Post on 01-Nov-2014
description
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