Mining endophytic bacteria and fungi for polyketide biosynthetic genes
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Mining endophytic bacteria and fungi for polyketide biosynthetic genes
Mining endophytic bacteria and fungi for polyketide biosynthetic genes Gabriel Padilla Departamento de Microbiologia Instituto de Ciências Biomédicas Universidade de São Paulo Topics • • • • • • Endophytic microorganisms Prospecting a culture collection Polyketide genes PKS I Fungi PKS II Actinobacteria PKS III Actinobacteria 3 700 strains 65% bacteria 30% fungi 5% archaea 99% Culture Culture Collection LGM ESALQ LGS ICB NIB UMC Bioprospection of endophytic microorganisms Endophytics are bacteria and fungi which live within plant tissues for all or part of their life cycle and cause no apparent infection Curr. Sciences 90:1309 (2006) WHY TO STUDY ENDOPHYTIC MICROORGANISMS? Poorly biotechnologically explored They form a significant part of the microbiota Compounds with low toxicity Diverse biological activity (antibacterial, antifungic, antitumoral, antiviral, antioxidant, imunossupressor, inseticide and control of diabetes and malaria). Taxol (Plaxitaxel): produced byr Taxomyces andreanae, endophytic fungus from Taxus brevifolia. Biotechnological applications of endophytic microorganisms Taxol Polyketides PKS Structure Function Biosyntesis of polyketides KS AT ER ACP KS DH KS ketosynthase AT acyl transferase, ACP acyl carrier protein DH dehydratase ER enoylreductase, KR ketoreductase TE thiosesterase MeT metyltransferase AT ACP KR CH3 MeT PKSs classification Depending of the kind of organism and FAS structural organization (analogy) Type I Mammals Type II Bacteria AT AT KR KS DH ACP ER Type III Actinobacteria KR KS DH ACP ER 10 PKS type I Modular 11 PKS type I Interactive MT CYC MT MT MT DH KR Lov F Lov B NR : Non- reduced PR: Parcial reduced HR: Highly reduced Bioprospection of polyketides in endophytics Fungi Fusarium sp Actinomycetes Endophytic bacteria and fungi (ESALQ/Piracicaba – isolates from plants with economical importance ) PKS I Juan Diego Rojas Lovastatins X Filaments fungi PKS II PKS III Erik Saenz Antracyclines X Actinomycetes Fabiana Andrieli Flavonoids X Actinomycetes 1. Isolation Brazilian Sugar Cane Leaves 5 isolates Roots 5 isolates Rhizosphere 7 isolates 14 Isolates 13 Strain 23 > Homology Cladosporium sp. 42 48 196 Fusarium sp. 209 P. pinophilum 118 234 290 145 251 P. ochrochloron 253 Dothideomycetes Diaphorte sp. C33 Trichoderma sp. F. neocoras Paraphaeosphaeria sp. Giberella sp. 41 Aspergillus sp. Saccharicola bicolor 77 P. rapai 24 Phomopsis sp. TH4 15 Polyporus tuberaster Epicoccum sp. Epicoccum sp. 2. ITS sequences analysis Penicillium janthinellu strain AY373921 F42EN Penicillium raperi strain NRRL 2674 (AF033433) F24EN Aspergillus oryzae strain D4 (EF488389) Eurotiomycetes Aspergillus flavus strain (AM745114) F196EN Basidiomycota 5% Penicillium aculeatum strain (EU076930) Penicillium pinophilum strain (EF488397) Arthrographis cuboidea strain (AB213444) Mitosporic Ascomycota 5% Eurotiomycetes 21% Scytalidium lignicola strain (DQ093703) Mitosporic Ascomycota Woollsiaroot (AY230788) F33EN F234EN Fusarium sp. NRRL29124 (AF158314) Gibberella moniliformis strain (EU151483) Fusarium neoceras strain NRRL20 (FNU61682) F48EN Fusarium sp. FVS3 (DQ885388) Hypocreomycetidae; Fusarium sp. NRRL25483 (FSU61695) Sordariomycetes 26% Pseudofusarium purpureu (EU301058) F41EN Sordariomycetes Trichoderma ovalisporum strain (EU280118) Ascomycota 95% Trichoderma intricatum strain (EU264002) Trichoderma k oningiopsi strain GJS 04-373 (DQ323437) Phomopsis sp. Vega425 (EU002927) F275EN F145EN Ascomycota Sordariomycetidae F253EN Phomopsis sp. ARGA5 (AY705847) Dothideomycetes 43% Diaporthe helianthi (AJ312351) Cladosporium cladosporioides strain (AY463365) F13EN Dothideomycetes Cladosporium tenuissimu strain ATCC 38027 (AF393724) Cladosporium cladosporioides strain STE-U 3683 (AY251074) Epicoccum nigrum strain (AB369423) Epicoccum nigrum CBS (AJ279448) FTH2EN FC33EN Epicoccum nigrum strain (EU272495) F251EN ITS 1F and ITS 4R Phoma herbarum strain (AB369456) Ampelomyces sp. Po63 (AY513943) Ampelomyces sp. Vega607 (EF672292) Cochliobolus intermediu straim (AF071327) Curvularia intermedia strain (AF163078) Dothideomycetes F247EN Curvularia brachyspora strain (AF212308) F77EN Dothideomycete sp. (EU680559) Paraphaeosphaeria pilleata strain (AF250821) Paraconiothyrium sporulosum strain (AB303549) Saccharicola taiwanensi strain ATCC 38204 (AF439464) Saccharicola bicolor strain ATCC 42652 (LBU04203) F118EN F209EN Resinicium bicolor strain (DQ340321) Resinicium rimulosum strain FP-102843 (DQ826546) Basidiomycota F290EN Resinicium saccharicola strain (DQ826549) White et al, 1990 0.05 16 Multiple alignment of the Keto- synthase sequences 17 Phylogenetic analysis of KS sequences Melanins Non Reducing PKS * * * * Pigments Reducing PKS * * * * Amplified with CmeT primers 18 Multiplce Alignment of C- methyl transferase sequences Motif I Motif II Motif III 19 Phylogenetic analysis of CmeT sequences Monascus purpureus PKSCT Botryotinia fuck eliana PKS17 Penicillium marneffei CMT Cochliobolus heterostrophus PKS21 Non reducing PKSs Cochliobolus heterostrophus PKS23 Botryotinia fuck eliana PKS16 Cochliobolus heterostrophus PKS22 PKSN Alternaria solani 77 CMT Cochliobolus heterostrophus PKS6 Phoma CMT 41 CMT Gibberella moniliformis FUM1 Cochliobolus heterostrophus PKS11 24 CMT Reducing PKSs 209 CMT 234 CMT Penicillium citrinum mlcA Aspergillus terreus LNKS Monascus pilosus mk A Aspergillus clavatus EKS Botryotinia fuck eliana PKS2 Aspergillus terreus LDKS Penicillium citrinum mlcB Streptomyces globisporus CMT 20 0.2 21 Aflatoxinas e pigmentos T-toxin (nonaketide)-like PKS/NRPS hibrido Lovastatin nonaketide-like 13 Melaninas 251 Cladosporium sp. C33 Epicoccum sp. 234 Phoma sp. TH4 Epicoccum sp. Phomopsis sp. 253 Diaphorte sp. Alinhamento múltiplo das seqüências de aminoácidos do domínio C-met Aligment of Cmet SAM dependents Kagan and Clarck, 1994 Comparative tridimensional modelling 2qo3 2yr0 •Transferase •B cetoacil-ACP synthase •beta-ketoacyl-[acyl carrier protein] synthase II from Streptococcus pneumoniae, tricyclic form •Transferase •Metil transferase SAM dependent •Crystal structure of hypothetical methyltransferase2 ttha0223 from Thermus thermophilus hb8 Alinhamento múltiplo das estruturas secundárias das sequências de aminoácidos do domínio KS 145KS vs 1OXH * RMSD = 1.719Å Alinhamento múltiplo das estruturas secundárias de aminoácidos do domínio C-met Martin 2002 24Cmet vs 2YQZ Motivo II Motivo III RMSD=0.245 Å 26I Motivo 4. Prospecting for Statins H L 24 24A At TLC of extracts from strain 24 At 24 41 77 145 209 290 L H TLC of endophytic extracts Dichlorometan: Etyl Acetate: Methanol 7:3:1 H: Lovastatin hydroxy acid L: Lovastatin lactone 27 Detector A-246 nm lovh Pd lovh Pd2 1250 Lovastatin Hydroxy acid 1250 18min. 1000 Spectrum at time 17.79 min. 1000 Lambda max : 239 247 224 226 206 Lambda min : 244 225 232 210 200 500 mAU mAU 750 250 mAU 750 500 17.79 min 500 250 250 0 500 0 200 250 300 350 400 450 250 500 0 nm -250 0 10 20 30 40 50 60 Minutes Detector A-254 nm lov k pd lov k pd1 200 Lovastatin Lactone Spectrum at time 22.97 min. 1500 22.97 min 1500 150 1000 mAU 500 mAU 1000 Lambda max : 239 247 229 226 219 Lambda min : 244 230 227 220 213 100 500 0 100 mAU 150 23 min. 200 0 50 50 200 250 300 350 400 450 500 nm 0 mAU -250 0 0 28 10 20 30 Minutes 40 50 Chromatogram Strain 24 (extract) 25 Detector A-265 nm a241 a241 20 25 15 20 15 mAU 10 mAU 23 min. 5 10 5 lov k pd lov k pd1 0 150 0 200 Detector A-254 nm 0 200 10 20 30 40 50 60 100 mAU 150 50 100 50 0 mAU Minutes 0 10 20 30 Minutes 40 50 29 NMR 1H from extract strain 24 Acquisition Time (sec) Number of Transients 2.2774 128 Frequency (MHz) Original Points Count 200.13 8192 Nucleus Points Count 1H 8192 Sweep Width (Hz) Temperature (degree C) 24.000 3597.12 TMS 1.26 0.94 2.04 0.98 2.39 7.5 7.0 6.5 6.0 1.09 1.00 5.5 4.91 5.97 5.35 5.21 5.18 5.09 6.84 6.80 6.78 7.16 7.15 7.07 1.18 0.89 1.32 2.36 2.32 3.97 3.62 1.09 3.45 3.59 3.50 3.56 3.52 1.16 1.13 Chloroform-d 0.62 5.0 3.65 4.5 4.0 3.5 Chemical Shift (ppm) 3.74 3.0 2.5 2.0 1.5 1.0 0.5 300 Atividade biológica Biological Activity of extracts strain 24 Mucor sp. Roze et al, 1998 Chamilos et al, 2006 145 Candida albicans 77 Ergosterol Chin et al, 1997 Lorenz et al, 1990 Kelvin, 2003 PKS Type II in Actinobacteria E coli ESPA26 ESPG1P1 59 45 100 50 100 ESPA28 ESPA23 ESPA30 ESPA8 S hawaiiensis ESPA10 4 ESPA01 34 ESPA18 92 ESPA32 S macrosporeus 38 ESPB6P4 61 ESPB5P3 ESPG10P4 9 68 ESPA11P2 99 ESPH11P2 S atrovirens 82 ESPC07 98 35 Streptomyces sp ESPA9 88 ESPA19 S capoamus 12 ESPE10 6 100 13 84 Phylogenetic analysis of 16SrRNA DNA 93 82 76 A. Streptomyces 74,5%. B. Nocardiopsis 19,1% C. Nocardia 4,3% D. Bacillus 2,1% 100 100 36 10 A 100 ESPH4 3 96 ESPA4P1 S griseus S aureus 80 ESPB01 82 ESPA21 99 ESPA25 ESPH4P3 S tsukiyonensis S flavomacrosporus 43 ESPC6P2 19 ESPA82 19 ESPA3P1 25 ESPA12.1 58 ESPH4P4 S variabilis 87 ESPC73 57 ESPF7P4 2 ESPA12P2 ESPA16 ESPA02 S flavidofuscus 99 ESPA27 99 N dassonvillei 15 ESPA17 37 ESPA11 55 ESPA07 86 70 10 ESPA22 ESPA03 75 31 ESPA05 N alba 41 Nocardiopsis sp ESPA04 Nocardia puris 99 ESPA14 100 Nocardia sp ESPG44 B cereus Bacillus sp 100 B subtillus ESPS29 D B C fabB E coli grhA Streptomyces sp Rubromycin ESPA12P2 Angucycline I jadJ S venezuelae 69 mtmP S argillaceus 99 ESPB5P3 100 KS S chrysomallus subsp fumigatus Aurelic 68 Acid ESPH4P3 SimA1 S antibioticus 97 73 ESPA19 31 99 urdA S fradiae 84 aur1D S aureofaciens Angucycline II 61 ESPB1 100 50 PGA Streptomyces sp 90 gra orf1 S violaceoruber 99 actI S coelicolor 88 KS S eurocidicus Naphthaquinone 55 ESPA14 44 31 aknB S galilaeus 100 dpsA S peucetius 87 Anthracycline I cosB S olindensis 100 KS S purpurascens snoaI S nogalater 100 KS S cinnamoneus 15926 Anthracycline II ESPA4 ESPE10 100 ESPA17 100 ESPA22 81 90 ESPA27 ESPS29 ND 31 ESPA2 96 ESPA7 59 ESPA3 56 ESPA5 ESPH4 3 100 ESPC73 95 KS S triostinicus 65 82 Ru2 S filipinensis ESPH4P4 100 62 ESPB6P4 whiES S aureofaciens 43 ESPC7 100 KS S violaceoruber 100 33 whiE S coelicolor ESPA3P1 52 ESPA21 100 ESPA10 Spore pigment 34 99 ESPA25 KS Streptomyces sp 100 KS Streptomyces sp 15MN1 85 ESPA11 97 whiEI S avermitilis curA S curacoi KS S macrosporeus 86 51 ESPA11P2 100 ESPH11P2 64 ESPA12 1 54 ESPG10P4 81 KS gene phylogenetic tree Controls: KS gene (2) whiE (1) (ND) No determined 10 Antibiotic E coli fabB ESPA11P2 S neyagawaensis concanamycin A 50 S noursei nystatin 83 S diastaticus 31 89 S nodosus amphotericin 97 68 95 Streptomyces sp heptaene1 S natalensis pimaricin 49 ESPA4 99 S avermitilis oligomycin 100 99 97 ESPS29 ESPA3 ESPC7 100 Saccharopolyspora erythraea 65 ESPA14 93 Streptomyces sp heptaene2 100 ESPA23 Frankia sp 100 ESPA4P1 100 KS type I 67 ESPB6P4 100 ESPA32 93 86 100 ESPA9 ESPA18 75 ESPA17 ESPA28 Sorangium cellulosum sorA 100 ESPA26 ESPA12P2 44 100 ESPA30 100 41 ESPA25 71 25 ESPE10 67 ESPA21 ESPA10 100 30 ESPB1 S griseus subsp griseus 96 Sorangium cellulosum sorB S albus oxazolomycin 100 54 85 S verticillus bleomycin S virginiae virginiamycin 100 Acidovorax citrulli ESPA8 100 ESPA5 100 ESPA12 1 ESPG10P4 100 Polyketide synthase type III Biosynthetic superfamily enzymes (15 types) all with homology with CHS; Superior plants (1970): Chalcona synthase (CHS) catalise the flavonoids biosynthesis. CHS structure alfafla (green). THNS structure from de S. coelicolor (red ). Cysteine catalytic site (bars and spheres). Metabolites synthesized by PKS type III Plants S. griseus e S. coelicolor Amycolatopsis Biosynthesis of antimicrobials of vancomycin group Biosynthesis of melanine and other metabolites Bioprospection of endophytic Actinomycetes 46 endophytic actinomycetes + 4 Streptomyces control (Streptomyces sp, Nocardia sp e Nocardiopsis sp) Extraction of genomic DNA (Method Salting out: Prospiech & Newmann, 1995) Detection of rppA-like (PKS III) genes in endophytics Controles Endofítico (A82-50) 600 pb Transcription analysis by RT-PCR endofíticos 600 pb THNS in Streptomyces THNS enzyme (1,3,6,8-tetrahidroxynaphtalene synthase) ou RppA = PKS III (Streptomyces) (Li et al, 2007). Biosynthesis: 1° part:: THNS catalize malonil-CoA condensation to form na intermediary polyketide. 2° part: THNS catalize two condensations (Claisen and Aldol) to form the metabolite THN. THN: melanin metabolism and backbone of metabolites (flavioline). Biological activity (antibiotic, antitumoral) rppA-like genes between the endophytics 40 Endophytic Enzyme C7 THNS A12-1(31) RppA A82(50) RppA JR1 STS e CHS JR3 CHS H43/C73 STS e CHS BLAST p - GenBank 7/21 Organism/C Funçtion ód. S. coelicolor Condensation (pdb1U0M) enzyme S. lividans “ (BAB91445.1 ) S. “ antibioticus (BAB94443.1 ) Streptomyces “ sp (ZP06276870 .1) S. griseus “ (BAA33495.1 ) Streptomyces “’ sp (ZP06276870 .1) % identid. /similarid. 92 % 89% 84% 90% 90% 91% Endophytic a.a. sequences aligment with THNS of S. coelicolor Catalytic triade residues Othe catalytic residues High variability regions Insertions or delitions 41 S. coelicolor B THNS structure A A 180o THNS Homodímers (KS monomers) A and B chains; catalytic site in colours 42 B Structural model 43 S. coelicolor THNS Hypothetic JR1 THNS Catalytic residues (Cys, His, Asn) Other catalytic residues Structural analysis 44 THNS_S.coel Endofítico JR1 THNS_S.coel Endofítico JR1 THNS_S.coel Endofítico JR1 PGFEDRNKVYEREAKSRVPAVIQRALDDAELLATDIDVIIYVSCTGFMMP ----------------------------------------------FLMP SLTAWLINEMGFDSTTRQIPIAQLGCAAGGAAINRAHDFCTAYPEANALI SLTAWLINTMGFRAGTRQLPIAQLGCAAGGAAINRAHDFCRAYPGSNVLI * VACEFCSLCYQPTDLGVGSLLCNGLFGDGIAAAVVRGRGGTGVRLERNGS VSCEFCSLCYQPTDIGVGSLLSNGLFGDAISAAVVRGEGGTGMSLERNGS * * * YLIPKTEDWIMYDVKATGFHFLLDKRVPATMEPLAPALKELAGEHGWDAS HLVPDTEDWISYAVRDTGFHFLLDKRVPGTMEMLAPVLKDIVDLHGWTVP * DLDFYIVHAGGPRILDDLSTFLEVDPHAFRFSRATLTEYGNIASAVVLDA AMDFFIIHAGGPRILDDLCHYLDLPMEMFRYSRATLTERGNIARS----* * * LRRLFDEGGVEEGARGLLAGFGPGITAEMSLGCWQTADVRRGIRQDVTRT -------------------------------------------------- THNS_S. coel Endofítico JR1 AARGVSRRVRQA 312 ------------ THNS_S.coel Endofítico JR1 THNS_S.coel Endofítico JR1 THNS_S.coel Endofítico JR1 50 4 100 54 150 104 200 154 250 199 300 Catakytic site conserved in both structures THNS S. coelicolor Hypothetic JR1 THNS THNS de S. coelicolor (1U0M) PKS III de S. lividans (ZP05527630.1) 45 Endofítico C7 RppB de S. antibioticus (BAB91444.1) Endofítico A12-1(31) PKS III deS. avermitilis (NP828307.1) RppA S. erythraea (AAL78053.1) Endofítico A82(50) THNS S. cinnamonensis (CAL34084.1) Bacteria NapB1 de S. aculeolatus (ABS50451.1) RppA de S. antibioticus (BAB91443.1) PKS III de S. griseus (BAA33495.1) Endofítico H43/C73 Endofítico JR1 Endofítico JR3 PhlD de P. fluorescens (AAB48106.1) PKS III de S. coelicolor (CAB94644.1) Monophyletic group PKS III de S. coelicolor (CAC17488.1) CHS de Medicago sativa (AAA02824.1) Naregenin-CHS de M. sativa (CAA48226.1) CHS de Solanum tuberosum (AAB67735.1) CHS de Nicotiana tabacum (AAK49457.1) Plants Neurospora crassa (XP324158.1) Gibberella zeae (EAA72166.1) Magnaporthe grisea (EAA50884.1) Aspergillus oryzae (BAD97391.1) A. oryzae (BAD97394.1) A. oryzae (BAD97390.1) A. oryzae (BAD97392.1) Fungi rDNA 16S identification Endo E coli ESPA26 ESPG1P1 59 45 100 50 100 ESPA28 ESPA23 ESPA30 ESPA8 phytic S hawaiiensis ESPA10 ESPA01 34 ESPA18 92 ESPA32 S macrosporeus 38 ESPB6P4 61 ESPB5P3 ESPG10P4 9 68 ESPA11P2 99 ESPH11P2 S atrovirens 82 ESPC07 98 35 Streptomyces sp ESPA9 88 ESPA19 S capoamus 12 ESPE10 6 4 100 13 93 80 82 C7 H43/C 7-3 ESPH4 3 ESPA4P1 96 84 46 A12-1 S griseus S aureus ESPB01 82 99 ESPH4P3 100 S tsukiyonensis S flavomacrosporus 43 ESPC6P2 19 ESPA82 19 ESPA3P1 25 ESPA12.1 58 ESPH4P4 S variabilis 87 ESPC73 57 ESPF7P4 2 100 ESPA12P2 ESPA16 ESPA02 S flavidofuscus 99 ESPA27 99 N dassonvillei 15 ESPA17 37 ESPA11 55 ESPA07 86 70 10 ESPA22 ESPA03 75 31 ESPA05 N alba 41 Nocardiopsis sp ESPA04 Nocardia puris 99 ESPA14 100 Nocardia sp ESPG44 100 B cereus 36 Bacillus sp 100 B subtillus ESPS29 (31) ESPA21 ESPA25 A82 76 95-100% 92-94% 91% 85-90% 80-84% ----------- specie genus order class phylo (50) B1 JR1 Lasher et al., 2009 JR3 Sequências mais similares do NCBI (BLASTn) Streptomyces sp. 102111 % ident . 99 % S. costaricanus 83% S. fumanus 97% S. spiralis * 99% Streptomyces sp. BM-2 Streptomyces sp. MB-D-1 Streptomyces sp. MB-D-1 10 rDNA 16S 98% 98% 96% Antimicrobial activity e e e a b a b b c d a e c c d d Endofphytic A12-1 (31) Endophytic A82 (50) Control semeada no centro em meio N.A (4 dias/28°C). semeada no centro em meio N.A (4 dias/28°C). Meio NA, Sem endofítico, 37°C/24h. a e b= B. subtilis, c= Proteus sp, d= Salmonella, e= E. coli Culture conditions 37°C/24h Research Team • • • • • • • • • • Juan Diego Rojas Karen Massini Fabiana Andrieli Erik Saenz Renata Furlan Welington Araújo Lara Duraes Sette Massuo Jorge Kato Nidia Yoshida CNPq, CAPES, FAPESP