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Genome and proteomic analysis of industrial fungi. Scott E. Baker Pacific Northwest National Laboratory BMS Annual Scientific Meeting: Exploitation of Fungi Manchester, UK September 6, 2005. Products: Fuels and chemicals. Products: Fuels and chemicals. Petroleum. Petroleum refinery.

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Genome and proteomic analysis of industrial fungi

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Genome and proteomic analysis of industrial fungi l.jpg

Genome and proteomic analysis of industrial fungi

Scott E. Baker

Pacific Northwest National Laboratory

BMS Annual Scientific Meeting: Exploitation of Fungi

Manchester, UK

September 6, 2005


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Current and future routes to fuels and chemicals l.jpg

Products: Fuels and chemicals

Products: Fuels and chemicals

Petroleum

Petroleum

refinery

Complex biomass: Agricultural products and “waste”

Bio-refinery

Current andfutureroutes to fuels and chemicals

Petroleum products

Biobased products

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Filamentous fungi inside the bio refinery l.jpg

Products: Fuels and chemicals

Bio-refinery

Filamentous fungi inside the Bio-refinery

Fungal fermentationand catalysis

Fungal fermentationand catalysis

Simple sugars

Complex biomass: Agricultural products and “waste”

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The world of the mycologist…

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Publicly available and pending fungal genome sequence databases

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Why fungal genomics?

  • Genome sequence paints a high level picture of organism biology

  • Genome sequence is a platform for discovery

  • Genome sequence enables other high throughput discovery tools (proteomics, transcriptomics, etc)

  • A genome project can unite/revive a research community

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http://www.aspergillus.man.ac.uk/indexhome.htm?secure/sequence_info/index.php~main

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Why a public Aspergillus niger genome project?

  • A bioprocess organism

    • First citric acid process reported in 1917 with wildtype ATCC 1015 Aspergillus niger strain

    • Highly efficient fermentation of glucose to citric acid

  • A protein production organism

    • Source of important enzymes

    • Industrial protein producer

  • Sequenced twice by industry

    • Public access to sequence with restriction

  • Large economic footprint

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ATCC 9029NRRL 3122

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ATCC 9029NRRL 3122

~10X

~80X

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PNNL A. niger strain (sequenced by Integrated Genomics)

Phylogeny from Robert A. Samson, Jos A.M.P. Houbraken, Angelina F.A. Kuijpers, J. Mick Frank and Jens C. Frisvad. 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50:45-61.

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The DOE Aspergillus niger genome project

  • Proposed to the US Department of Energy Microbial Genome Program by the PNNL Fungal Biotechnology team

  • Collaboration with DOE’s Joint Genome Institute

  • Current status

    • Final coverage: ~8X shotgun

    • Production sequenced to 4X and QC assembly performed, 8X sequencing to be completed by September 14th, assembly and automated annotation to follow

    • EST libraries constructed from RNA isolated from citric acid production and complex biomass digestion conditions~25,000 to be sequenced

    • Annotation: In collaboration with JGI/LANL

    • Public release: Target of December 1st

  • Other Aspergillus niger genomes

    • ATCC 9029: low coverage, sequenced by Integrated Genomics – Sequence available on request. Contact Scott Baker or Jon Magnuson (scott.baker@pnl.gov or jon.magnuson@pnl.gov)

    • CBS 513.88: DSM – announced public release at Asilomar FGC

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Integrated Genomics JGI

  • Date

  • Method

  • Coverage

  • Genomic library insert size

  • Contigs or scaffolds

2000

Shotgun

No finishing

~4-6X

1-2kb

>9000

contigs

2005

Shotgun

Plus finishing

~8X

3kb

8kb

40kb

<100

scaffolds

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The qc a niger atcc 1015 assembly 4x coverage l.jpg

The “QC” A. niger ATCC 1015 assembly – 4X coverage

  • total number of scaffolds: 118

  • total length of scaffolds: 35634017

  • N50 scaffold number: 6

  • N50 scaffold size: 1931570

  • total number of contigs: 1646

  • total length of contigs: 34162656

  • N50 contig number: 215

  • N50 contig size: 47636

  • Total: 243,688 reads

    • 3 kb: 105,065 = 43.1%

    • 8 kb: 118,655 = 48.7%

    • 40 kb: 19,968 = 8.2%

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The “QC” A. niger ATCC 1015 assembly – 4X coverage

  • Over 40 that encode ketosynthase and acyl-transferase domains(i.e. PKSs and FASs)

  • Mat-1-1(alpha box)

  • ~95% of the of the genome is found in 24 scaffolds – 1.5 scaffolds/chromosome arm

  • EST coverage/annotation

    • Genencor to release ~7500 EST sequences from several different growth condition libraries

    • JGI will sequence 25,000 ESTs

    • The Fungal Genomics program at Concordia University will contribute ~12,000 ESTs

  • Annotation jamboree tentatively scheduled for April 2006, following the European Conference on Fungal Genetics

  • Limited gap closure or “finishing” is planned by JGI-LANL

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What’s next? A multi-gene phylogeny and more genomes from Aspergillus section Nigri

PNNL A. niger strain (sequenced by Integrated Genomics)

DOE MGP Proposed July 14th

Phylogeny from Robert A. Samson, Jos A.M.P. Houbraken, Angelina F.A. Kuijpers, J. Mick Frank and Jens C. Frisvad. 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50:45-61.

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Genome sequence…so what?


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General Procedure for Proteomics

Lyse cells

Digest with trypsin

  • Perform

  • LCQ

  • analysis

Isolate proteins

Separate in one or more dimensions reverse phase, ion exchange

Run data through

peptide identifying

program (SEAQUEST)

MS

Raw Data

Identify unique peptide = identify parent ORF

MS/MS

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Quantitative proteomics

  • Used for comparison of biological samples generated by two or more different experimental conditions

  • Current technologies utilize isotopic labeling strategies

    • ICAT

    • Metabolic labeling

    • Pairwise comparison

  • Our goal: Generate a quantitative proteomic methodology using statistical analysis of raw MS abundance data and that does not use isotopic labeling

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MASIC: A program for measuring ion peak intensity and area

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There are lies, there are damn dirty lies and there are statistics…

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Peptides from sample protein

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Statistical analysis I

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Statistical analysis II

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Relative quantitation with confidence intervals (95%)

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“Global” proteomic summary chart

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Proteomics summary and future directions

  • Using statistical analysis of raw mass spec data we have developed a methodology for relative quantitation of proteins across multiple samples

  • Future experiments:

    • Time course experiment – Citric acid production in Aspergillus niger

    • Strain comparison – Trichoderma reesei QM6a vs Rut-C-30

    • Other comparisons – “Production” strains vs. wildtype

    • Internal standards for greater quality control

    • Isotopic peptides for “absolute” quantitation

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Acknowledgements

PNNL Fungal Biotechnology

Ziyu Dai

Jon Magnuson

Chris Wend

Ellen Panisko

Ken Bruno

Kyle Fowler

Kelly Vincent

Bob Romine

Beth Hofstad

Mark Butcher

Katie Panther

Dennis Stiles

Linda Lasure

PNNL Proteomics QC Analysis Team

Don Daly

Kevin Anderson

Matt Monroe

Phylogenetic analysis

Rob SamsonCBS

Jens Frisvad DTU

Dave GeiserPenn State

DOE JGI A. niger ATCC 1015 genome

Dan DrellErika Lindquist

Diego MartinezPaul Richardson

Dan Rokhsar

Chris Detter…the list continues to grow!

David Bruce

Secreteome analysis

Adrian TsangConcordia U

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Future genomes…

Two “lower” fungi through the JGI CSP

Piromyces

Phycomyces (led by Luis Corrochano)

Phycomyces fungi, showing sporangiophores (fruiting bodies) in the wild type and color mutants. Photo by Tamotsu Ootaki.

Piromyces sp E2.. Photo by Johannes Hackstein.

http://www.jgi.doe.gov/sequencing/why/CSP2006/piromyces.html

http://www.jgi.doe.gov/sequencing/why/CSP2006/Pblakesleeanus.html

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