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

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