPLAQIMFAVHSQKDLGRFELEGIQSEPIASKAYTRFDVEFHLFQQAD
1523
1492
1487
1488
Electronic Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2013
FvBEAS 1434 VSALQP---GSRDLSSTPLAQLIFAVHSQKDLGRFKFQGLESVPVPSKAYTRFDMEFHLFQETD 1494
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1524
1493
1488
1489
1495
GLNGYLNFAAELFKLETMQNVVSVFLQILRHGLEQPKSLISVLPLTDGLKELDSMGLLKIHRGL
GLTGYINFATELFKVETIQNVVSVFLQILRHGLEHPQTLISVVPLTDGLAELRSMGLLEIKK-V
KLSGNFVFAADLFKLENIQNVVNVFYEILRQGIDQPQTPIAVLPLTDGLAELRSMGLLEIEK-A
GLKGSCNFATDLFKPETIQNVVSVFFQILRHGLDQPETCISVLPLTDGVEELRRLDLLEIKR-T
SLKGSVNFADELFKMETVENVVRVFFEILRNGLQSSRTPVSILPLTDGIVTLEKLDVLNVKH-V
1587
1555
1550
1551
1557
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1588
1556
1551
1552
1558
EYQRDSSLVDIFRSQVATCPDTIAVIDSSARLTYAQLDHQSNLLEAWIRRKGLPAESLVGVLSP
EYPRDSSVVDVFRTQVASYPDTLAVVDSSSRLTYAELDHQSDLLATWLRQQNLPTEALVVVLAP
EYPRESSVVDVFRDQVAAHSDALAVTDSSSRLTYAELDRKSDQLATWLRQQNMAAETVVGVLAP
NYPRDSSVVDVFREQAAANPEVIAVTDSSSRLTYAELDNKSELLSRWLRRRNLTPETLVSVLAP
DYPRESSLADVFQTQVSAYPDSLAVVDSSCRLTYTELDRQSDILAGWLRRRSMPAETLVAVFAP
1651
1619
1614
1615
1621
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1652
1620
1615
1616
1622
RSCETIIAFLGILKANLAYLPLDPKSPVSRMRDVLSDLPGHTIILLGSDVAAPDLELPCLELVR
RSCETIITFLGILKANLAYLPLDIRSPITRMRDVLSTLPGRTIALLCSDEVAPDFQLPSIELVR
RSCQTIIAFLGILKASLAYLPFDVNIPAARLQAVLSELSGHKLVLLGSDTTAPKVQLSDVEFVR
RSCETIVAYVGILKANLAYLPLDVRSPVTRMKDILSSVSGNTIVLMGSGVEDPGFDLPQLELVR
RSCETIVAFFGVLKANLAYLPLDVRSPSARVQDILSGLSGPTIVLIGHDTAPPDIEVTNVEFVR
1715
1683
1678
1679
1685
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1716
1684
1679
1680
1686
ISDALKS--GASAVNGSETTD--LSAPSANSLAYVLYTSGSTGRPKGVMVEHRAIVRLVQRGVI
IADALEEAAGMTSLNGHEHVP--VPSPSPTSLAYVLYTSGSTGRPKGVMIEHRAIVRLARSDII
IKDTLEH----RDLNGHADVV--AASPSAKSLAYVIFTSGSTGRPKGVMVEHRSIIRLAKESNL
ITDTFDE----T----IEDVQ-DSPQPSATSLAYVVFTSGSTGKPKGVMIEHRAIVRLVKSDNF
IRDALND----SNADGFEVIEHDSTKPSATSLAYVLYTSGSTGRPKGVMIEHRVIIRTVTSGCI
1775
1745
1736
1734
1745
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1776
1746
1737
1735
1746
PNFPPLRGAIMAHLFNTVFDGATYEIFLMLLNGGTLVCIDYLTTLSPKALETVFLREGINCAIM
PDYRPACGDTMAHMFNTAFDGATYEIYTMLLNGGTLVCVDYMDTLSPKSLEAVFKKEQVNATIM
ISKLPT-AVTVGHLSSIAFDAAIWEVYTALLNGGTLVCIDYMTILDSKALEATFAREQVQAVLI
PGFPSP--ARMSNVFNPAFDGAIWEINWMLLNGGTVVCIDYLTTLDGKELAAVFAKERVNAAFF
PNYPSE--TRMAHMATIAFDGASYEIYSALLFGRTLVCVDYMTTLDARALKDVFFREHVNAASH
1839
1809
1799
1796
1807
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1840
1810
1800
1797
1808
TPALLK-LYLANARDGLKGLDMLMVAGDRFDPQDAVEAQTLVRG-DCYNAYGPTENGVMSTLYK
APALLK-LYLADARDALKGLDVLISGGDRFDPQDAVDAQSLVRG-SCYNGYGPTENGVFSTVYK
TPALIK-QCLADAPAILAALDVLVSGGDRFDGQDAIAAQALVRL-GVYNAYGPTENGVMSTIYK
APAMLK-LYLVDAREALKNLDFLIVGGERFDTKEAVEAMPLVRG-KIANIYGPTEAGIISTCYN
VTSSSQDVPLRVPRRLSRTLMFFFLVVTDSTAPDALDAQGLYQGVQCYNGYGPTENGVMSTIYP
1901
1871
1861
1858
1871
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1902
1872
1862
1859
1872
IDTSDSFINGVPLGRAIDNSGAYITDPNQQLVGPGVLGELIVTGDGLARGYTDPALDRDRFVQV
VDKNDPFVNGVPLGRAVNNSGAYVVDRNQQLVGPGIIGELVVTGDGLARGYTERAFDQNRFTQL
VTGNDSFINGVPIGRAISNSGAYIMDPNQQLVPAGVMGELVVTGDGLARGYTDSTLDIDRFVQV
IPKDEAYTNGVPIGGSIYNSGAYVMDPNQQLVGLGVMGELVVTGDGVGRGYTNPELNKNRFIDI
IDSTESFINGVPIGRALNNSGAYVVDPEQQLVGIGVMGELVVTGDGLARGYSDKALDENRFVHI
1965
1935
1925
1922
1935
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
1966
1936
1926
1923
1936
VINGESVRAYRTGDRMRYRAGQDCLFEFFGRMDFQFKIRSNRIESAEVEAAILSHPLVRDAAIV
KVEGQSVRGYRTGDRVRYRVGEG-LIEFFGRMDFQFKIRSNRIEAGEVEAAILSHPAVRNAAVI
NIDGQLVTAYRTGDRVRYRAGDG-QIEFFGRMDQQVKIRGHRIELAEVERAILSQNSVRDAVVV
TIEGKTFKAYRTGDRMRARVGDG-LLEFFGRMDNQFKIRGNRIEAGEVESAMLSLKNVLNAAIV
TVNDQTVKAYRTGDRVRYRIGDG-LIEFFGRMDTQFKIRGNRIESAEIEAALLRDSSVRDAAVV
2029
1998
1988
1985
1998
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2030
1999
1989
1986
1999
VVGVQEEQEP-EMVGFVVAA--DDAVEQEATDNQVEGWQELFESSMYNGID-AISPSALGKDFT
LR-VEEKLEP-EIVGFVVAEH-DDTAEQEEAGDQVEGWQAFFESTTYTELD-TVSSSEIGKDFK
IQ-NQEGQEP-EMVGFIVTQD-DHSTEQDEAGNHVEGWGDLFESSTYADIN-TIDQSAL-HDFT
VRGGGEDEGPLEMVGFIVADDKNDTTEEEETGNQVEGWQDHFESGMYSDISTAVDQSAIGNDFK
LQQN-EDQAP-EILGFVVADHDHSENDKGQSANQVEGWQDHFESGMYSDIG-EIDPSTIGSDFK
2089
2058
2047
2049
2059
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2090
2059
2048
2050
2060
GWTSMYDGSEIDKSEMQEWLDDTIHTLRDGHVPGHVLEIGTGTGMILFNLGS-VESYVGLEPTK
GWTSMYDGNEIDKAEMQEWLDDTIHTLTDGQALGHVLEIGTGSGMVLFNLGSGLQSFVGLEPSK
GWTSMYDGSLINKAEMQEWLDDTIHTLLDGQAPGHVLEIGTGSGMVMFNLGTGLQSYVGLEPSR
GWTSMYDGKDIDKGEMQEWLDDAIHTLHNGQIPRDVLEIGTGSGMILFNLNPGLNSYVGLDPSK
GWTSMYDGSQIDFDEMHEWLGETTRTLHDNRSLGNVLEIGTGSGMILFNLDSRLESYVGLEPSR
2152
2122
2111
2113
2123
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2153
2123
2112
2114
2124
SAVEFVNKAIKTLPNLAGRAEVHTGTATDIDQLSGLRPDLVILNSVVQYFPTVEYLTRVVDALV
SAAAFVNNAIKSTPALAGKAQVFVGTATDTNKLDDLHPDLVIFNSVLQYFPTRDYLERVVDALV
SAAAFVNSTIKSIPALAGKAEVHVGTATDISRLSGLCPELVVINSVMQYFPTPEYLVEVVDTLA
SAVEFVNRAVESSPKFAGKAKVHVGMATDVNKLGEVHPDLVVFNSVVQYFPTPEYLAEVIDGLI
SAAAFVNKATESIPSLAGKAKVQVGTATDIGQVDDLHPDLVVLNSVIQYFPSSEYLAEIADTLI
2216
2186
2175
2177
2187
BbBEAS 2217 RIRGVKRLFFGDVRSQALHRQFLAACAMHALG--KTATRDDVRRYMAEREEREEELLVEPAFFT 2278
BbBSLS 2187 HLRSAKRIFFGDVRSYATNRHFLAARAIYTLG--NHTTKDEVRKKMAEMEEREEEFLVEPAFFT 2248
XsBSLS 2176 RIPGVKRLFFGDVRTHATNRHFLAARALHALG--DKSTKESLRQKIAELEEREEELLVDPAFFT 2237
Electronic Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2013
FeESYN 2178 AIPSVKRIFLGDIRSYATNGHFLAARAIHTLGTNNNATKDRVRQKIQELEDREEEFLVEPAFFT 2241
FvBEAS 2188 HLPNVQRIFFGDVRSQATNEHFLAARAIHTLG--KNATKDDVRQKMAELEDMEEELLVEPAFFT 2249
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2279
2249
2238
2242
2250
ALMNRHPNLIQHVEILPKNIRATNELSAYRYAAVVHLRDPESAARPVYPIAADDWVDFQASQMR
TLVNRLPD-VRHVEIIPKNMQATNELSAYRYAAVVHLRGSDELTRPVHPIKMDDWVDFQASHMH
TLATRLPDQVEHVEILPKKMRATNELSAYRYAAVVHMRGSEERVQPVYPINDNDWVDFQASQMD
TLKERRPDVVKHVEIIPKNMKATNELSAYRYTAVVHLRDET--DEPVYHIEKDSWVDFEAKQMD
SLKDRFPGLVEHVEILPKNMEAVNELSAYRYAAVVHVRGSLG-DELVLPVEKDDWIDFQANQLN
2342
2311
2301
2303
2312
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2343
2312
2302
2304
2313
SDVLREYLRLSAGADTVAVCNIPYEKTIFERLIVESLDDNTGSDAPQSRLHGRSLDGAPW-ISA
KDALREYLRLAENTKTVAISNIPYGKTIFERQVVESLDE-TSEDAPHA-----SLDGAAW-ISA
RDALLYLLQRSADASTVAISNIPYSKTILERHVVESLDD--NGDNARS-----SLDGAAW-FSA
KTALLDHLRLSKDAMSVAVSNITYAHTAFERRIVESLDEDSKDDTKGT------LDGAAW-LSA
QKSLGDLLK-SSDAAIMAVSKIPFEITAFERQVVASLNSN--------------ID--EWQLST
2405
2368
2357
2360
2359
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2406
2369
2358
2361
2360
VRSDAESRASLSVPDLVQLAAESGFQVQVSAARQWSQSGALDAVFHRRHASSSQPTMRTLFQFP
VRSDAKARSSLSVPDLVLLAKETGFRVEVSAARQWSQSGALDAVFHRYPA---EPGVRTLFQFP
VRSDAERCASLSVTDLVQLGEEAGFRVEVSCARQWSQSGALDAVFHHYSP--AYKGARVLIRFP
VRSEAENRASLTVPDILEIAKEAGFRVEVSAARQWSQSGALDAVFHHFPP---SSTDRTLIQFP
IRSSAEGDSSLSVPDIFRIAGEAGFRVEVSSARQWSQNGALDAVFHHC-----CSQGRTLVNFP
2469
2429
2419
2421
2418
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2470
2430
2420
2422
2419
DDNALRASATLTNRPLQRLQRRRVAAQIRERLQTLVPSYMIPAKIVVLDQMPLNANGKVDRKEL
TDNDVRMSAPLTNQPLQRLQKRRVAVQVREWLQDRIPSYMIPSHIVALDQMPLNTSGKVDRKEL
TDDQARRPATLTNRPLQRLQSRRFVSQVREELQVLLPSYMIPARIVVVDQMPLNASGKVDRKEL
TDNELRSSLTLANRPLQKLQRRRAALQVREKLQTLVPSYMVPPNIVVLDTMPLNTNGKIDRKEL
TDHHLRGSDLLTNRPLQRLQNRRIAIEVRERLRSLLPSYMIPSNIVVLDKMPLNANGKVDRKEL
2533
2493
2483
2485
2482
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2534
2494
2484
2486
2483
ARRARTTTMTKKKKPQRLASEPACPISDIEVALCEEATATFGMQVGISDHFFKLGGHSLLATKL
SRQAKAIKKVQKSAPP---TAPAFPLSEVEVMLCEELTKTFEMDVNITDDFFQLGGHSLLATRL
ARRAQVVPKSEAAPAQ------VVPLNEIEVMLCEEFADVFGIDVVVTDNFFKLGGHSLMATKL
TRRARTLPKQQTAAPV-----PDFPISDIEITLCEEATEVFGMKVEISDHFFQLGGHSLLATKL
SRRAKVVPKQQTAAPL-----PTFPISEVEVILCEEATEVFGMKVDITDHFFNLGGHSLLATKL
2597
2554
2541
2544
2541
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2598
2555
2542
2545
2542
ISRVGDRLKARLTVKDVFDHPIFSELAIVIREGLQN---VVPVALNGGG-QAKQGSAGVVAPRN
VARISHRLGARLTVKDVFDYPVFSELADIIRQQLASKNTLLPTASAGGGGQDKKESAG-VAPTT
AARISRRLDARVSVKEVFDQPIVADLAASIRRGLASRN-LTPTTTFSGH--LGQVVSARVAPRN
ISRIQHRLHVRVTVKDVFDSPVFADLAVIIRQGLAMQN---PVAEGQDK----QGWSSRVAPRT
ISRIDQRLKVRITVKDVFDHPVFADLASVIRQGLGLQQ---PVSDGQG-----QDRSAHMAPRT
2657
2617
2602
2601
2597
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2658
2618
2603
2602
2598
EMETMLCEEFANVLGMDVGVTDN-FFDLGGHSLMATKLAARIGRRLNTTISVKEVFEHPIVFQL
DMEAMLCEEFANILGMDVGITDN-FFDLGGHSLMATRLAARIGHRLNTTISVKDIFSHPVIFQL
DIEAVLCKEFADVLGVLTLVSLTAFFDLGGHSLMATKLAARLSRRLDAPISVKDIFDYPVPSKL
EVEKMLCEEFAAGLGVPVGITDN-FFDLGGHSLMATKLAVRIGRRLIRHHS-QGHLRLPCAFQL
ETEAILCDEFAKVLGFQVGITDN-FFDLGGHSLMATKLAVRIGHRLDTTVSVKDVFDHPVLFQL
2720
2680
2666
2663
2660
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2721
2681
2667
2664
2661
ANSLEL-GQLESDR--VKHTMLADYTAFQLLSVEDLQGFLQNEISPQLECAHGGIQDVYPATHM
SAKLEV-SQLESSSG-GTDIKMPDYTAFQLIPAADAEKFMQDHIYPQINFSQDMVQDVYLATHL
ANHIHI-TQFESYDL-TNGIPAADGASFQLLAPEDPLAFIEREISPQLQYSSDTILDVYPATQM
AKKLES-SHSKSYEESGDDIQMADYTAFQLLDLEDPQDFVQSQIRPQLDSCYGTIQDVYPSTQM
AIALDNLVQSKTNEIVGG-REMAEYSPFQLLFTEDPEEFMASEIKPQLE-LQEIIQDIYPSTQM
2781
2742
2728
2726
2722
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2782
2743
2729
2727
2723
QKAFLCDASTGHPKPLVPFYIDFPPDS--DCSTLVEACSSLVKRFDMFRTVVVEAAGELYQVVL
QQCFLRDV-FGRPKPLVPFYVEFPPDS--NPHTLATACTSLVDKYDIFRTIFVEAEGNLYQVVL
QRLFLRNPRTGHPWTITPFCIDFPVDS--DCAKLAKACTSLVQHFDMFRTVFLVAAGQLHQVVL
QKAFLFDPTTGEPRGLVPFYIDFPSN--ADAETLTKAIGALVDKLDMFRTVFLEAAGDLYQVVV
QKAFLFDHTTARPRPFVPFYIDFPSTSEPDAAGLIKACESLVNHLDIFRTVFAEASGELYQVVL
2843
2803
2790
2788
2786
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2844
2804
2791
2789
2787
EHFDLQIDVVETEENVHAATNDFVDRILEVPVHLGQPLIQFTILKQASS-VRVLLCLSHALYDG
KHLNLDIDVVETDANVHKTSSDLVDAIAKEPVRLGQPMIQVKVLKQTSS-VRVLLWLSHALYDG
KQVDVPIEIVETEENINSATRAFLDLDLEKPVRLGEPVLRIAILKTKASPLRIILRMSHALYDG
EHLNLPIETIETEKNVNTATGDYLDVHGKDPVRLGHPCIQFAILKTASS-VRVLLRMSHALYDG
SCLDLPIQVIETEDNINTATNEFLDEFAKEPVRLGHPLIRFTIIKQTKS-MRVIMRISHALYDG
2906
2866
2854
2851
2849
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
2907
2867
2855
2852
2850
LSLEHVVRDLHMLYKGRSLLPANQFSRYMQYMDHTRKAGCDFWRDVIQDTPITVLGHVDAGGRE
LSWEHIVRDLHILSKERSLPPATQFSRYMQYVDHTRGPGCDFWRDVLQNAPITNLSDAGSGGRP
IELEQIVRSLHVLYSGGSLPTPPKFARYMEHMANSRKDGYEFWRSVLQNSSMTSIESASSNARQ
LSFEYIVRGLHVLYSGRNLPPPTQFARYMQYAAHSREEGYPFWREVLQNAPMTVLHDTNNGMSE
LSLEHVVRKLHMLYNGRSLLPPHQFSRYMQYTADGRESGHGFWRDVIQNTPMTILSDDT--VVD
2970
2930
2918
2915
2911
BbBEAS 2971 LEVEAARTLHATKIISIPLQAVRSSI--ITQATVFNAACALVLSRETGAKDVVFGRIVSGRQGL 3032
BbBSLS 2931 TKAGDPRVWHAGKVISGPSQAIRSS---ITQATVFNAACAIVLSKETGTDNVVFGRIVSGRQGL 2991
Electronic Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2013
XsBSLS 2919 KEVGPVGTCRASKGISIPLQGSADSR--ITQATVFATACALMLSKETKSRDVTFGRIVSGRQGL 2980
FeESYN 2916 QEMPASKAVHLSEVVNVPAQAIRNSTN--TQATVFNTACALVLAKESGSQDVVFGRIVSGRQGL 2977
FvBEAS 2912 GNDATCKALHLSKIVNIPSQVLRGSSNIITQATVFNAACALVLSRESDSKDVVFGRIVSGRQGL 2975
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3033
2992
2981
2978
2976
PVSWQNIVGPCTNAVPVRARIIDDDDDNHRQMLRDMQDQYLLSLPFETLDFDEVRRSCTNWPAT
PVRWQNIIGPCTNAVPVRA-VVDAHG-NHQQMLRDLQEQYLLSLPYETIGFDEIKRSCTDWPDS
PITCQHVVGPCTNTIPVRL-CMDENP-NLRELLRKMQDQYLDSLPFETLGFDDLKKNCTEWSDE
PVVWQDIIGPCTNAVPVHARVDDGNP---QRIIRDLRDQYLRTLPFESLGFEEIKRNCTDWPEE
PVEYQDIVGPCTNAVPVRAHIESSDY---NQLLHDIQDQYLLSLPHETIGFSDLKRNCTDWPEA
3096
3053
3042
3038
3036
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3097
3054
3043
3039
3037
ANNYACCVTYHDFSYHPESEMEQQRVEMGVLARKDALLKEEPVYDLGIAGEVEPDGVHLQVTVV
ARNYGCCVTYQNFEYHPESEVDQQRVEMGILAKKAELIKEEPLYNVAIAGEVEPDGVHLQVTVV
TTTYGCCIVYQNFNFHPESQVEEQRIQIGVLSREDQLANEASIHDLVLAGSPEPDGQHLHVTVV
LTNFSVCVTYHNFEYHPESEVDNQKVEMGVLAKYVELSENEPLYDLAIAGEVEADGVNLKVTVV
ITNFSCCITYHNFEYHPESQFEQQRVEMGVLTKFVNIEMDEPLYDLAIAGEVEPDGAGLKVTVI
3160
3117
3106
3102
3100
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3161
3118
3107
3103
3101
AKTRLFSEERAAYLMEEVCRLFESLNSAL
VDSQLFSQEGATHLMEQVCNTFQALNASL
ANRQLCDDSRVKHMLEELCEMIETLSKALRDPYISDTNPANPESSLVAPVEIPRTPTNGFAPDI
AKARLYNEARIRHVLEEVCKTFNGLNEAL
AKTQLFGRKRVEHLLEEVSKTFEGLNSSL
3189
3146
3170
3131
3129
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3190
3147
3171 SSESRIHILGVGNLGKFVAHALRKRHPQLPITLLFHRADLVSEWDAAVKAITCVTSGVSDKSTG
3132
3130
3189
3146
3234
3131
3129
BbBEAS
BbBSLS
XsBSLS
ESYN
FvBEAS
3190
3147
3235 FDVEFLSNSTGHKTLIKHLIVATRTYMTVSAVDLVKRRLESTSSILFLQNGMGKNRHRAMNDVK
3132
3130
3189
3146
3298
3131
3129
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3190
3147
3299 SRPSYWAGVCSCGVYPTSPFSIVHAGRGPIVLGRVLEEANDSPESTSAASVAQDFMIQRLLETP
3132
3130
3189
3146
3362
3131
3129
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3190
3147
3363 DLEATLTTPNDIKEVQLQKLIVNSMINPLTVVFRCNNGQLFGQPARLALMRTLLDEAGEIVRAI
3132
3130
3189
3146
3426
3131
3129
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3190
3147
3427 LPQESQKSGNSQLTNENLLAAVLRVAEMTGECSSSMLQDVQAGRRTEIDHINGYLALQGKRLGL
3132
3130
3189
3146
3490
3131
3129
BbBEAS
BbBSLS
XsBSLS
FeESYN
FvBEAS
3190
3147
3491 PYSNIETLVNMVKQGRAIVNGDIQSLFKMSGIKPMDLGKVLKAGSSNGAVAAYGKR
3132
3130
3189
3146
3546
3131
3129
Fig. S1
Amino acid sequence alignment of five fungal CODSs. BbBEAS: GenBank accession number
ACI30655; BbBSLS: GenBank accession number ACR78148; XsBSLS: GenBank accession number
ABR28366; FvESYN: GenBank accession number Q00869; FvBEAS: GenBank accession number
AGC31490. The intermodular M1-M2 linker is boxed.
Electronic Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2013
BbBEAS
BbBSLS
VibH
1087 GLELKVSDIFQNPTLARLQAVMSGDSTPSTITTPFATIPASTWDGPVEQSYSQGRLWFLDQLDI 1150
1066 GIQLKVSDIFQNPTLASLQAAIGGSS--MTVTS----IPALALDGPVEQSYSQGRLWFLDQLEI 1123
1
MLLAQKPFWQRHLAYP 16
BbBEAS
BbBSLS
VibH
1151 GAVWYLIPYAVRMRGALNIDALRAALLALEQRHETLRTTFENQNGVGVQIVHQRLAKELKIIDA 1214
1124 GANWYTIPYAVRLRGPLDVDALNRALLALEKRHETLRTTFEDQDGVGVQIIHETLLDQLRIINA 1187
17 HINLDTVAHSLRLTGPLDTTLLLRALHLTVSEIDLFRARFSAQ---GELYWHP-FSPPIDYQDL 76
BbBEAS
BbBSLS
VibH
1215 SSHGDDGYLQPLEQEQTTPFDLTCEAGWRAS--LICVGEDHHVLSIVMHHIVSDGWSIDVLRQE 1276
1188 D-HAD--YVQLLKQEQTAPFNLASESGWRVS--LIRLDDDDNILSIVMHHIISDGWSIDVLRRE 1246
77 SIHLEAEPLAWRQIEQDLQRSSTLIDAPITSHQVYRLSHSEHLIYTRAHHIVLDGYGMMLFEQR 140
BbBEAS
BbBSLS
VibH
1277 LGQLYAAVLHGDEDPLSAVSPLPIQYRDFSMWQRR-QQVAEHDRQLQYWRKQLADCSPAKLPTD 1339
1247 LGQLYAAALHGADLFGSALSPLPIQYRDFSVWQKQDAQVAEHERQLQYWQKQLADCSPAKLPTD 1310
141 LSQHYQSLLSG-QTPTAAFKPYQSYLEEEAAYLTS----HRYWQDKQFWQGYLR-EAPDLTLTS 198
BbBEAS
BbBSLS
VibH
1340 FPRPPLLSGDAGSVPVEISGELFQKLHRFCNVTSTTPFAVLLAAFRAAHYRLTGVDDAVVGTPI 1403
1311 FHRPALLSGKATTVPVTITSELYYRLQEFCSTFNTTSFVVLLATFRAAHYRLTGVDDAVIGTPI 1374
199 ATYDPQLS-HAVSLSYTLNSQLNHLLLKLAN-ANQIGWPDALVALCALYLESAEPDAPWLWLPF 260
BbBEAS
BbBSLS
VibH
1404 ANRNRPELERLIGFFVNTQCMRITVDDD-DTFEGLVRQVRRTTTEAFENEDVPFERVVSAMLPA 1466
1375 ANRNRHELENLIGFFVNTQCMRITINEDEDTFESLVRQVRSTTTAAFEHEDVPFERVVSAMLP- 1437
261 MNRWGSVAANVPGLMVNS---------------------------------LPLLRLFAQQTS- 290
BbBEAS
BbBSLS
VibH
1467 GGGSRDLSQTPLAQLIFAVHSQENLGKFELEGLESEPVANKAYTRFDAEFHLFQTRDGLNGYLN 1530
1438 --GSRDLSQNPLAQLVFAIHSHKDLGKFELEALESEPLQNEVYTRFDAEFHFFQAPDGLTGYIN 1499
291 --LGNYLKQS--GQAIRSLYLH---GRYRIEQIE-------------------Q--D--QG-LN 323
BbBEAS
BbBSLS
VibH
1531 FAAELFKLETMQNVVSVFLQILRHGLEQPKSLISVLPLTDGLKELDSMGLLKIHRGLEYQRDSS 1594
1500 FATELFKVETIQNVVSVFLQILRHGLEHPQTLISVVPLTDGLAELRSMGLLEIKK-VEYPRDSS 1562
324 AEQSYFMSP--------FINILP--FESP-------HFADCQTELKVLASGSAEG-------IN 363
BbBEAS
BbBSLS
VibH
1595 LVDIFRSQVATCPDTIAVIDSSARLTYAQLDHQSNLLEAWIRRKGLPAESLVGVLSPRSCETII 1658
1563 VVDVFRTQVASYPDTLAVVDSSSRLTYAELDHQSDLLATWLRQQNLPTEALVVVLAPRSCETII 1626
364 FT--FR---GS-PQHELCLDITADLASYPQSH-------WQS----------------HCERFP 398
BbBEAS
BbBSLS
VibH
1659 AFLGILKANLAYLPLDPKSPVSRMRDVLSDLPGHTIILLGSDVAAPDLELPCLELVRISDALKS 1722
1627 TFLGILKANLAYLPLDIRSPITRMRDVLSTLPGRTIALLCSDEVAPDFQLPSIELVRIADALEE 1690
399 RFFEQLLARFQQVEQD----VAR----LLAEPAALAATTSTRAIAS
436
Fig. S2
Amino acid sequence alignment of BbBEAS, BbBSLS and a stand-alone NRPS condensation
domain VibH (GenBank accession number AAD48879). The intermodular M1-M2 linker is boxed.
Electronic Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2013
A
B
[M+NH4]+
815.0
801.0
[M+NH4]+
100
100
80
80
[M+H]+
60
[M+K]+
821.9
805.9
20
[M+Na]+
40
[M+Na]+
819.9
40
20
[M+K]+
835.8
783.9
60
797.9
[M+H]+
0
0
780
800
820
780
m/z
C
800
820
840
m/z
D
[M+NH4]+
843.0
829.0
[M+NH4]+
100
100
80
80
[M+H]+
[M+H]+
[M+Na]+
[M+K]+
40
833.9
849.9
[M+K]+
20
863.9
40
826.0
60
811.9
60
847.9
[M+Na]+
20
0
0
800
820
840
m/z
820
840
860
m/z
E
909.1
[M+H]+
926.1
80
931.0
[M+NH4]+ [M+Na]+
100
60
40
[M+K]+
947.0
20
0
900
920
940
m/z
Fig. S3 ESI-MS spectra of beauvericin (A), beauvericin A (B), beauvericin B (C), beauvericin C (D) and
bassianolide (E).