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Mat Kadi Tora Tutti Uniti Ai Ai Ai Malaltiatur MAT KADI TORA TUTTI UNITIUS 20180265545A1 AI AI AI MALALTIATUR ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2018/ 0265545 A1 CRUZ MORALES et al. (43 ) Pub . Date : Sep . 20 , 2018 ( 54 ) GENETIC SYSTEM FOR PRODUCING A Publication Classification PROTEASES INHIBITOR OF A SMALL (51 ) Int . Cl. PEPTIDE ALDEHYDE TYPE CO7K 5 / 065 (2006 .01 ) C12N 15 /63 @(71 ) Applicant: Centro de Investigación y de Estudios (2006 .01 ) Avanzados del Instituto Politécnico C12N 15 /70 (2006 .01 ) 2 ) U . S . Cl. Nacional, Ciudad de México , CDMX CPC .. CO7K 5 / 06078 (2013 . 01 ) ; C12N 15 / 70 (MX ) ( 2013 .01 ) ; C12N 15 /635 (2013 . 01 ) @(72 ) Inventors : Pablo CRUZ MORALES , Irapuato (MX ) ; Francisco BARONA GÓMEZ , (57 ) ABSTRACT Guanajuato (MX ) ; Hilda Eréndira The present invention describes a new peptide aldehyde RAMOS ABOITES , Salamanca (MX ) produced naturally by Streptomyces lividans 66 , which we have called livipeptin . Using genome mining of natural @( 73 ) Assignee : Centro de Investigación y de Estudios products , we predicted that SL10883 - 5 genes encode an Avanzados del Instituto Politécnico unprecedented biosynthetic system , unusually small ( 4 . 6 Nacional , Ciudad de México , CDMX Kbp ) , which produces an acylated peptide aldehyde . (MX ) Because of the chemical characteristics of the predicted compound , we postulated its anti - proteolytic activity , which (21 ) Appl . No .: 15 /537 , 125 we confirmed by identifying and purifying this compound through metabolic profiles of HPLC and MS of the mutated ( 22 ) PCT Filed : Dec . 14 , 2015 strain lacking these three genes and the wild strain . To this ( 86 ) PCT No. : PCT/ IB2015 /059566 objective , we identified the conditions wherein these genes are strongly expressed . The livipeptin was purified and its $ 371 ( c ) ( 1 ) , inhibitory activity on the proteolytic activity of selected (2 ) Date : Jun . 16 , 2017 proteases was demonstrated in vitro . The design of an expression cassette for its heterologous expression is also ( 30 ) Foreign Application Priority Data described plus its use for heterologous protein production . Dec . 16 , 2014 (MX ) .. .. MX / A / 2014 /015560 Specification includes a Sequence Listing . Patent Application Publication Sep . 20 , 2018 Sheet 1 of9 U S 2018 / 0265545 A1 Figure 1 Acylated Amino - acylated A ppp un 9 RSDOR* trotimenet CHANH - NH 1 NH, NH I J NM- p er a la La vallen en R $ * argue he way Figure 6 Gene Predicted Function SL10883 Non - ribosomal peptide synthetase SL10884 Leucyl/ Phenylalanyl - tRNA -protein transferase SL10885 N - Acetyltransferase SL10886 O -methyltransferase , family 2 SL10887 Butyryl -CoA dehydrogenase SL10888 TenA family transcriptional regulator SL10889 Major facilitator superfamily MFS1 SL10890 Putative cytochrome P450 monooxygenase SLI0891 Pyridoxamine 5 '- phosphate oxidase -related , FMN -binding SL10892 Unknown SL10893 CsoR - like DUF156 Heavy metal- associated domain - containing secreted SL10894 protein Patent Application Publication Sep . 20 , 2018 Sheet 2 of9 U S 2018 / 0265545 A1 Figure 2 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * SPA Producer Strain Target Protease class Reference * * * * * * * * * * * * * * * * * P : + 44 * * * * * * * * * * * * * * * * * 44 - * * * * * * * * * * Streptomyces Yokosukanensis * * * * * * * * Antipain MC829 - AS1, Streptomyces Trypsin , plasmin , Cystein y Serine Suda et al, 1972 ; mauvecolor papain ( Thiol proteases Umezawa et al, 1972 , Streptomyces hygroscopicus Chimostatin MC521 - C8 , Streptomyces ChimoTrypsin , Cystein y Serine Umezawa et al, 1970 ; lavendulae MC524 -C1 Papain (Thiol proteases Tatsuta et al, 1973 , ???? ??????????? . Trypsin , plasmin , Leupeptins Streptomyces roseus MA839 -A1 Papain , 20s Serine/ Cystein /threon Aoyagi et al, 1969 proteosome subunit ine proteases * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Streptomyces griseoruber MD 469 . Umezawa et al, 1973 , Elastatinal CG8 Elastase Serine - proteases Okura et al, 1975 Streptomyces testaceus Hamada Pepsina, Pepstatin et Okami, Streptomyces gastricsina Aspartatil -proteases Umezawa et al, 1970 , argenteolus var . Toyonakensis cathepsina D y Morishima et al, 1970 - ?? . ?? . ? . ????????? - - - - - renina Murao y Watanabe , 1977 ; MAPI' s Streptomyces nigrescens WT- 27 Subtilisin Alkaline proteases Watanabe et al, 1979 ( alpha ) & 1982 (Beta ) * * * * * * * * * * * * * * * * * * * *Streptomyces * * * * * * * * * * * * * * * * * *sp * * * *, *NRRL * * * * * * * * * -* F6652 * * * * * * * * *, * YYYYYY 12 V * * * * * * * * * * * * * * * * * * * * Flavopeptin Streptomyces flavogriseus ATCC Papain , calpain Cystein - proteases Chen et al , 2013 33331 Nerfilin Streptomyces halstedii 2723- SV2 Cathepsina , Papain Cystein -proteases Hirao et al, 1995 - RRENY * * * * * * * * * * * * * * * * * * * * * YA Staccopins Staphylococcus tanabeensis Calpain , Papain Cystein -proteases Saito et al, 1987 Strepin Streptomyces tanabeensis ( SAB Trypsin , Papain , Cystein y Serine 934 ) calpain ( Thiol proteases Ogura et al , 1985 - ?? - ?? ?? “ ?????? * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 20s proteosome Tyropeptins Kitasatospora sp . MK993 - F2 subunit Treonin -proteases Momose et al, 2001 ( a & b ) * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Papain , Ficain y Tyrostatin Kitasatospora sp . Cepa 55 carboxyl Carboxyl- proteinases / Oda et al, 1989 proteinases Cystein -proteases - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Bacillus laterosporus Laubach Trombin , Factor Xa , Serine -proteases / Tiol Bacithrocins NR2988 Trypsin y Papain proteases Kamiyama et al 1994 Cystein y Serine Thiolstatin Bacillus cereus EY - 21 Papain , Trypsin ( Thiol proteases Murao et al, 1985 - - - - - - - - Acetyl- Leu - arginal Dipeptidyl or Unnidentified strain BMG520 - yF2 y aminopeptidase lll, Cystein y Serine Nishikiori et al, 1984 ; Caricastatin Nigrosabulum novosp . Papain , Ficain , (Thiol proteases Murao et al , 1987 . * * * * * * * * * * * * * * ? ?? - ??? ?? ?. ?. ? . * .* * . * * * * * Bromelin * * * * * * * * * * ** * * * * * * * * * * ** * * * * * * ** * * * * * * * * * * * * * * ** * ? ? . ?????????????? . * * * * * * 20s proteosome Threonyl- proteases PenFellutamides Penicillum fellutanum subunit Shigemori et al, 1991 HIV - 1 asparty GE20372 Streptomyces sp . ATCC 55925 protease Aspartyl -proteasee Stefanelli et al, 1995 Nostosins Nostoc sp . cepa FSN Trypsin Seryl- proteases Liu et al 2014 Figure 10 SLI0883 SL108 www Control Patent Application Publication Sep . 20 , 2018 Sheet 3 of9 U S 2018 / 0265545 A1 Figure 3 they How to HVO NE R . SO NH R * * * * S Alpha MAPI : MW 595 . 69 Flavopeptin MW = 786 .01 - 856 , 14 R - CHICHT} ? Chymnostatin A (valine ) : MW 59367 R - R Beta MAPI : WW 595 .69 RqCMH 2 + 1 12 * 5 - 6 - 7 - 8 -9 = C7MCHICH ? Chymostatin B fleucine ): M 607, 7 R = O # * * S GE20372 A : MW 611. 69 RECH ,CH (CH3 ) , or CH (CH ) CH , CHE RECH ( CH3CH2CH2 Chymostatin C {Isoleucine ): MW 607 . 7 R - OH * * RGE20372 B : MW 611. 69 om * INNEN INW 0. H er NWYM. RFCH2CH (CH3 ) ; R - CH (CH3 ) 2 ; Tyrestatin A ( valine ): MW 511. 60 RECH2CH2CH3; R = CH - CH (CH3 ) 2 Tyroslatin B (leucinaj : MW 511. 60 O . OG R - CH2CH (CH3 ) 2 R - CH2CH (CH3 )2 Tyropepin ( leuore) : MW 525 .03 S pain } } } { { { { { ? HANH AN NIH OOHAN NH NE NA NH " 07 .0 R 4 Staccopin P1; MW 545 , 7 R = OH , Staccopin PZ : MW 5817 Strepin P - 1 : MW 504 . 6 MHES HANH Elastatinal: MW 512 . 56 HaNi ANH NH NA O A 0227 NH NH NH R = CH2CH ( CH3 ) Bacithain A : MW 399 .49 R - CH (CH3 ) 2 Bacithrocin 8 : M 375 .46 Acety - Leu Arginal: MW 333 . 39 R = CH Loupeptin : MW 425 . 55 R = CH2CHE Bacitracin C : MW 361. 43 R = COHCOH Leupeptin if : MW 440 .58 R - CH3 Thiostatin D : WW 347 . 41 0 NH DNEVNA.O os OHO 0 Malla modernoR - B , Marilin MW 495 , 6 Feate} } ?? $ 5 Nostoso A : MW 449 . 5438 R = OH , Nertain it MW 5116 Fa{ { c } } } 527, 65 Patent Application Publication Sep . 20 , 2018 Sheet 4 of9 U S 2018 /0265545 A1 Figure 4 FlavA ; GI: 357410558 ; Sila _ 1335 FlavB ; Gl: 357410557 ; Sfla _ 1334 w AACile ANgingin CATICIATED i ilele gin CATCval/ lle ANDphe | - 7 - - . - . - . - .- . - M . - W wwwr. 25 Kb le le Vallile R1 = C5 - R2 = CH , OH NRPS I MbtH protein Hypothetical protein 0 N -acyl transferase G5CN HAN Gin Gin Fe Figure 5 .1 33697766Frankia sp 158103254Detaicu Frankiasimbioa EDE471min 331152851FrenkiaatACX14 140XWalajajope 912OSUS 2? 5/????????? ?????? 12 = herritorialitna742621 Frankia sp0d5 ? ???? ne139925092 Nocasines soJOG44 ?????S pons natomintang Hoe mire96294496934 1. 2 jambul SA HICU 049 ostatomas123428 Brevibacterium
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