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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. Greg Challis Department of Chemistry. Overview. Overview of available approaches

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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

Greg Challis

Department of Chemistry


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

wild type

X

mutant

Song et al.,J. Am. Chem. Soc. (2006), 128, 14754

Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269


Expression of pathway specific activator / comparative metabolic profiling

host + activator

host - activator

Bergman et al., Nat. Chem. Biol. (2007) 3, 213-217


host

Heterologous gene expression / comparative metabolic profiling

host + genes

host - genes

Hornung et al., ChemBioChem (2007) 8, 757-766


Prediction of physicochemical properties

predicted precursors

hu

Banskota et al., J. Antibiot., (2006) 59, 533-542


*

*

*

*

labelled predicted precursor

“Genomisotopic” approach

Gross et al.Chem. Biol. (2007) 14, 53-63


In vitro pathway reconstitution

purified

enzymes

predicted precursors

Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023


Supernatant of

soil organism A

(stimulating compound)

Soil organism B

(antibiotic producer)

Diffusible compound from a soil organism induces another organism to generate a new antibiotic activity

  • 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

Ueda et al., J. Antibiotics, 2000

Addition of a soil metabolite to Streptomyces avermitilis

induces it to produce a cryptic metabolite

H. Vlamakis, P. Straight, M. Fischbach


Example 1: isolation of a novel

cryptic NRPS product


A new S. coelicolor NRPS gene cluster

cchI

cchJ

cchH

cchB

cchA

Non-ribosomal peptide synthetase (cchH)

MbtH-like protein (cchK)

Flavin-dependent monooxygenase (cchB)

Formyl-tetrahydrofolate-dependent formyl transferase (cchA)

Esterase (cchJ)

Export functions

Ferric-siderophore import

Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114


Prediction of substrates and possible products for the S. coelicolor cryptic NRPS

Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114


Mutant

A435 / nm

Wild type

Retention time / min

Gene KO / comparative metabolic profiling targeting predicted properties

cchH

X


Mass spectrometric analysis of coelichelin

ESI-FTICR-MS

C21H39N7O11

ESI-MS-MS


NMR analysis of Ga-coelichelin complex


2D-NMR analysis of Ga-coelichelin complex

HMBC

ROESY


Molecular modelling of Ga-coelichelin


Structure of coelichelin


Assembly of a tetrapeptide by a trimodular NRPS

cchJ

cchH


S. fungicidicus

S. fungicidicus + cch cluster

S .coelicolor M145

Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269

Heterologous expression of the cch cluster in Streptomyces fungicidicus


Example 2: isolation of novel

products of a cryptic iterative PKS


Archetypal type III PKS products from bacteria


Mechanism of 3,5-DHPA-CoA assembly by DpgA

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

Moore, Noel and coworkers, unpublished


X

EIC 197 M145

EIC 183 M145

EIC 197 Dsco7221

EIC 183 Dsco7221

Identification of a new S. coelicolor type III PKS products by genome mining

sco7221


Song, Barona-Gomez, Corre, Xiang, Udwary, Austin, Noel, Moore and Challis, J. Am. Chem. Soc. (2006), 128, 14754

Petersen, Zahner, Metzger, Freund and Hummel, J. Antibiot. (1993) 46, 1126-1138

Structures of the products

Germicidin B

(MW = 182)

Germicidin C

(MW = 182)

Germicidin A

(MW = 196)

Isogermicidin B*

(MW = 182)

Isogermicidin C*

(MW = 182)

Isogermicidin A*

(MW = 196)


Biosynthetic origins of germicidin A in S. coelicolor

EIC 197

EIC 202


Proposed mechanism 1 for germicidin assembly


Proposed mechanism 2 for germicidin assembly


Heterologous expression of sco7221 in Streptomyces venezualae ISP5230

PermE*

gcs (sco7221)

EIC 197 ISP5230

EIC 183 ISP5230

EIC 197 ISP5230 + sco7221

EIC 183 ISP5230 + sco7221


Fatty acid biosynthesis in Streptomyces coelicolor and E. coli compared


EIC 197 M511

EIC 183 M511

EIC 197 YL/ecFabH

EIC 183 YL/ecFabH

Analysis of germicidin production in S. coelicolor YL/ecFabH

PermE*

+

fabD

oriT

aac(3)IV

fabC

fabB

ecfabH


Proposed mechanism for germicidin assembly


Ser

Cys

X-ray structure of germicidin synthase

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

sco5222

sco5223

Sesquiterpene synthase

Cytochrome P-450

sco5222 overexpressed in E. coli with N-terminal His6 and purified from CFE

Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023


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


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