Intro GC Protein detection Nitrilases NHases PKS THM

Computational analysis of metagenomic data: delineation ofcompositional features and screens for desirable enzymes

Konrad U. Forstner

Bork Group, EMBL

Promotionskolloquium

04. Februar 2009

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

For the microbial ecologist, what can be cultured is the basis of hisconception of what exists. This is exactly like learning aboutanimals from visiting zoos.

Carl Woese

Intro GC Protein detection Nitrilases NHases PKS THM

Intro GC Protein detection Nitrilases NHases PKS THM

Intro GC Protein detection Nitrilases NHases PKS THM

Intro GC Protein detection Nitrilases NHases PKS THM

Intro GC Protein detection Nitrilases NHases PKS THM

Great plate count anomaly

Less than 1% of the microbes can be cultured under standardconditions.

Intro GC Protein detection Nitrilases NHases PKS THM

Metagenomics=

culture independent approaches

Intro GC Protein detection Nitrilases NHases PKS THM

Workflow of metagenomics sequencing

Intro GC Protein detection Nitrilases NHases PKS THM

Selected metagenomic data sets

Intro GC Protein detection Nitrilases NHases PKS THM

Challenges

Usually a low coverage

Dominant species

Short sequences

Data size

⇒ storage/memory/CPU intensive⇒ software not developed for that

No standard protocols

⇒ hard to compare

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

GC analysis

GC content = percentage ofGuanine-Cytosine bp in theDNA/RNA

influences a.o.

Melting temperature of DNA/RNACodon usage

Intro GC Protein detection Nitrilases NHases PKS THM

GC analysis - huge difference between soil and ocean water

Foerstner et al., 2005

Intro GC Protein detection Nitrilases NHases PKS THM

GC analysis - further data confirms statement

Raes et al., 2007

Intro GC Protein detection Nitrilases NHases PKS THM

GC analysis - possible influencing factors

Nitrogen availability

Genome size

Ultraviolet light exposure andrepair mechanism

Codon usage of pioneers

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

Metagenomics data sets as resources of biotech enzymes

Many microbial enzymes areessential tools in e.g. the chemical,pharma and food industries

Searching in metagenomic datasets might reveal new potentmembers of known enzymes classes

Intro GC Protein detection Nitrilases NHases PKS THM

Protein detection and classification workflow

Intro GC Protein detection Nitrilases NHases PKS THM

Nitrilases

Nitrile + water carboxylic acids + ammonia

One protein

Application in the chemical industry

Stereo- and regio-specific conversion of nitriles

Intro GC Protein detection Nitrilases NHases PKS THM

Nitrilases - new members and subfamilies found

Raes et al., 2007

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

NHases

Nitril hydratases (NHases)

Nitrile + water amide

Two domains

Application in the chemical industry

Acrylamide >30,000 tons/yearNicotinamide >3500 tons/year

Waste water treatment

Intro GC Protein detection Nitrilases NHases PKS THM

NHases - tree of the α domain

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

NHases - Monosiga brevicollis’ taxomony

Intro GC Protein detection Nitrilases NHases PKS THM

NHases - in Monosiga brevicollis

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I

Polyketide synthases (PKS) create a heterogeneous group ofsecondary metabolites

The synthesis is similar to the fatty acid synthesis

Multiple domains

We focused on polyketide synthases type I (PKS I)

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - polyketide synthesis steps

This picture of this slide is removed due to copyright restriction.Jenke-Kodama et al., 2005

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - examples of polyketides

Erythromycin Oleandomycin Aflatoxin B1

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - tree of the AT domain HMM hits

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - tree overview

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - hit distribution

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

PKS I - PKSs per genome

Foerstner et al., 2008

Intro GC Protein detection Nitrilases NHases PKS THM

Table of content

1 Introduction

2 GC content analysis

3 Protein detection workflow

4 Nitrilases

5 Nitril hydratases

6 Polyketide synthases I

7 Take home messages

Intro GC Protein detection Nitrilases NHases PKS THM

Take home messages

Metagenomics ...

... might help us to explore the complete microbial world

... still has many technical challenges

... can reveal the environmetal influence on genomic features

... can help discover new enzymes

Intro GC Protein detection Nitrilases NHases PKS THM

Acknowledgements

Peer Bork

Thomas Dandekar

Lars Steinmetz

Toby Gibson

The whole Bork group esp. JeroenRaes and Takuji Yamada

Christian von Mering

Melly

My friends and family

Intro GC Protein detection Nitrilases NHases PKS THM

Image sources/attribution - part 1/2

Orangutan Houston Zoo http://flickr.com/photos/billtex48/2178056762/ by (Bill and Mavis) -B&M

Opel Zoo 07.07.2007 http://flickr.com/photos/lamberty/754218458 by frijolito75

Giraffe http://flickr.com/photos/abelle/280246250/ by A.Bell

Snuggling http://flickr.com/photos/buckwoo/2421562192/ by Ken W!

Delicious Dead Bee and Hungry Ants http://flickr.com/photos/hamed/176176998/ by Hamed Saber

hundreds of fish swarm a soft coral head http://flickr.com/photos/g-na/370131126/ by g-na

hunt is on http://flickr.com/photos/doug88888/2930690305/ by doug88888

Long-billed Curlew http://flickr.com/photos/mikebaird/3011987508/ by mikebaird

145ps 01087.jpg http://flickr.com/photos/ricephotos/2679758872/ by IRRI Images

Polymicrobic biofilm epifluorescencehttp://commons.wikimedia.org/wiki/File:Polymicrobic_biofilm_epifluorescence.jpg

The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical PacificRusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. PLoS Biology Vol. 5, No. 3, e77doi:10.1371/journal.pbio.0050077

green farm http://flickr.com/photos/nakae/204037619/ by nakae

Acid Mine Drainage http://flickr.com/photos/savethewildup/400614071/ by savethewildup

blue ocean http://flickr.com/photos/coolskipper/27242821/ by coolskipper

Digestive system http://commons.wikimedia.org/wiki/File:Digestive_system_whitout_labels.svg

by Mariana Ruiz Villarreal

Pg166 bioreactor http://commons.wikimedia.org/wiki/File:Pg166_bioreactor.jpg

Intro GC Protein detection Nitrilases NHases PKS THM

Image sources/attribution - part 2/2

Big Drop-Off [...] http://flickr.com/photos/ctsnow/113339176/ by ctsnow

Sphaeroeca-colony http://commons.wikimedia.org/wiki/File:Sphaeroeca-colony.jpg by Dhzanette

Ocean view http://flickr.com/photos/provoost/399669002/ by Sjors Provoost

The hurdles http://flickr.com/photos/29621494N02/3060466344/ by paula fisher

Erythromycin http://de.wikipedia.org/w/index.php?title=Datei:Erythrommycin_A_B_C.svg byYikrazuul

Aflatoxin B1 http://de.wikipedia.org/w/index.php?title=Datei:

Aflatoxin_B1.svg&filetimestamp=20070113042046 by Bryan Derksen

Oleandomycin http://en.wikipedia.org/wiki/File:Oleandomycin.png by Edgar181

Tool rack http://en.wikipedia.org/wiki/File:Oleandomycin.png by L. Marie

Collaboration http://flickr.com/photos/fncll/145149313/ ChrisL AK

Base pair AT http://commons.wikimedia.org/wiki/File:Base_pair_AT.svg

Base pair GC http://commons.wikimedia.org/wiki/File:Base_pair_GC.svg

Intro GC Protein detection Nitrilases NHases PKS THM

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Document version 1.0 2009/02/04