Microarray Analysis of Recombinant

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Microarray Analysis of Recombinant Aspergillus
nidulans overexpressing the UPR inducing
transcription factor hacA
Karin Lanthaler Manda Gent
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OBJECTIVES IN GAPSIA
Establish full transcriptome and proteome
profiles of all the biological systems
Evaluate this data with respect to recombinant
protein secretion and hyphal development
Identify common features in gene regulation
amongst different recombinant strains
Study evolution of strains in chemostat
cultivation
Establish transcriptome profile of evolved
mutants vs. parental strain under equivalent
culture conditions in order to identify genes up
or down regulated in the evolved strains
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KEY PLAYERS IN THE UNFOLDED PROTEIN RESPONSE
Transcription factor hacA, which gets spliced by
the transmembrane kinase ireA upon accumulation
of unfolded/misfolded proteins in the
Endoplasmatic Reticulum (ER)
The UPRE (Unfolded Protein Response Element)
consensus sequence has been determined by Harm
Mulder (DANISCO)
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NEW STRAINS IN GAPSIA
Aspergillus nidulans 12 (pyrG/pantho-) control
strain
Aspergillus nidulans 31 (pyrG/pantho-)
hacA-gpd promoter
Aspergillus nidulans 35 (pyrG/pantho-)
hacA-gpd promoter
Aspergillus nidulans 110 (pyrG/pantho-)
ireA-gpd promoter
Aspergillus nidulans 251 (pyrG/pantho-)
ireA-gla promoter
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Aspergillus nidulans A1004 (wt) on 8 gl-1
glucose 15 mll-1 VAS T30C pH5.5
Glucose depleted
Log2 ratio of 2 batch samples (dye flip approach)
CDW gl-1 ?
CO2 ppm
Log2 ratio of 2 batch samples
time h
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Aspergillus nidulans A1004 (wt) on 8 gl-1
glucose 15 mll-1 VAS T30C pH5.5 D
ca 0.1 h-1
Log2 ratio of 2 batch samples (dye flip approach)
Log2 ratio of 2 batch samples
CO2 ppm ___
CDW gl-1 fermenter ?
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Variation of expression
Batch Chemostat
Log2 ratio of 2 chemostat samples (dye flip
approach)
Log2 ratio of 2 batch samples (dye flip approach)
Log2 ratio of 2 batch samples
Log2 ratio of 2 chemostat samples
Log ratio of two dye-flips from batch or
chemostat fermentations Hybridisation array
technology coupled with chemostat culture in
Aspergillus nidulans. Gent M.E., Lanthaler K.,
Zeef L, Robson G.D., FangY. and Oliver S.G.
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Statistical comparison hacA overexpressing A.
nidulans 31 and control 12
Expression profiling of genes upregulated in the
same way as hacA yields 146 genes.
73 genes (50) are of unknown function
73 genes (50) are of known function or
hypothetical proteins
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Genes upregulated like hacA3
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In Silico and in Vivo comparison of
hacA overexpressing A. nidulans strain 31
Visual examination of the upregulated spots on
the slides showed many UPR related genes being
overexpressed in response to hacA overexpression
Many of those genes carry the UPRE as determined
by in silico analysis of the A. nidulans genome
at DANISCO
Unfortunately many of those genes are cut out
after statistical analysis
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THANKS TO
Susan Madrid
Kamilla Engelberth-Rasmussen
Geoff Robson and Stephen Oliver