Analysis of Tubulysin Biosynthetic Genes in Archangium gephyra

Archangium gephyra ᮹ tubulysin ᔾ⧊ᖒ ᮁᱥᯱ ᇥᕾ

↽ᵝ᪅^1,2, ၶ┽ᵡ¹, v݅ᬕ², ᯕᱶᵝ², ʡᩢ⦥², ᯕ⦥Ǎ², ᱶᰍᬊ², ᳑Ğᩑ¹*

1⪙ᕽݡ⦺Ʊᔾ໦Ŗ⦺ŝ

2ີ⎶ᜅ⒱ᨕີऽ(ᵝ)

Received: April 26, 2021 / Revised: May 13, 2021 / Accepted: May 14, 2021

ᕽ ು

⡍ᮁඹᖙ⡍ᨱᕽၙᖙᗭš(microtubule)ᮡᖙ⡍᮹ᇥᩕ^,

ᖒᰆ^, ᯕ࠺ᨱđᱶᱢᯙᩎ⧁ᮥ⦽݅^[1]. ၙᖙᗭšᮡ⛽ᇩฑ

(tubulin)ᮝಽǍᖒࡹᨕᯩᮝ໑^, ⛽ᇩฑ᮹ᵲ⧊ŝ⧕ℕෝ☖

⧕ʑ܆ᮥᙹ⧪⦽݅^. ᯕ్⦽ᵲ᫵⦽ʑ܆ᮝಽᯙ⧕ၙᖙᗭš

ᮡ⧎ᦵᱽ}ၽ᮹ᵝ᫵┡Āᯕࡹᨕ᪵݅^. ၙᖙᗭš᮹ʑ܆ᮥ

ᱡ⧕⦹۵ ݡ⢽ᱢᯙ ⧎ᦵྜྷḩಽ۵ paclitaxel, vinblastine, epothilone ॒ᯕᯩ݅^[2].

TubulysinᮡᱱᧂᖙɁᨱ᮹⧕ᔾᔑࡹ۵ᯕ₉ݡᔍᔾญ⪽

ᖒྜྷḩಽ⛽ᇩฑ᮹ᵲ⧊ᮥᱡ⧕⦹ᩍၙᖙᗭš᮹⧕ℕෝⅩ

௹⧉ᮝಽ៉ᖙ⡍ࠦᖒᮥᅕᯕ۵ߑ(Fig. 1) [3−5], paclitaxel, vinblastine, epothilone ᅕ݅ࠥ⭉ᦍ޵vಆ⦽⪽ᖒᮥӹ┡

ԕ۵äᮝಽᅕŁࡹᨩ݅[6]. Tubulysinᮡ݅᧲⦽ᦵᖙ⡍ᵝᨱ ݡ⧕⧎ᦵ⪽ᖒᮥᅕᯕḡอ^{[7, 8]} v⦽ᖙ⡍ࠦᖒᮝಽᯙ⧕↽ ɝᨱ۵⧎ℕ᧞ྜྷᱲ⧊ℕ(antibody-drug conjugate)᮹┲ᰍ᧞

ྜྷಽšᝍᮥၼŁᯩ݅^[9−^12].

Tubulysinᮡ ݅ᖐ }᮹ ᦥၙיᔑ(L-pipecolic acid, L- isoleucine, L-valine, L-cysteine, L-phenylalanine ੱ۵ ^L- Analysis of Tubulysin Biosynthetic Genes in Archangium gephyra

Juo Choi^1,2, Taejoon Park¹, Daun Kang², Jeongju Lee², Yungpil Kim², Pilgoo Lee², Gregory J. Y. Chung², and Kyungyun Cho¹*

1Department of Biotechnology, Hoseo University, Asan 31499, Republic of Korea

2MECOX CureMed Co., Seoul 06744, Republic of Korea

Tubulysins are a group of bioactive secondary metabolites from myxobacteria exhibiting strong anticancer activity against various cancer cell lines. In this study, we describe the identification of putative tubulysin biosynthetic gene clusters (tubA~tubF) in the genome sequences of two tubulysin-producing myxobacterial strains, Archangium gephyra MEHO_002 and MEHO_004. The inactivation of the putative tubulysin biosyn- thetic genes resulted in a tubulysin-production defect. The DNA sequences of the A. gephyra MEHO_002 and MEHO_004 tubulysin biosynthetic genes were 97% identical, and the amino acid sequences of the encoded proteins shared a similarity of 97−100%. The nucleotide sequences of the tubulysin biosynthetic gene clusters in MEHO_002 and MEHO_004 were 86% identical to that in Cystobacter sp. SBCb004 known as a tubulysin-producing myxobacterium, and the organization of the clusters was identical except for the lack of a tubZ gene in the clusters in MEHO_002 and MEHO_004. The amino acid sequences of the proteins encoded by each gene were 88-97% similar to those encoded by SBCb004, and the domain compositions of the proteins were also identical.

Keywords: Archangium gephyra, tubulysin, myxobacteria, secondary metabolite

*Corresponding author

Tel.: +82-41-540-5627, Fax: +82-41-540-9538

E-mail: [email protected] Fig. 1. The structure of tubulysin A and B.

tyrosine)ŝࢱ݉᭥᮹ ^acetateಽ⧖ᝍǍ᳑aᯕ൉ᨕᲙᯩ۵ ߑ^, ᯕ۵ ⁵}᮹እญᅕᗽ⠊┡ᯕऽ⧊ᖒ⬉ᗭ(non-ribosomal peptide synthetase, NRPS) ༉ऩŝ ²}᮹⡕ญ⍡┡ᯕऽ

⧊ᖒ⬉ᗭ(polyketide synthase, PKS) ༉ऩᨱ ᮹⧕ᔾ⧊ᖒ

ࡽ݅[13]. Tubulysinᮡ ᱱᧂᖙɁ Angiococcus disciformis, Archangium gephyra, Cystobacter sp.ᨱᕽ ᇥญࡹᨩᮝ໑^,

⩥ᰍʭḡ ²³}᮹ᯱᩑᮁࠥℕॅᯕ᦭ಅᲙᯩ݅[5]. Tubulysin ᔾ⧊ᖒ ᮁᱥᯱǑᮡ A. disciformis An d48ŝ Cystobacter sp. SBCb004ಽᇡ░ၾ⩡ᲙᅕŁࡹᨩ݅(Fig. 2) [5, 13]. ⦹ḡ อ A. gephyra᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮡᦥḢʭḡᅕ Łࡹḡᦫᦹ݅^. ᅙᩑǍᨱᕽ۵ᮁᱥℕᇥᕾᮥ☖⧕ǎԕ☁

᧲ᨱᕽᇥญࡽ A. gephyra Ɂᵝॅ᮹ tubulysin ᔾ⧊ᖒᮁᱥ

ᯱǑᮥ┱ᔪ⦹ᩍᇥᕾ⦹ᩡ݅^.

ᰍഭ ၰ ႊჶ

Ɂᵝ ၰ ႑᧲ ᳑Õ

A. gephyra MEHO_001 (=KYC2615)ŝ ^MEHO_003

(=KYC4066)ᮡᅙᩑǍ❡ᯕǎԕ☁᧲ᨱᕽᇥญ⦽Ɂᵝᯕ݅

[14]. A. gephyra MEHO_002۵ A. gephyra MEHO_001᮹ ᇥᔑᄡᯕᵝᯕ݅. A. gephyra MEHO_004, MEHO_005,

MEHO_007ᮡᅙᩑǍᨱᕽᱽ᳑⦹ᩡ݅^. ᱱᧂᖙɁ᮹ᯝၹᱢ

ᯙ႑᧲ᮥ᭥⧕ᕽ۵ ^VY/3 ႑ḡ^[15]ෝᔍᬊ⦹ᩡᮝ໑^, ႑᧲⇵

⇽ྜྷᱽ᳑ෝ᭥⧕ᕽ۵ ^CYS ႑ḡ^[15]ෝᔍᬊ⦹ᩡ݅^. ༉ुɁ ᵝ۵ ³²Ⳅᨱᕽ႑᧲⦹ᩡ݅^.

⥭௝ᜅၙऽ ၰ Ɂᵝ᮹ ᱽ᳑

A. gephyra MEHO_004۵ A. gephyra MEHO_003 Ɂᵝ ᮹ᇥᔑᄡᯕᵝಽ^, ʑ᳕ᨱ᦭ಅḥႊჶ^[16]ᨱ঑௝ᖁၽ⦹ᩡ

݅^. ຝᱡ᧝ᔾ⩶Ɂᵝᯙ A. gephyra MEHO_003ᮥ ^CYS ᧂ ℕ႑ḡᨱᕽĥݡ႑᧲⦽⬥^{, CYS} ⠪❱႑ḡᨱࠥั⦹ᩍ ^S-ᬕ

࠺ᖒᯕᔢᝅࡽɁᵝෝᖁၽ⦽݅ᮭ^, ݅᜽ᧂℕ႑ḡᨱ႑᧲⦹

ᩍᇥᔑᖒᰆ⦹۵äᮝಽ⪶ᯙࡽɁᵝෝ↽᳦ ^MEHO_004

ಽ໦໦⦹ᩡ݅^.

A. gephyra MEHO_005۵ ^MEHO_002 Ɂᵝ᮹ ^tubB ᮁᱥ

ᯱᨱ ^pJO115 ⥭௝ᜅၙऽෝᔞ᯦⦹ᩍᱽ᳑⦹ᩡ݅^{. pJO115}

⥭௝ᜅၙऽ۵ ᪍ญŁەⓕ౩᪅❑ऽ 5′-CTCAACAACGGA TGCTGTTC-3′᪡ 5′-TCACGTAATCGCCATACTGC-3′ෝ

⥥௝ᯕນಽᔍᬊ⦹Ł A. gephyra MEHO_002 Ɂᵝ᮹ᮁᱥ ℕ ^DNAෝᵝ⩶ᮝಽᵲ⧊⬉ᗭᩑᘥၹ᮲(polymerase chain reaction, PCR)ᮥᝅ᜽⦹ᩍ᨜ᮡ ^tubB ᮁᱥᯱ᮹ԕᇡ ^DNA

᳑b^{(507 bp)}ᮥ ^pCR2.1 ᄂ░(Promega, USA)ᨱᔞ᯦⦹ᩍ ᱽ᳑⦹ᩡ݅^. ᯕভ ^DNA ᳑bᯕaḥ ^tubB ᮁᱥᯱ᮹ᱥᔍႊ

⨆ᯕᄂ░᮹ ^lacZ ᮁᱥᯱᱥᔍႊ⨆ŝၹݡႊ⨆ᮝಽᔞ᯦ࡽ

äᮥᖁၽ⦹ᩡ݅^.

A. gephyra MEHO_007ᮡ ^MEHO_004 Ɂᵝ᮹ ^tubB ᮁᱥ

ᯱᨱ ^pJO118 ⥭௝ᜅၙऽෝᔞ᯦⦹ᩍᱽ᳑⦹ᩡ݅^{. pJO118}

⥭௝ᜅၙऽ۵ ᪍ญŁەⓕ౩᪅❑ऽ 5′-CTCAACAACGGA TGCTGTTC-3′᪡ 5′-TCACGTAATCGCCATACTGC-3′ෝ

⥥௝ᯕນಽᔍᬊ⦹Ł A. gephyra MEHO_004 Ɂᵝ᮹ᮁᱥ ℕ ^DNAෝᵝ⩶ᮝಽ ^PCRᮥᝅ᜽⦹ᩍ᨜ᮡ ^tubB ᮁᱥᯱ᮹ ԕᇡ ^DNA ᳑b^{(507 bp)}ᮥ ^pCR2.1 ᄂ░ᨱᔞ᯦⦹ᩍᱽ᳑⦹

ᩡ݅^. ᯕভ ^DNA ᳑bᯕaḥ ^tubB ᮁᱥᯱ᮹ᱥᔍႊ⨆ᯕᄂ

░᮹ ^lacZ ᮁᱥᯱᱥᔍႊ⨆ŝၹݡႊ⨆ᮝಽᔞ᯦ࡽäᮥᖁ

ၽ⦹ᩡ݅^.

ᱽ᳑⦽⥭௝ᜅၙऽ۵ᱥʑ⃽Ŗᮥ☖⧕ᱱᧂᖙɁɁᵝᨱࠥ

᯦⦹ᩡᮝ໑, 50 μg/ml aӹษᯕᝁᯕॅᨕᯩ۵႑ḡᨱᕽ႑

᧲⧉ᮝಽ៉ᔢ࠺ᰍ᳑⧊ᨱ᮹⧕⥭௝ᜅၙऽa ^tubB ᮁᱥᯱ

ᨱᔞ᯦ࡽɁᵝෝᖁᄥ⦹ᩡ݅^. ⥭௝ᜅၙऽaᱶ⪶⯩ ^tubB ᮁ ᱥᯱᨱᔞ᯦ࡹᨩ۵ḡ۵ᄂ░ ^DNA ᇡ᭥ᨱᔢᅕᱢᯙ᪍ญŁ ەⓕ౩᪅❑ऽ 5′-ACTGGCCGTCGTTTTACA-3′᪡ᩝᔪℕᔢ ᮹ ᔞ᯦ᇡ᭥ ᇡɝᨱ ᔢᅕᱢᯙ ᪍ญŁەⓕ౩᪅❑ऽ ^5′- GCAGCAGATCGAACATGAC-3′ෝ⥥௝ᯕນಽᔍᬊ⦹Ł^, ⩶ ḩᱥ⪹ࡽɁᵝ᮹ᮁᱥℕ ^DNAෝᵝ⩶ᮝಽᔍᬊ⦽ ^PCRᮥ☖

⧕⪶ᯙ⦹ᩡ݅^. ႑᧲⇵⇽ྜྷ᮹ ᱽ᳑

Ɂᵝॅᮥ 1% Amberlite XAD-16 ౩ḥ(Sigma, USA)ᮥք

ᮡ ^CYS ႑ḡᨱᕽ ⁷ᯝ࠺ᦩḥ┶႑᧲⦽अ౩ḥᮥ⫭ᙹ⦹ᩡ

݅^. ౩ḥᮥ᷾ඹᙹಽ ³⫭ᖙ⃺⦽अ^, ౩ḥྕí᮹ ⁵႑^(v/w)ᨱ

⧕ݚ⦹۵ ^100% ີ┥᪍ಽ ³⫭⇵⇽⦹Ł^, ⫭ᱥ᷾ၽʑෝᔍᬊ

⦹ᩍÕ᳑⦹ᩡ݅^. ᷾ඹᙹ᪡ᨱ❙ᦥᖙ▭ᯕ✙ෝ ^1:1 እᮉ^(v/v) ಽᕿᨕքᮡ⬥^, ᯹⮵ॅᨕᵡ݅ᮭ^, ᨱ❙ᦥᖙ▭ᯕ✙⊖ᮥ⫭ ᙹ⦹ᩍ⫭ᱥ᷾ၽʑಽÕ᳑⦹Ł ^100% ີ┥᪍ᨱᬊ⧕⦹ᩡ݅^.

High Performance Liquid Chromatography (HPLC) Tubulysin᮹ ᇥᕾᮥ ᭥⧕ᕽ۵ Zorbax SB-C18 ⋝ౝ

Fig. 2. The growth of Archangium gephyra MEHO_003 and MEHO_004 in liquid medium. The wild type strain MEHO_003 and the dispersion mutant MEHO_004 were cultured in the CYS medium for 5 days.

(4.6 × 150 mm, 5 μ^m)ᮥᰆ₊⦽ Agilent 1260 VL Infinity Series HPLC ᜽ᜅ▽ᮥᔍᬊ⦹ᩡ݅(Agilent Technologies, USA). ᯕ࠺ᔢ ^A ᬊๅಽ 0.1% trifluoroacetic acid (FTA)ෝ

⧉ᮁ⦽᷾ඹᙹ^, ᯕ࠺ᔢ ^B ᬊๅಽ۵ ^{0.1% FTA}ෝ⧉ᮁ⦽ᦥ ᖙ☁ܩ✙ตᮥᔍᬊ⦹ᩡ݅^. ᬊๅ᮹ᮁᗮᮡ 0.5 ml/minᮝಽ ⁰− 5ᇥe ^{40% B} ᬊๅ^{, 5}−²⁵ᇥe ⁴⁰ᨱᕽ ^{45% B} ᬊๅʑᬙʑ^, 25−³⁰ᇥe ⁴⁵ᨱᕽ ^{100% B} ᬊๅʑᬙʑ^{, 30}−³⁵ᇥe ^100%

B ᬊๅ^{, 35}−⁴⁰ᇥe ^{40% B} ᬊๅಽᬊญ⦹ᩡ݅^. ᬊญᧂᮡ

photo diode array (PDA) á⇽ʑෝᔍᬊ⦹ᩍ ^{254 nm}ᨱᕽ á⇽⦹ᩡ݅^.

Tubulysin᮹ ᇥญෝ ᭥⧕ᕽ۵ Agilent Zorbax SB-C18 PrepHT ⋝ౝ(21.2 × 250 mm, 7 μ^m)ᮥᔍᬊ⦹ᩡ݅^. ᯕ࠺ᔢ A۵ ^{0.1% TFA}ෝ⧉ᮁ⦽᷾ඹᙹ^, ᯕ࠺ᔢ ^B۵ ^{0.1% TFA}ෝ

⧉ᮁ⦽ᦥᖙ☁ܩ✙ตᮥᔍᬊ⦹ᩡ݅^. ᮁᗮᮡ ^{6 ml/min}ᮝಽ 0−⁶⁰ᇥe ⁴⁰−^{50% B} ᬊๅʑᬙʑ^{, 60}−⁷⁰ᇥe ^{100% B} ᬊ ๅ^{, 70}−⁷¹ᇥe ¹⁰⁰−^{40% B} ᬊๅʑᬙʑ^{, 71}−⁸⁰ᇥe ^40%

B ᬊๅႊჶᮝಽᇥญෝ⦹ᩡ݅^.

High-resolution Tandem MS ᇥᕾ

Tubulysin᮹ tandem MS ᇥᕾᮡ⦽ǎʑⅩŝ⦺ḡᬱᩑǍ ᬱ^(KBSI)ᨱ ᮹഑⦹ᩍ 15T FT-ICR ḩపᇥᕾʑ^(solariX XR^TM system, Bruker Daltonics, USA)ෝᔍᬊ⦹ᩍᯕ൉ᨕ Ჭ݅^.

DNA ᕽᩕ ᇥᕾ

ᮁᱥℕ ^DNA᮹ᇥญ۵ᯝၹᱢᮝಽ᦭ಅḥႊჶᮥᔍᬊ⦹

ᩡ݅^[17]. ᮁᱥℕ ^DNA᮹ᩝʑᕽᩕđᱶᮡษⓍಽᲁᨱ ^Whole Genome De novo Sequencingᮥ᮹഑⦹ᩍᝅ᜽⦹ᩡ݅^. ᯕ₉ ݡᔍᔑྜྷᔾ⧊ᖒᮁᱥᯱ᮹ᇥᕾᮡ antiSMASH (antibiotics

& Secondary Metabolite Analysis SHell) ⥥ಽəఉ^[18]ᮥ ᯕᬊ⦹ᩡ݅^. ᕽᩕ ᔢ࠺ᖒ ᇥᕾᮡ ၙǎ ǎพᔾྜྷᱶᅕᖝ░

(NCBI)᮹ ^BLAST ⥥ಽəఉ^[19]ᮥᔍᬊ⦹ᩡŁ^, ݉႒ḩࠥີ

ᯙᇥᕾᮡ ^NCBI᮹ CD-Search ⥥ಽəఉ^[20]ᮥᔍᬊ⦹ᩡ݅^. A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ ^tubulysin ᔾ⧊ᖒᮁᱥᯱǑ᮹ ^DNA ᕽᩕᮡ ^NCBI᮹ GenBank DNA ᕽ

ᩕ ߑᯕ░ᄁᯕᜅᨱ ᱽ⇽ࡹᨩ݅(Accession numbers, MW489865 and MW489866).

đŝ ၰ Łₑ

A. gephyra᮹ tubulysin ᔾ⧊ᖒ ᮁᱥᯱ ┱ᔪ

A. gephyra MEHO_001ŝ A. gephyra MEHO_003ᮡ PCR á⇽ᨱ᮹⧕ tubulysin ᔾ⧊ᖒᮁᱥᯱෝaḥäᮝಽ ᖁᄥࡹᨩᮝ໑^{, LC-MS} ᇥᕾᨱ᮹⧕ tubulysin A᪡ ^Bෝᔾᔑ

⦹۵ äᮝಽ ⪶ᯙࡽ Ɂᵝॅᯕ݅[14]. A. gephyra MEHO_

001ŝ ^MEHO_003 Ɂᵝ۵݅ෙݡᇡᇥ᮹᧝ᔾᱱᧂᖙɁॅ

ŝษ₍aḡಽᧂℕ႑᧲᜽ᖙ⡍ॅᯕ᮲Ḳࡹ۵✚ᖒᮥᅕᯙ

݅^. ᯕ్⦽✚ᖒᮡၽ⬉᳑ෝᯕᬊ⦽ݡప႑᧲ᨱᰆᧁaࡹ໑^, Ɂᵝ}ᖁᮥ᭥⦽ᮁᱥᯱ᯲᳑ࠥᇩa܆⦹í⦽݅^. ᱱᧂᖙ Ɂᮡ ^S-ᬕ࠺ᖒᨱšᩍ⦹۵ᮁᱥᯱaᇩ⪽ᖒ⪵ࢁĞᬑᇥᔑ

⦹ᩍᖒᰆ⦽݅^[21]. ᅙᩑǍ❡ᮡᧂℕ႑᧲ŝᱶᨱᕽᯱᩑᱢ

ᮝಽ ^S-ᬕ࠺ᖒᯕ ᔢᝅࡽ Ɂᵝෝ ᖁၽ⦹۵ ႊ᜾ᮝಽ

MEHO_001 Ɂᵝ᮹ᇥᔑᄡᯕᵝෝᇥญ⦹ᩍ ^MEHO_002ಽ

໦໦⦹ᩡŁ, MEHO_003 Ɂᵝ᮹ ᇥᔑᄡᯕᵝෝ ᇥญ⦹ᩍ MEHO_004ಽ໦໦⦹ᩡ݅^{. MEHO_002}᪡ ^MEHO_004 Ɂᵝ

۵ ^CYS ᧂℕ႑ḡᨱᕽ႑᧲⧁Ğᬑ᧝ᔾ⩶Ɂᵝ᪡ݍญ᪥

ᱥ⯩ᇥᔑ⦹ᩍᖒᰆ⦹ᩡ݅^{(Fig. 2).}

Tubulysin ᔾ⧊ᖒ ᮁᱥᯱǑᮥ ┱ᔪ⦹ʑ ᭥⦹ᩍ ^A.

gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝᨱݡ⧕ᮁᱥℕᕽ

ᩕᇥᕾᮥᝅ᜽⦹ᩡ݅^. ᧝ᔾ⩶Ɂᵝݡᝁᇥᔑᄡᯕᵝ᮹ᮁᱥ ℕෝᇥᕾ⦽ᯕᮁ۵ᇥᔑᄡᯕᵝॅᯕᔢݡᱢᮝಽ݅൉ʑᛍ ᬑ໑⨆⬥ᮁᱥᯱ᯲᳑ၰྜྷḩᔾᔑᯕᯕॅɁᵝॅᮥݡᔢ ᮝಽᯕ൉ᨕḩäᮝಽᔾb⧩ʑভྙᯕᨩ݅^{. MEHO_002}᪡ MEHO_004 Ɂᵝಽᇡ░ᮁᱥℕ ^DNAෝᇥญ⦽⬥^{, Whole} Genome De novo Sequencingᮥᝅ᜽⦽đŝ^{, MEHO_002} Ɂᵝ᮹Ğᬑᨱ۵ ¹⁵} ^contigಽǍᖒࡽⅾ 13,208,011 bp᮹

ᮁᱥℕᩝʑᕽᩕᮥ᨜ᮥᙹᯩᨩᮝ໑, MEHO_004 Ɂᵝ᮹Ğ ᬑᨱ۵ ⁸} ^contigಽǍᖒࡽⅾ 13,119,373 bp᮹ᮁᱥℕᩝ ʑᕽᩕᮥ᨜ᮥᙹᯩᨩ݅^.

antiSMASH ⥥ಽəఉᮡᔾྜྷ᮹ᮁᱥℕᱶᅕಽᇡ░݅᧲

⦽᳦ඹ᮹ᯕ₉ݡᔍᔑྜྷᔾ⧊ᖒᮁᱥᯱǑᮥၾ⩡ԕ໑^, ᮁᱥ

ᯱ bbᨱ ݡ⦽ ᔢᖙ ᇥᕾࠥ ᱽŖ⦹۵⥥ಽəఉᯕ݅^[18].

antiSMASH ⥥ಽəఉᮥᯕᬊ⦹ᩍ ^MEHO_002 Ɂᵝᮁᱥℕ ᨱ᳕ᰍ⦹۵ᯕ₉ݡᔍᔑྜྷᔾ⧊ᖒᮁᱥᯱǑᮥᇥᕾ⦽đŝ^,

tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮝಽ⇵ᱶࡹ۵ᮁᱥᯱǑᯕʙᯕ

a aᰆ ʕ ^contigᨱ ᳕ᰍ⦹۵äᮝಽ ӹ┡ԍ݅^{(Fig. 3A).}

MEHO_004 Ɂᵝ᮹ Ğᬑᨱࠥษ₍aḡಽ ʙᯕaaᰆʕ

contigᨱ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮝಽ⇵ᱶࡹ۵ᮁᱥᯱǑ ᯕ᳕ᰍ⦹۵äᮝಽᇥᕾࡹᨩ݅(Fig. 3B). Tubulysin ᔾ⧊ᖒ

ᮁᱥᯱǑᮝಽ⇵ᱶࡹ۵ᮁᱥᯱǑᮡ᧞ ^{38 kb} Ⓧʑಽ ⁸}ᮁ ᱥᯱಽǍᖒࡹᨕᯩ۵ߑ^{(Fig. 3),} bᮁᱥᯱaᦵ⪙⪵⦹۵݉ ႒ḩ᮹ᦥၙיᔑᕽᩕᯕ Cystobacter sp. SBCb004᮹⛽ᇩ௝

ᯕᝁᔾ⧊ᖒᮁᱥᯱaᦵ⪙⪵⦹۵݉႒ḩॅ᮹ᦥၙיᔑᕽ

ᩕŝ ^88% ᯕᔢᮁᔍ⦹໑^, ࠥີᯙǍᖒࠥ࠺ᯝ⦹ᩡᮝအಽᯕ

ॅᮥbb tubA, orf2, orf1, tubB~Fಽ໦໦⦹ᩡ݅^.

Tubulysin ᔾ⧊ᖒ ᮁᱥᯱ᮹ ⪶ᯙ

A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ᩝᔪℕᨱ

᳕ᰍ⦹۵ᮁᱥᯱॅᯕᝅᱽ tubulysin ᔾ⧊ᖒᨱšᩍ⦹۵ḡ ᝅ⨹ᱢᮝಽ᷾໦⦹ʑ᭥⦹ᩍ ^tubB ᮁᱥᯱԕᇡ ^DNA ᳑b

ᮥ aḥ ^pJO115᪡ ^pJO118 ⥭௝ᜅၙऽෝ ᱽ᳑⦹ᩍ MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ᩝᔪℕᨱbbᔞ᯦⧉ᮝ ಽ៉ ^tubB ᮁᱥᯱaᇩ⪽ᖒ⪵ࡽ ^MEHO_005᪡ ^MEHO_007 Ɂᵝෝᱽ᳑⦹ᩡ݅^. ᱽ᳑⦽Ɂᵝॅᮥ ^CYS ႑ḡᨱᕽ႑᧲⦹

ᩍ႑᧲⇵⇽ྜྷᮥᱽ᳑⦹Ł ^HPLCಽᇥᕾ⦽đŝ^, ࢱɁᵝ᮹ ႑᧲⇵⇽ྜྷ༉ࢱᨱᕽ tubulysin A᪡ ^Bᨱ⧕ݚ⦹۵⦝Ⓧa ᔍ௝ḥäᮥ⪶ᯙ⧁ᙹᯩᨩ݅^{(Fig. 4).}

ᔍ௝ḥ⦝Ⓧa ^tubulysinᯙḡ⪶ᯙ⦹ʑ᭥⦹ᩍ ^MEHO_

004 Ɂᵝ᮹႑᧲⇵⇽ྜྷᨱᕽ tubulysin Aᨱ⧕ݚ⦹۵ ^peak

ྜྷḩᮥᇥญ⦹ᩍ 15T FT-ICR ḩపᇥᕾʑෝᔍᬊ⦹ᩍḩప

ᮥᇥᕾ⦹ᩡ݅^{. 1}₉ ᯕ᪉⪵⦽ྜྷḩᮡ ^[M+H]+᮹ ^m/z sᯕ 844.452655ಽ ^{0.19 ppm}ᨱᕽᇥᯱ᜾ᯕ ^C43H₆₆N₅O₁₀Sಽ⇵ ᱶࡹᨩᮝ໑^, ᯕ۵⦝Ⓧᨱݡ⦽࠺᭥ᬱᗭၙᖙǍ᳑ᇥᕾᮥ

☖⧕ᕽ ⪶ᯙࡹᨩ݅^. ঑௝ᕽ ⧕ݚ ྜྷḩ᮹ ᇥᯱ᜾ᮡ

C₄₃H₆₅N₅O₁₀Sಽ tubulysin Aᯝäᮝಽᩩᔢࡹᨩ݅^. ᯕ⦝Ⓧ

ྜྷḩᮥ ²₉ᯕ᪉⪵⦹ᩍᇥᕾ⦹ᩡᮥভ ^m/z sᯕ 742.384353, 504.216237, 239.175383, 211.180471ᯙ᳑b⦝Ⓧॅᯕá

⇽ࡹᨩ۵ߑ^, ᯕ్⦽᳑b᧲ᔢᮡʑ᳕ᨱ᦭ಅḥ tubulysin A ᮹᳑b᧲ᔢ(MoNA ID, CCMSLIB00000478582)ŝ⯂ᔍ⦹

ᩡ݅^. ᯕᨱ޵⧕^, ᳑b⦝Ⓧ᮹ᇥᯱపᮥʑⅩಽᰍǍᖒ⦽ྜྷ ḩ᮹Ǎ᳑ࠥ tubulysin Aᯙäᮝಽᇥᕾࡹᨩ݅^{(Fig. 5).} ঑௝

ᕽᯕ్⦽đŝ۵⥭௝ᜅၙऽᔞ᯦ᨱ᮹⧕ᔍ௝ḥ⦝Ⓧྜྷḩ

Fig. 3. Tubulysin biosynthetic gene clusters. Tubulysin biosynthetic gene clusters from A. gephyra MEHO_002 (Accession number, MW489865), A. gephyra MEHO_004 (MW489866), Angiococcus disciformis An d48 (AJ620477) [13], and Cystobacter sp. SBCb004 (GU002154) [5] are shown. The inverted triangles indicate where plasmids were inserted in the MEHO_005 and MEHO_007 strains.

Fig. 4. HPLC chromatograms of the culture extracts. (A) A.

gephyra MEHO_002 and MEHO_005 strains. (B) A. gephyra MEHO_004 and MEHO_007 strains.

ᮡ ^tubulysinᯕ໑^, ᅙᩑǍᨱᕽၽčࡽ ^tubB~F ᮁᱥᯱǑᯕ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᯥᮥḢᱲᱢᮝಽᅕᩍᵝᨩ݅^.

Tubulysin ᔾ⧊ᖒ ᮁᱥᯱǑ ᇥᕾ

A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ ^tubulysin ᔾ⧊ᖒᮁᱥᯱǑ(tubA~tubF)ᮡ ^DNA ᩝʑᕽᩕᯕᕽಽ ^97%

࠺ᯝ⦹ᩡ݅^. ᯕॅ ᩝʑᕽᩕᮡ Cystobacter sp. SBCb004᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᩝʑᕽᩕŝ۵ ^86% ࠺ᯝ⦹ᩡᮝ ໑, A. disciformis An d48᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑŝ ۵ ^75% ࠺ᯝ⦹ᩡ݅^. ঑௝ᕽ A. gephyra MEHO_002᪡ MEHO_004 Ɂᵝ᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮡ ^DNA ᩝ ʑᕽᩕᨱᕽᕽಽๅᬑᮁᔍ⦹ḡอ Cystobacter sp. SBCb004

᮹ tubulysin ᔾ⧊ᖒ ᮁᱥᯱǑŝ۵ ^14% ݅෕໑ ^A.

disciformis An d48᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑŝ۵ ^25%

݅ෙäᮝಽӹ┡ԍ݅^. ⦹ḡอᮁᱥᯱǍᖒᮡๅᬑᮁᔍ⦹ᩍ tubZ ᮁᱥᯱෝᱽ᫙⦹Ł۵ ^tubAಽᇡ░ ^tubFʭḡ᮹ᮁᱥᯱ

Ǎᖒᯕ࠺ᯝ⦹ᩡ݅^{(Fig. 3).}

A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ ^tubulysin ᔾ⧊ᖒᮁᱥᯱॅᯕᦵ⪙⪵⦹۵݉႒ḩॅ᮹ᦥၙיᔑᕽᩕᮡ

ᕽಽ ⁹⁷−^100% ᮁᔍ⦹ᩡ݅(Table 1). ᯕॅ᮹ᦥၙיᔑᕽᩕ

ᮡ Cystobacter sp. SBCb004᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑ

ᩝʑᕽᩕŝ ⁸⁸−^97% ᮁᔍ⦹ᩡᮝ໑^{(Table 2}᪡ ^{3), A.}

disciformis An d48᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑŝ۵ ⁷⁰− 89% ᮁᔍ⦹ᩡ݅(Table 1S). ⦹ḡอb݉႒ḩᮥǍᖒ⦹۵ࠥ Fig. 5. The tandem MS data of tubulysin A from A. gephyra MEHO_004.

Table 1. Comparison between the tubulysin biosynthetic genes of Archangium gephyra MEHO_002 and MEHO_004.

A. gephyra MEHO_002

Identity/Similarity (%) A. gephyra MEHO_004

Gene Product size (aa) Gene Product size (aa)

tubA 431 96/99 tubA 430

orf2 219 99/100 orf2 219

orf1 394 99/100 orf1 394

tubB 1,498 98/98 tubB 1,498

tubC 2,622 98/98 tubC 2,622

tubD 3,509 98/98 tubD 3,509

tubE 1,159 98/98 tubE 1,159

tubF 2,831 97/97 tubF 2,827

orf18 367 96/98 orf18 367

orfD 120 95/95 orfD 120

Identity/Similarity: Identity and similarity of the amino acid sequences between the proteins encoded by A. gephyra MEHO_002 and MEHO_004.

ີᯙ Ǎᖒᮡ ࠺ᯝ⦽ äᮝಽ ᇥᕾࡹᨩ݅. A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ ^tubB~F ᮁᱥᯱॅᮡ

Cystobacter sp. SBCb004᪡࠺ᯝ⦽ࠥີᯙǍᖒᮥaḥእ

ญᅕᗽ⠊┡ᯕऽ⧊ᖒ⬉ᗭ^(NRPS), ⡕ญ⍡┡ᯕऽ⧊ᖒ⬉ᗭ

(PKS) ੱ۵ ^NRPS᪡ ^PKS᮹⪝ᖒℕෝᦵ⪙⪵⦹۵äᮝಽᇥ ᕾࡹᨩ݅^{(Table 2}᪡ ^3). əญŁ tubA, orf1, orf2 ᮁᱥᯱॅࠥ

࠺ᯝ⦽݉႒ḩᮥᦵ⪙⪵⦹۵äᮝಽᇥᕾࡹᨩ݅^.

A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ ^tubulysin ᔾ⧊ᖒ ᮁᱥᯱǑŝ Cystobacter sp. SBCb004 ၰ ^A.

disciformis An d48᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑ᮹aᰆⓑ

₉ᯕ۵ ^tubZ ᮁᱥᯱ᮹᳕ᰍᯕ݅. Cystobacter sp. SBCb004

᪡ A. disciformis An d48ᨱ۵ ^tubZ ᮁᱥᯱa ᯩḡอ ^A.

gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝᨱ۵ᨧ݅^{(Fig. 3,} Table 2᪡ ^{3). tubZ} ᮁᱥᯱ۵ lysine cyclodeaminase ᮁᔍ

݉႒ḩᮥᦵ⪙⪵⦹۵ᮁᱥᯱಽ ^lysineᮝಽᇡ░ tubulysin ᔾ

⧊ᖒ᮹᜽᯲ྜྷḩᯙ pipecolic acidෝอऽ۵äᮝಽ⇵ᱶࡹ

Łᯩ݅[13, 22]. Cystobacter sp. SBCb004᪡ A. disciformis An d48᮹ ^tubZ ᮁᱥᯱෝᇩ⪽ᖒ⪵᜽┅۵Ğᬑᔾᔑࡹ۵ᮁ

ࠥℕ᮹᳦ඹ᪡ᔾᔑపᯕݍ௝ḡʑ۵⧕ࠥᩍᱥ⯩ ^tubulysin

ᯕᔾᔑࡽ݅^{[5, 23].} ঑௝ᕽ݅ෙᮁᱥᯱᨱ᮹⧕ᦵ⪙⪵ࡹ۵

lysine cyclodeaminase᮹ʑ܆ᨱ᮹⧕ pipecolic acidaŖɪ

ࡹʑࠥ⦹۵äᮝಽᱽᦩࡹᨩ݅^. ᮁᱥℕᕽᩕᇥᕾᨱ᮹⦹໕

A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ۵ᩝᔪℕ᮹݅

ෙ᭥⊹ᨱ Cystobacter sp. SBCb004᮹ ^TubZ ݉႒ḩŝᦥၙ

יᔑᕽᩕᯕ ⁴⁴−^50% ᮁᔍ⦽ࢱ}᮹ lysine cyclodeaminase

ᮁᔍ݉႒ḩᮥᦵ⪙⪵⦹۵ᮁᱥᯱෝaḡŁᯩ۵äᮝಽӹ

┡ԍ݅^. ঑௝ᕽ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᨱ ^tubZ ᮁᱥᯱ

aᨧᨕࠥ tubulysin ᔾᔑᨱྙᱽaᨧᮥäᮝಽ⇵ᱶࡽ݅

A. gephyra᮹⢽ᵡɁᵝᯙ ^{DSM 2261T}Ɂᵝ᮹ᮁᱥℕᨱ۵ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᯕᨧ݅^{. MEHO_002}᪡ ^MEHO_

004᮹ᮁᱥℕᨱᕽ۵ tubulysin ᔾ⧊ᖒᮁᱥᯱǑ(tubA~tubF) ŝ orf18, orfDᯕ ^{DSM 2261T} ᮁᱥℕ᮹ AA314_01909᪡

AA314_01912 ᮁᱥᯱᨱ⧕ݚ⦹۵ᮁᱥᯱॅᔍᯕᨱᔞ᯦ࡹ

ᨕᯩ݅^{. orf18}ᮡ patatin-like proteinᮥᦵ⪙⪵⦹໑ ^orfD۵ hypothetical proteinᮥ ᦵ⪙⪵⦹۵ äᮝಽ ᇥᕾࡹᨩ݅^. tubulysin ᔾ⧊ᖒᨱšಉ⦹ᩍ ^orf18ŝ ^orfD᮹ʑ܆ᮡ᦭ಅᲙ

ᯩḡᦫ݅^. ⦽⠙^{, MEHO_002}᪡ ^MEHO_004᮹ᮁᱥℕᨱᕽ ۵ ^{DSM 2261T} ᮁᱥℕ᮹ AA314_01910ŝ AA314_01911

ᮁᱥᯱᨱ ⧕ݚ⦹۵ ᮁᱥᯱa ᳕ᰍ⦹ḡ ᦫ۵݅^. ঑௝ᕽ MEHO_002᪡ ^MEHO_004 Ɂᵝᨱᕽ۵ᯕࢱᮁᱥᯱෝ⡍⧉

⦹۵ 1,867 bp DNA ᇡ᭥a tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮥa

ḥ ᧞ ^{40 kb} Ⓧʑ᮹ ^DNAಽ Ʊℕࡽ äᮝಽ ⇵ᱶࡽ݅^.

AA314_01910ŝ AA314_01911 ᮁᱥᯱ۵ hypothetical proteinᮥᦵ⪙⪵⦽݅^.

Table 2. Comparison between the tubulysin biosynthetic genes of A. gephyra MEHO_002 and Cystobacter sp. SBCb004.

A. gephyra MEHO_002 Cystobacter sp. SBCb004

Gene Product size

(aa) Predicted function PKS/NRPS motif predicted by antiSMASH

Identity/

Similarity (%)

Gene Product size (aa)

tubA 431 NRPS C 81/88 tubA 432

orf2 219 hypothetical protein 76/87 orf2 219

- - - tubZ 386

orf1 394 hypothetical protein 91/97 orf1 394

tubB 1,498 NRPS C-A(pip)-nMT-PCP 84/89 tubB 1,520

tubC 2,622 NRPS C-A(ile)-PCP-C-A(val)-nMT-PCP 85/90 tubC 2,626

tubD 3,509 PKS/NRPS KS-AT(mal)-KR-DH-ER-ACP-C-A(cys)-PCP 85/90 tubD 3,511

tubE 1,159 NRPS C-CAL-PCP 86/90 tubE 1,161

tubF 2,831 PKS KS-AT(mal)-KR-cMT-KR-DH-ER-ACP-TE 82/88 tubF 2,843

- - - orf17 337

orf18 367 patatin-like protein 80/89 orf18 368

orfD 120 hypothetical protein 60/72 orfD 124

A, adenylation domain; aa, amino acid; ACP, acyl-carrier protein domain; AT, acyltransferase; C, condensation domain; CAL, co- enzyme A ligase domain; cMT, carbon methyltransferase; cys, cysteine; DH, dehydrase; ER, enoylreductase domain; ile, isoleucine;

KR, ketoreductase domain; KS, ketosynthase domain; mal, malonate; nMT, nitrogen methyltransferase; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl-carrier protein domain; pip, pipecolic acid; PKS, polyketide synthase; TE, thioesterase domain; val, valine.

Identity/Similarity: Identity and similarity to the corresponding proteins encoded by Cystobacter sp. SBCb004.

MEHO_002᪡ ^MEHO_004 Ɂᵝ۵ tubulysin ᔾ⧊ᖒᮁᱥ

ᯱǑ᮹ ^DNA ᩝʑᕽᩕᯕᕽಽ ^97% ࠺ᯝ⦹໑ᮁᱥℕᕽᩕࠥ

ᱥၹᱢᮝಽ ๅᬑ ᮁᔍ⦹݅^. ⦹ḡอ ^MEHO_002 Ɂᵝ۵ argyrin ᔾ⧊ᖒᮁᱥᯱǑᮥaᲙ ^argyrinᮥᔾᔑ⦹۵ၹ໕^, MEHO_004 Ɂᵝ۵ ^argyrin ᔾ⧊ᖒᮁᱥᯱෝaḡḡᦫᦥ argyrinᮥᔾᔑ⦹ḡᦫ۵݅۵ᱱᯕ݅෕݅(Fig. 4). Argyrin

ᮡᱱᧂᖙɁŝႊᖁɁᨱᕽᇥญࡽ cyclic octapeptide ĥᩕ᮹

ྜྷḩಽ⧎ᦵŝ⧎ᖙɁ⪽ᖒᮥᅕᯙ݅[24, 25]. MEHO_002

᪡ ^MEHO_004 Ɂᵝ᮹ tubulysin ᔾᔑᙹᮉᮡ ᮁᔍ⦽ߑ^, HPLC ᇥᕾ᜽ᔢݡᱢᮝಽ ฯᮡ᧲ᯕᔾᔑࡹ۵ ^argyrinᯕ tubulysinŝᮁᔍ⦽᭥⊹ᨱᕽᇥญࡹʑভྙᨱ ^argyrinᮥᔾ ᔑ⦹ḡᦫ۵ ^MEHO_004 Ɂᵝa ^tubulysinᮥᔾᔑ⦹ᩍᇥญ

⦹Łᯱ⧁ভ޵ᮁญ⧁äᮝಽᅕᯙ݅^.

đುᱢᮝಽ^, ᅙᩑǍᨱᕽ۵ᮁᱥℕᇥᕾᮥ☖⧕ A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱ

Ǒᮥ ┱ᔪ⦹ᩡᮝ໑^, ᮁᱥᯱ ᇩ⪽ᖒ⪵ෝ ☖⧕ ᯕॅᯕ tubulysin ᔾ⧊ᖒᮁᱥᯱᯥᮥ⪶ᯙ⦹ᩡ݅. Tubulysinᮡv ಆ⦽⧎ᦵ⪽ᖒᮝಽᯙ⧕᮲ᬊᮥ᭥⦽݅᧲⦽ᩑǍaᯕ൉ᨕ ḡŁ ᯩ۵ ྜྷḩᯕ݅^{. MEHO_002}᪡ ^MEHO_004 Ɂᵝ᮹ tubulysin ᔾᔑᙹᮉᮡ ⥭௝ᜅⓍᨱ ႑᧲⦹۵Ğᬑ ^0.2−^1.0 mg/l ᙹᵡᮝಽʑ᳕ᨱ᦭ಅḥɁᵝॅ᮹ᔾᔑᙹᮉ^[22]ŝᮁᔍ

⦹íๅᬑԏ݅^. ┱ᔪࡽᮁᱥᯱ۵⨆⬥ᮁᱥᯱ᯲᳑ᨱ᮹⦽

tubulysin ᔾᔑᙹᮉ᮹᷾ݡၰᮁࠥℕᔾᔑᮥ᭥⦽ᵲ᫵⦽

ᰍഭಽᔍᬊࢁäᮝಽʑݡ⦽݅^.

᫵ ᧞

Tubulysinᮡ݅᧲⦽ᦵᖙ⡍ᵝᨱݡ⧕v⦽⧎ᦵ⪽ᖒᮥᅕ

ᯕ۵ᱱᧂᖙɁᮁ௹ᯕ₉ݡᔍᔾญ⪽ᖒྜྷḩᯕ݅^. ᅙᩑǍᨱ ᕽ۵ ^tubulysinᮥᔾᔑ⦹۵ࢱɁᵝ᮹ᱱᧂᖙɁ Archangium gephyra MEHO_002᪡ ^MEHO_004᮹ᮁᱥℕᇥᕾᮥ☖⧕

tubulysin ᔾ⧊ᖒᮁᱥᯱॅಽ⇵ᱶࡹ۵ᮁᱥᯱǑᮥၽč⦹

ᩡᮝ໑^, ⥭௝ᜅၙऽᔞ᯦ᨱ᮹⦽ᮁᱥᯱᇩ⪽ᖒ⪵ෝ☖⧕ᯕ

ॅᮁᱥᯱॅᯕ tubulysin ᔾᔑŝḢᱲᩑšࡹᨕᯩᮭᮥ⪶ ᯙ⦹ᩡ݅. A. gephyra MEHO_002᪡ ^MEHO_004 Ɂᵝ᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑ(tubA~tubF)ᮡ ^DNA ᩝʑᕽᩕᯕ

ᕽಽ ^97% ࠺ᯝ⦹ᩡᮝ໑^, ᦵ⪙⪵⦹۵݉႒ḩॅ᮹ᦥၙיᔑ

ᕽᩕࠥᕽಽ ⁹⁷−^100% ᮁᔍ⦹ᩡ݅^{. MEHO_002}᪡ ^MEHO_

004 Ɂᵝ᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑᮡ tubulysin ᔾᔑᱱ ᧂᖙɁᮝಽ ᦭ಅḥ Cystobacter sp. SBCb004᮹ ^tubulysin ᔾ⧊ᖒᮁᱥᯱǑŝ ^DNA ᩝʑᕽᩕᯕ ^86% ࠺ᯝ⦹ᩡ݅^. ᮁᱥ

ᯱǑ᮹Ǎᖒᮡ ^tubZ ᮁᱥᯱa᳕ᰍ⦹ḡᦫ۵݅۵ᱱᮥᱽ᫙

⦹Ł۵ ^SBCb004᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱǑǍᖒŝ࠺ᯝ

⦹ᩡ݅^. bᮁᱥᯱaᦵ⪙⪵⦹۵݉႒ḩ᮹ᦥၙיᔑᕽᩕᮡ

Cystobacter sp. SBCb004᮹ tubulysin ᔾ⧊ᖒᮁᱥᯱaᦵ

⪙⪵⦹۵݉႒ḩॅŝ ⁸⁸−^97% ᮁᔍ⦹ᩡᮝ໑^, b݉႒ḩॅ᮹ Table 3. Comparison between the tubulysin biosynthetic genes of A. gephyra MEHO_004 and Cystobacter sp. SBCb004.

A. gephyra MEHO_004 Cystobacter sp. SBCb004

Gene Product size

(aa) Predicted function PKS/NRPS motif predicted by antiSMASH

Identity/

Similarity (%)

Gene Product size (aa)

tubA 430 NRPS C 81/89 tubA 432

orf2 219 hypothetical protein 77/87 orf2 219

- - - tubZ 386

orf1 394 hypothetical protein 91/97 orf1 394

tubB 1,498 NRPS C-A(pip)-nMT-PCP 84/89 tubB 1,520

tubC 2,622 NRPS C-A(ile)-PCP-C-A(val)-nMT-PCP 85/90 tubC 2,626

tubD 3,509 PKS/NRPS KS-AT(mal)-KR-DH-ER-ACP-C-A(cys)-PCP 85/90 tubD 3,511

tubE 1,159 NRPS C-CAL-PCP 86/90 tubE 1,161

tubF 2,827 PKS KS-AT(mal)-KR-cMT-KR-DH-ER-ACP-TE 82/88 tubF 2,843

- - - orf17 337

orf18 367 patatin-like protein 81/89 orf18 368

orfD 120 hypothetical protein 62/72 orfD 124

A, adenylation domain; aa, amino acid; ACP, acyl-carrier protein domain; AT, acyltransferase; C, condensation domain; CAL, co- enzyme A ligase domain; cMT, carbon methyltransferase; cys, cysteine; DH, dehydrase; ER, enoylreductase domain; ile, isoleucine;

KR, ketoreductase domain; KS, ketosynthase domain; mal, malonate; nMT, nitrogen methyltransferase; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl-carrier protein domain; pip, pipecolic acid; PKS, polyketide synthase; TE, thioesterase domain; val, valine.

Identity/Similarity: Identity and similarity to the corresponding proteins encoded by Cystobacter sp. SBCb004.

ࠥີᯙǍᖒࠥ࠺ᯝ⦹ᩡ݅^.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

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