1. Intro GC Protein detection Nitrilases NHases PKS THM
Computational analysis of metagenomic data: delineation of
compositional features and screens for desirable enzymes
Konrad U. F¨rstner
o
Bork Group, EMBL
Promotionskolloquium
04. Februar 2009

2. 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

3. 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

4. Intro GC Protein detection Nitrilases NHases PKS THM
For the microbial ecologist, what can be cultured is the basis of his
conception of what exists. This is exactly like learning about
animals from visiting zoos.
Carl Woese

5. Intro GC Protein detection Nitrilases NHases PKS THM

6. Intro GC Protein detection Nitrilases NHases PKS THM

7. Intro GC Protein detection Nitrilases NHases PKS THM

8. Intro GC Protein detection Nitrilases NHases PKS THM

9. Intro GC Protein detection Nitrilases NHases PKS THM
Great plate count anomaly
Less than 1% of the microbes can be cultured under standard
conditions.

10. Intro GC Protein detection Nitrilases NHases PKS THM
Metagenomics
=
culture independent approaches

11. Intro GC Protein detection Nitrilases NHases PKS THM
Workflow of metagenomics sequencing

12. Intro GC Protein detection Nitrilases NHases PKS THM
Selected metagenomic data sets

13. 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

14. 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

15. Intro GC Protein detection Nitrilases NHases PKS THM
GC analysis
GC content = percentage of
Guanine-Cytosine bp in the
DNA/RNA
influences a.o.
Melting temperature of DNA/RNA
Codon usage

16. Intro GC Protein detection Nitrilases NHases PKS THM
GC analysis - huge difference between soil and ocean water
Foerstner et al., 2005

17. Intro GC Protein detection Nitrilases NHases PKS THM
GC analysis - further data confirms statement
Raes et al., 2007

18. Intro GC Protein detection Nitrilases NHases PKS THM
GC analysis - possible influencing factors
Nitrogen availability
Genome size
Ultraviolet light exposure and
repair mechanism
Codon usage of pioneers

19. 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

20. Intro GC Protein detection Nitrilases NHases PKS THM
Metagenomics data sets as resources of biotech enzymes
Many microbial enzymes are
essential tools in e.g. the chemical,
pharma and food industries
Searching in metagenomic data
sets might reveal new potent
members of known enzymes classes

21. Intro GC Protein detection Nitrilases NHases PKS THM
Protein detection and classification workflow

22. 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

23. Intro GC Protein detection Nitrilases NHases PKS THM
Nitrilases - new members and subfamilies found
Raes et al., 2007

24. 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

25. 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/year
Nicotinamide >3500 tons/year
Waste water treatment

26. Intro GC Protein detection Nitrilases NHases PKS THM
NHases - tree of the α domain
Foerstner et al., 2008

27. Intro GC Protein detection Nitrilases NHases PKS THM
NHases - Monosiga brevicollis’ taxomony

28. Intro GC Protein detection Nitrilases NHases PKS THM
NHases - in Monosiga brevicollis
Foerstner et al., 2008

29. 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

30. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I
Polyketide synthases (PKS) create a heterogeneous group of
secondary metabolites
The synthesis is similar to the fatty acid synthesis
Multiple domains
We focused on polyketide synthases type I (PKS I)

31. 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

32. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I - examples of polyketides
Erythromycin Oleandomycin Aflatoxin B1

33. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I - tree of the AT domain HMM hits
Foerstner et al., 2008

34. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I - tree overview
Foerstner et al., 2008

35. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I - hit distribution
Foerstner et al., 2008

36. Intro GC Protein detection Nitrilases NHases PKS THM
PKS I - PKSs per genome
Foerstner et al., 2008

37. 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

38. 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

39. Intro GC Protein detection Nitrilases NHases PKS THM
Acknowledgements
Peer Bork
Thomas Dandekar
Lars Steinmetz
Toby Gibson
The whole Bork group esp. Jeroen
Raes and Takuji Yamada
Christian von Mering
Melly
My friends and family

40. 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 epifluorescence
http://commons.wikimedia.org/wiki/File:Polymicrobic_biofilm_epifluorescence.jpg
The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. PLoS Biology Vol. 5, No. 3, e77
doi: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

41. 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 by
Yikrazuul
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

42. Intro GC Protein detection Nitrilases NHases PKS THM
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Document version 1.0 2009/02/04