The Eli and Edythe L. Broad InstituteA Collaboration of Massachusetts Institute of Technology, Harvard University and affiliated Hospitals, and Whitehead Institute for Biomedical Research

Project 2: Bioinformatics and Systems Modeling

Jeremy Zucker

Specific Aims of Project 2

• Finish the Neurospora assembly • Continue to improve the structural

annotation.• Curate the functional annotation, with

particular emphasis on metabolic enzymes• Develop scientific insights by using system

models to integrate data from Projects 1 and 3

New Assembly/Annotation

• NC7 =>NC10• Contigs: 251 =>20• Genes: 9826=>9734• Transcripts: 9846=>9908• Exons: 27208=>26625

• New genes: 530• Removed genes: 631 • Merged genes: 24• Split genes: 86

• 3869 UTR changes

This is the final assembly!

Heather Hood, Zehua Chen

But the annotation continues…

• RNAseq!– New genes– UTR’s– Alternative

splicing– Noncoding

RNAs

Matt Sachs, Brian Haas

From genome annotation to functional annotation

Pathway predictor

Enzyme predictor

• 267 Pathways• 1701 enzymatic reactions• 1455 compounds• 4000+ community annotations (thanks Heather and CAP participants!)• 853 literature citations

fungicyc.broadinstitute.org:1555

Summary of BioCyc Capabilities• Knowledge Management System for Neurospora

Community– Literature citations– Evidence codes

• Metabolic Pathways and gene regulation• Omics viewer

– RNA expression data– Flux predictions– Metabolite measurements– Protein expression

• Enables system modeling

From functional annotation to system modeling

Pathway predictor

Enzyme predictor

Model

Predictions

Omics data

Biofuels from Neurospora?

• Growing interest for obtaining biofuels from fungi

• Neurospora crassa has more cellulytic enzymes than Trichoderma reesei

• N. crassa can degrade cellulose and hemicellulose to ethanol [Rao83]

• Simultaneous saccharification and fermentation means that N. crassa is a possible candidate for consolidated bioprocessing

Xylose

Ethanol

Effects of Oxygen limitation on Xylose fermentation in Neurospora crassa

Zhang, Z., Qu, Y., Zhang, X., Lin, J., March 2008. Effects of oxygen limitation on xylose fermentation, intracellular metabolites, and key enzymes of Neurospora crassa as3.1602. Applied biochemistry and biotechnology 145 (1-3), 39-51.

Xylose

Pyruvate

TCA Ethanol

Respiration Fermentation

Glycolysis

0 2 4 6 8 10 12 140

10

20

30

40

50

60

70

Ethanol production vs Oxygen level

Oxygen level (mmol/L*g)

Etha

nol c

onve

rsio

n (%

)Low O2

Intermediate O2

High O2

Glycolysis

Xylose degradationPentose phosphate

Aerobic respirationFermentation

TCA Cycle

Model of Xylose Fermentation

Xylose

Oxygen

Ethanol

ATP

Two paths from xylose to xylitol

Glycolysis

Xylose degradationPentose phosphate

Aerobic respirationFermentation

TCA Cycle

Oxygen=5

ATP=16.3

NADPHRegeneration

NADPH &NAD+

Utilization

HighOxygen

NAD+ Regeneration

Glycolysis

Xylose degradationPentose phosphate

Aerobic respirationFermentation

TCA CycleEthanol

LowOxygen

Oxygen=0

Glycolysis

Xylose degradationPentose phosphate

Aerobic respirationFermentation

TCA CycleEthanol

IntermediateOxygen

OptimalEthanol

NADPH &NAD

Utilization

Oxygen=0.5

ATP=2.8

NAD Regeneration

NADPHRegeneration

All O2 used to regenerate

NAD used in first step

Glycolysis

Xylose degradationPentose phosphate

Aerobic respirationFermentation

TCA CycleEthanol

IntermediateOxygen

OptimalEthanol

NADPH &NAD

Utilization

Oxygen=0.5

ATP=2.8

NAD Regeneration

NADPHRegeneration

All O2 used to regenerate

NAD used in first step

BottleneckPyruvate

decarboxylase

Improve NADHenzyme

Future Goals

• Proceed pathway by pathway with the genome-scale metabolic reconstruction

• Apply gene expression and gene regulation data under a variety of different conditions

• Focus on a short list of high confidence predictions that can be experimentally validated.

AcknowledgementsBroad InstituteJames GalaganBruce Birren

Brian HaasAaron BrandesMatt HennLi Jun MaChristina Cuomo

Carsten Russ

Broad Genome Sequencing Platform

Program ProjectHeather HoodJay DunlapKathy BorkovichLouise GlassMary-Anne NelsonMatt SachsGloria TurnerDick Weiss

Mark Farman

Many others…

Finishing TeamMargaret PriestHarindra ArachchiLynne AftuckMike Fitzgerald

Genome AssemblySarah YoungSean Sykes

Annotation TeamBrian HaasMike KoehrsenQian ZengTom Walk