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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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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

Greg Challis

Department of Chemistry

slide2

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
slide3

Overview of approaches

Corre and Challis, Chem. Biol. (2007) 14, 7-9

slide4

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

slide5

Expression of pathway specific activator / comparative metabolic profiling

host + activator

host - activator

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

slide6

host

Heterologous gene expression / comparative metabolic profiling

host + genes

host - genes

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

slide7

Prediction of physicochemical properties

predicted precursors

hu

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

slide8

*

*

*

*

labelled predicted precursor

“Genomisotopic” approach

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

slide9

In vitro pathway reconstitution

purified

enzymes

predicted precursors

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

slide10

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

slide11

Example 1: isolation of a novel

cryptic NRPS product

a new s coelicolor nrps gene cluster
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
Prediction of substrates and possible products for the S. coelicolor cryptic NRPS

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

slide14

Mutant

A435 / nm

Wild type

Retention time / min

Gene KO / comparative metabolic profiling targeting predicted properties

cchH

X

slide15

Mass spectrometric analysis of coelichelin

ESI-FTICR-MS

C21H39N7O11

ESI-MS-MS

heterologous expression of the cch cluster in streptomyces fungicidicus

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
slide22

Example 2: isolation of novel

products of a cryptic iterative PKS

slide24

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

3,5-DHPA-CoA

Tseng, McLoughlin, Kelleher and Walsh Biochemistry (2004) 43, 970-980

slide26

In vitro investigation of the products formed by Sco7221 from acyl thioesters + malonyl CoA

Moore, Noel and coworkers, unpublished

slide27

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

slide28

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)

slide32

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

slide34

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

slide36

Ser

Cys

X-ray structure of germicidin synthase

Gcs

AcpP

slide37

Example 3: a novel product of a

cryptic sesquiterpene synthase

in vitro investigation of the product formed by sco5222 from farensyl pyrophosphate
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

slide40

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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