greg challis department of chemistry lecture 2: methods for experimental identification of cryptic...
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Greg Challis
Department of Chemistry
Lecture 2: Methods for experimental identification of cryptic biosynthetic gene cluster products
Microbial Genomics and Secondary Metabolites Summer School, MedILS, Split, Croatia, 25-29 June 2007
Overview
• Overview of available approaches
• Identification of a S. coelicolor cryptic NRPS product
prediction of properties, gene KO / metabolic profiling
• Identification of S. coelicolor cryptic type III PKS products
gene KO / metabolic profiling
• Identification of a S. coelicolor cryptic terpene synthase product
in vitro reconstiution
Overview of approaches
Corre and Challis, Chem. Biol. (2007) 14, 7-9
Gene knockout / comparative metabolic profiling
X wild type
mutant
H2NNH
O
N
O
OH
NH
ONH2
NOH
O
NHO
O
O OH
H H
OH
O
O
HO
Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269
Song et al., J. Am. Chem. Soc. (2006), 128, 14754
Expression of pathway specific activator / comparative metabolic profiling
host + activator
host - activator
O
NH
OH
O
OH
R
aspyridones
Bergman et al., Nat. Chem. Biol. (2007) 3, 213-217
Heterologous gene expression / comparative metabolic profiling
host
host + genes
host - genes
NH
OO
O Cl
OMe
OOHOH
O
MeO
Hornung et al., ChemBioChem (2007) 8, 757-766
Prediction of physicochemical properties
predicted precursors h
NH
OO
OH
O OH
HO OH
OH
CO2HMeNH2N
NH
Banskota et al., J. Antibiot., (2006) 59, 533-542
“Genomisotopic” approach
labelled predicted precursor
** * *
NH
HNNH
O
NH
NH
HN
NH
NH
O
O
OO
OO
O
O
OH
HO
O OH
orfamides
Gross et al. Chem. Biol. (2007) 14, 53-63
In vitro pathway reconstitution
predicted precursors
purifiedenzymes
epi-isozizaene
Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023
H. Vlamakis, P. Straight, M. Fischbach
Addition of a soil metabolite to Streptomyces avermitilis induces it to produce a cryptic metabolite
Ueda et al., J. Antibiotics, 2000
Diffusible compound from a soil organism induces another organism to generate a new antibiotic activity
Supernatant of soil organism A
(stimulating compound)
Soil organism B(antibiotic producer)
• Paper discs containing extracts from the culture broth of organism A were placed adjacent to inoculated spots of organism B
•
• Organism B grew for 1 – 3 days
• Soft agar containing spores of Bacillus subtilis was overlain to indicate antibiotic production
Example 1: isolation of a novel cryptic NRPS product
A new S. coelicolor NRPS gene cluster
cchAcchBcchH
Flavin-dependent monooxygenase (cchB)
Non-ribosomal peptide synthetase (cchH)
Formyl-tetrahydrofolate-dependent formyl transferase (cchA)
MbtH-like protein (cchK)
Esterase (cchJ)
Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114
Export functions
Ferric-siderophore import
cchJcchI
Prediction of substrates and possible products for the S. coelicolor cryptic NRPS
O
NHNH2
OH
HN
OH
O
H
O
NOH
O
NH2
OH O
NHNH2
OH
HN
OH
O
H
O
HN
N
O
OHH
Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114
A E C A E C A
SO
HN
H2NO
N
H2N
OHO
H
S
Module 1 Module 2 Module 3
O
NH2HOH
S
OH
Gene KO / comparative metabolic profiling targeting predicted properties
cchH
X
-20
180
380
580
0 5 10 15 20 25 30 35 40
Retention time / min
Mutant
Wild type
-20
180
380
580
A4
35
/ n
m
408
+2 H
408+2H
307+2H
H2NNH
OOH
N
O
OH
NH
ONH2
NOH
O
NHO
O
O OH
H H
m/z
Mass spectrometric analysis of coelichelin
ESI-FTICR-MS
ESI-MS-MS
C21H39N7O11
NMR analysis of Ga-coelichelin complexAssignment H C
hfOrn1-C1 - 168.5 hfOrn1-C2 3.97, dd, 3.0 Hz, 3.0 Hz hfOrn1-C3 1.91, m; 1.50, m hfOrn1-C4 1.42, m; 1.50, m hfOrn1-C5 3.41, m; 3.51, m hfOrn1-NH2 - - hfOrn1-C(O)H 8.17, s 153.5 Thr2-C1 - 162.5 Thr2-C2 4.59, dd, 9.0 Hz, 9.0 Hz 58.0 Thr2-C3 3.82, m 66.0 Thr2-C4 1.14, d, 5.5 Hz 21.0 Thr2-NH 8.75, d, 9.0 Hz - Thr2-OH 5.08, d, 6.2 Hz - hOrn3-C1 - 173 hOrn3-C2 4.24, dd, 8.5 Hz, 11.0 Hz hOrn3-C3 1.76, m; ? hOrn3-C4 1.83, m; 1.83, m hOrn3-C5 3.45, m; 3.93, m hOrn3-NH 7.57, d, 8.5 Hz - hfOrn4-C1 - 170 hfOrn4-C2 4.15, dd, 11.0 Hz, 1.9 Hz hfOrn4-C3 1.64, m; 2.29, m hfOrn4-C4 1.96, m, 1.76, m hfOrn4-C5 3.76, m; ? hfOrn4-C(O)H 8.22, s 153.5 hfOrn4-NH2 - -
H2NNH
OOH
N
O
O
N
ONH2
NO
O
NO
O
Ga
O OH
H
H H
12
3
4
5
12
34
1
2345
12
3
4
5
hfOrn1Thr2
hOrn3
hfOrn4
2D-NMR analysis of Ga-coelichelin complex
H2NN
OOH
N
O
O
N
ONH2
NO
O
NO
O
Ga
O OH
H
H
HH
HH
H H
1
2
34
5
6
7
1
2
3
4
5
6 7
1
2 3
5
6
4
H2NN
O
N
O
O
N
ONH2
NO
O
NO
O
Ga
O OH
H
H
HH
HH
H H
H
HOH
H1
2
3
H
4
5
6
HMBC
ROESY
Molecular modelling of Ga-coelichelin
2.45
2.52
2.06d
H2NN
O
N
O
O
N
ONH2
NO
O
NO
O
Ga
O OH
H
H
HH
H
H H
HO H
2.58
1.83
2.46
Structure of coelichelin
H2NNH
O
N
O
OH
NH
ONH2
NOH
O
NHO
O
O OH
H H
OH
H2NNH
OOH
N
O
OH
NH2
NOH
O
O OH
H
proposedstructure
Assembly of a tetrapeptide by a trimodular NRPS
cchH
CchJH2N
NH
OOH
N
O
OH
NH
ONH2
NOH
O
NHO
O
O OH
H
H H
A E C A E C A
SH SO
N
HN
HOO
NHHOO
N
NH2
HO O
H
H
ON
H2N
HO
HO
SH
Module 1 Module 2 Module 3
cchJ
Heterologous expression of the cch cluster in Streptomyces fungicidicus
-50
350
750
1150
1550
-50
350
750
1150
1550
S. fungicidicus
S. fungicidicus + cch cluster
-50
350
750
1150
1550
S .coelicolor M145
Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269
Example 2: isolation of novelproducts of a cryptic iterative PKS
Archetypal type III PKS products from bacteria
HO OH
O
SCoA
OH
OAmycolatopsis
orientalis
DpgA4 x Me
O O
O
NH
O HN
O
NH
O
NHMe
Cl
O
HN
H
OH
NH
OCl
OH
NHO
OHHO
H
HO2C
HOH
O
HOHO
O
OH
OH2N
HO Me
NH2
O
HO OH
O
OHH2N
Mechanism of 3,5-DHPA-CoA assembly by DpgA
-O
O
SCoA
O
DpgA
-S
-O
O
SCoA
O
DpgA
S
O
-O
O
O
SCoA
O
DpgA
-S
-O
O
O
SCoA
OOOO
-O
O
SCoAHO
O O
O
SCoA
HO OH
3,5-DHPA-CoA
Tseng, McLoughlin, Kelleher and Walsh Biochemistry (2004) 43, 970-980
Type III polyketide synthases
In vitro investigation of the products formed by Sco7221 from acyl thioesters + malonyl CoA
R SCoA
O
SCoA
O
HO
O+
purifiedrecombinant
Sco7221O
O
HO R'
Moore, Noel and coworkers, unpublished
Identification of a new S. coelicolor type III PKS products by genome mining
sco7221
0 5 10 15 20 25 Time [min]0.0
0.5
1.0
1.5
2.0
6x10Intens.
7221_EtOAc_72_01_511.d: EIC 197 +All MS, Smoothed (0.3,1, GA)7221_EtOAc_72_01_511.d: EIC 183 +All MS, Smoothed (0.3,1, GA)WTM145_7_01_1360.d: EIC 183 ±All MS, Smoothed (1.1,1, GA)WTM145_7_01_1360.d: EIC 197 ±All MS, Smoothed (1.1,1, GA)
EIC 197 sco7221
EIC 183 sco7221
EIC 197 M145
EIC 183 M145
X
Structures of the products
O
O
HO
O
O
HO
Germicidin A(MW = 196)
Germicidin B(MW = 182)
O
O
HO
Isogermicidin B*(MW = 182)
O
O
HO
Isogermicidin A*(MW = 196)
O
O
HO
Germicidin C(MW = 182)
Song, Barona-Gomez, Corre, Xiang, Udwary, Austin, Noel, Moore and Challis, J. Am. Chem. Soc. (2006), 128, 14754Petersen, Zahner, Metzger, Freund and Hummel, J. Antibiot. (1993) 46, 1126-1138
O
O
HO
Isogermicidin C*(MW = 182)
Biosynthetic origins of germicidin A in S. coelicolor
OO
OH
NH2 O
OH
O
OH D
DD
DD/H
D
D D
D D
D D
[U-13C]Isoleucine [2H7]butyrate
0 5 10 15 Time [min]0.0
0.5
1.0
1.5
2.0
2.58x10
Intens.
D7GerBcollection_2_01_2303.d: EIC 197 +All MS, Smoothed (0.4,1, GA)D7GerBcollection_2_01_2303.d: EIC 202 +All MS, Smoothed (0.4,1, GA), Smoothed (0.4,1, GA), Smoothed (0.4,1, GA)
0 5 10 15 Time [min]0
2
4
6
87x10
Intens.
C13Gercollection_10_01_2311.d: EIC 197 +All MS, Smoothed (0.4,2, GA)C13Gercollection_10_01_2311.d: EIC 202 +All MS, Smoothed (0.4,2, GA)
196.93
201.95
213.91 223.92
+MS, 21.00-21.07min #(3004-3014)
0
2
4
6
8
7x10Intens.
180 190 200 210 220 m/z
196.93
201.94
218.91
223.93
+MS, 20.13-20.24min #(2884-2899)
0
1
2
3
7x10Intens.
180 190 200 210 220 m/z
EIC 202EIC 197
Proposed mechanism 1 for germicidin assembly
SCoA
O
GcsA
HS
-O
O
SCoA
O
GcsA
S
O
R2
SCoA
O
GcsA
HS
R2
R1R2
R1
R1
O
-O
O
SCoA
O
GcsA
S
OO
R2
R1
R4
R2
R1
O O O
R4
SCoA
O
O
R4
R1R2 OH
R3 R3
R3
R3 R3 R3
-CO2-CoASH -CoASH
-CO2 -CoASH
Proposed mechanism 2 for germicidin assembly
SCoA
O
FabH
HS
-O
O
S
O
FabH
S
O
R2
S
O
GcsA
HS
R2
R1R2
R1
R1
O
-O
O
SCoA
O
GcsA
S
OO
R2
R1
R4
R2
R1
O O O
R4
SCoA
O
O
R4
R1R2 OH
R3 R3
R3
R3 R3 R3
FabC FabC
-CO2
-CO2
-CoASH
-CoASH
-FabC-SH
Heterologous expression of sco7221 in Streptomyces venezualae ISP5230
0 2 4 6 8 10 12 14 16 Time [min]0
1
2
3
4
7x10Intens.
newSV5d_2_01_3370.d: EIC 183 +All MS, Smoothed (0.4,1, GA)newSV5d_2_01_3370.d: EIC 197 +All MS, Smoothed (0.4,1, GA)Svenezualaenew_2_01_2361.d: EIC 183 +All MS, Smoothed (0.4,1, GA), Smoothed (0.4,1, GA), Smoothed (0.4,1, GA)Svenezualaenew_2_01_2361.d: EIC 197 +All MS, Smoothed (0.4,1, GA), Smoothed (0.4,1, GA), Smoothed (0.4,1, GA)
EIC 197 ISP5230 + sco7221
EIC 183 ISP5230 + sco7221
EIC 197 ISP5230
EIC 183 ISP5230
gcs (sco7221)
PermE*
Fatty acid biosynthesis in Streptomyces coelicolor and E. coli compared
SCoA
O
-O
O
S
O
S
O
R2
R1R2
R1
OR3 R3
ACP ACP+
R1R2
R3
OH
O
Sc-FabH
R1 = R2 = Me, R3 = H
R1 = H, R2= R3 =H
R1 = Me, R2 = R3 = H
R1 = R3 = H, R2 = Me
SCoA
O
-O
O
S
O
S
OO
ACP ACP+Ec-FabH
O
OH
Analysis of germicidin production in S. coelicolor YL/ecFabH
EIC 197 YL/ecFabH
EIC 183 YL/ecFabH
EIC 197 M511
EIC 183 M511
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min]0
2
4
6
7x10Intens.
PSE2SMMmedium_3_01_3234.d: EIC 183 +All MS, Smoothed (0.4,1, GA)PSE2SMMmedium_3_01_3234.d: EIC 197 +All MS, Smoothed (0.4,1, GA)M145SMMmedium_4_01_3235.d: EIC 183 +All MS, Smoothed (0.4,1, GA)M145SMMmedium_4_01_3235.d: EIC 197 +All MS, Smoothed (0.4,1, GA)
O
O
HO
O
O
HO
fabD aac(3)IV fabC fabBoriT ecfabH+
OH
O
OH
O
OH
O
PermE*
Proposed mechanism for germicidin assembly
SCoA
O
FabH
HS
-O
O
S
O
FabH
S
O
R2
S
O
GcsA
HS
R2
R1R2
R1
R1
O
-O
O
SCoA
O
GcsA
S
OO
R2
R1
R4
R2
R1
O O O
R4
SCoA
O
O
R4
R1R2 OH
R3 R3
R3
R3 R3 R3
FabC FabC
-CO2
-CO2
-CoASH
-CoASH
-FabC-SH
X-ray structure of germicidin synthase
Ser
Cys
Gcs
AcpP
Example 3: a novel product of a cryptic sesquiterpene synthase
Cryptic sesquiterpene synthases of S. coelicolor
In vitro investigation of the product formed by Sco5222 from farensyl pyrophosphate
Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023
O
P
O
O O-
P
O-O O-
Sco5222
-PPi
epi-isozizaene
sco5222 overexpressed in E. coli with N-terminal His6 and purified from CFE
sco5222 sco5223
Sesquiterpene synthase Cytochrome P-450
Sco5223?
O
albaflavenone
Streptomyces albidoflavus
O2-H2O
Conclusions
• Several different approaches for the identification of the products of cryptic biosynthetic gene clusters have been developed in recent years
• Several novel bioactive metabolites have been discovered from well-studied microbes by these approaches
• Activation of silent cryptic gene clusters is a challenge that awaits generic solutions
• Genome mining is a promising approach for new bioactive metabolite discovery