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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


Greg challis department of chemistry

Overview cryptic biosynthetic gene cluster products

  • 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


Greg challis department of chemistry

Overview of approaches cryptic biosynthetic gene cluster products

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


Greg challis department of chemistry

Gene knockout / comparative metabolic profiling cryptic biosynthetic gene cluster products

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


Greg challis department of chemistry

Expression of pathway specific activator / comparative metabolic profiling

host + activator

host - activator

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


Greg challis department of chemistry

host metabolic profiling

Heterologous gene expression / comparative metabolic profiling

host + genes

host - genes

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


Greg challis department of chemistry

Prediction of physicochemical properties metabolic profiling

predicted precursors

hu

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


Greg challis department of chemistry

* metabolic profiling

*

*

*

labelled predicted precursor

“Genomisotopic” approach

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


Greg challis department of chemistry

In vitro metabolic profiling pathway reconstitution

purified

enzymes

predicted precursors

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


Greg challis department of chemistry

Supernatant of metabolic profiling

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


Greg challis department of chemistry

Example 1: isolation of a novel metabolic profiling

cryptic NRPS product


A new s coelicolor nrps gene cluster
A new metabolic profilingS. 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 metabolic profilingS. coelicolor cryptic NRPS

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


Greg challis department of chemistry

Mutant metabolic profiling

A435 / nm

Wild type

Retention time / min

Gene KO / comparative metabolic profiling targeting predicted properties

cchH

X


Greg challis department of chemistry

Mass spectrometric analysis of coelichelin metabolic profiling

ESI-FTICR-MS

C21H39N7O11

ESI-MS-MS



Greg challis department of chemistry

2D-NMR analysis of Ga-coelichelin complex metabolic profiling

HMBC

ROESY



Greg challis department of chemistry

Structure of coelichelin metabolic profiling



Heterologous expression of the cch cluster in streptomyces fungicidicus

S. fungicidicus metabolic profiling

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


Greg challis department of chemistry

Example 2: isolation of novel metabolic profiling

products of a cryptic iterative PKS



Greg challis department of chemistry

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

3,5-DHPA-CoA

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


Type iii polyketide synthases
Type III polyketide synthases metabolic profiling


Greg challis department of chemistry

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

Moore, Noel and coworkers, unpublished


Greg challis department of chemistry

X from acyl thioesters + malonyl CoA

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


Greg challis department of chemistry

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)


Greg challis department of chemistry

Biosynthetic origins of germicidin A in Moore and Challis, S. coelicolor

EIC 197

EIC 202




Greg challis department of chemistry

Heterologous expression of Moore and Challis, sco7221 in Streptomyces venezualae ISP5230

PermE*

gcs (sco7221)

EIC 197 ISP5230

EIC 183 ISP5230

EIC 197 ISP5230 + sco7221

EIC 183 ISP5230 + sco7221


Greg challis department of chemistry

Fatty acid biosynthesis in Moore and Challis, Streptomyces coelicolor and E. coli compared


Greg challis department of chemistry

EIC 197 M511 Moore and Challis,

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



Greg challis department of chemistry

Ser Moore and Challis,

Cys

X-ray structure of germicidin synthase

Gcs

AcpP


Greg challis department of chemistry

Example 3: a novel product of a Moore and Challis,

cryptic sesquiterpene synthase


Cryptic sesquiterpene synthases of s coelicolor
Cryptic sesquiterpene synthases of Moore and Challis, S. coelicolor


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


Greg challis department of chemistry

Conclusions farensyl pyrophosphate

  • 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