T. reesei - TNAU Genomics

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Protein quality control in the 

industrially - exploited fungal 

cell factory Trichoderma reesei 

Prof Helena Nevalainen, Dr Liisa Kautto, 

Dr Jasmine Grinyer and Dr Junior Te’o 


1


Prof Helena Nevalainen, brief biography 

FUNGAL BIOTECHNOLOGIST 

Research interests include molecular biology and functional 

proteomics of biotechnologically important filamentous fungi, in 

particular the industrially-exploited Trichoderma reesei. T. reesei offers 

a powerful cell factory for efficient expression of valuable gene 

products for various industrial and pharmaceutical applications. Other 

research interests feature fungal proteomics, biological control and 

molecular prospecting of the environment for novel bioactivities. 

Brief employment history: 

2009- Member of the Australian Research Council College of Experts 

2008- Head of the Department of Chemistry and Biomolecular Sciences, Macquarie 

University, Sydney 

2005- Professor of Biotechnology (Personal Chair) Department of Chemistry and 

Biomolecular Sciences, MU 

_____________________________________________________________________ 

1993- present Consultant to ROAL Ltd Finland 

1992- present Adjunct Professor (Docent) in Applied Microbial Genetics, University of 

Helsinki, Faculty of Science 

1989-1992 Head, Microbiological Department, Research Laboratories of ALKO Ltd 

Helsinki, Finland 


2


Trichoderma reesei as a cell factory 

• One of the best known cellulolytic fungi, producing 

extracellular proteins up to 100 g/L 

• Most of the secreted protein (~60%) is cellobiohydrolase I 

(CBHI) 

• Employed in industrial enzyme production 

• Developed for production of foreign gene products 


southdakotapolitics.blogs.com/.../index.html 

http://www.sustainpack.com/images/aap/pulp_ 

bleaching.jpg 

3


T. reesei has a eukaryotic 

protein-processing 

machinery 

Problem with foreign proteins: 

yields 100-1000 x lower than 

those of endogenous proteins 

Misfolded proteins are recognised 

by the cellular QC mechanisms 

(UPR and ERAD) and degraded 

by the proteasome 

UPR 

ERAD 


4


Our targets: 

The structure of the T. reesei proteasome (proteomics 

approach) 

Effect of overexpression of a dominant misfolded 

protein on gene expression (microarrays) 

Physiological effects of overexpression of a dominant 

misfolded protein (fluorescence and EM microscopy) 


5


The 26S proteasome, a proteolytic 

macromolecule 

Chymotrypsin-like (ChTL) – 5 

Trypsin-like (TL) – 2 

Peptidylglutamylpeptide hydrolyzing (PGPH) – 1 

Purified by 2-step chromatography: 


POROS Mono HQ - anion 

exchange 

Sephacryl S-500 HR - size 

exclusion 

Kautto et al. (2009) Prot. Expr. Purif. 67:156-163. 

6


2-DE analysis of the purified 26S proteasome 

q first dimension by pI 

q second dimension by mass 

q spots analysed by tandem mass spectrometry 

protein mass 

3 pI 10 

Protein spot from 

2-D gel 

Tryptic digestion & 

peptide extraction 

TYGGAAR EHICLLGR 

GPGFK 

GANR PSTTGVEMFR 

Unmodified and 

modified peptides 

Protein identification 

by database matching 

624.3 

769.8 

893.4 

994.5 

1056.1 

1326.7 

1501.9 

1759.8 

1923.4 

2100.6 

600 2200 

MALDI mass 

spectrometry 

or MS/MS 


7 

7


2D-map of the T. reesei 26 S proteasome 


8


Summary of protein identifications from the 

26S proteasome 

Total number of protein spots analysed 176 

Identifications by CSI 51 

Identifications from the in-house T. reesei 

database 

45 

20S particle subunits 14 

19S particle subunits 4 

PIPs (proteasome interacting proteins) 9 

Chaperones 7 

No association to the proteasome 13 

A 

B 

. 

C 

20 

S 

19S 


9


Making a misfolded 

protein (CBHI) by 

replacing cysteine 

residues with 

proline 

Disulfide bond First cysteine Location Second cysteine Location 

1 Cys4 L (Pca1-His11) Cys72 S (Asn70-Asp74) 

2 Cys19 S (Pro12-Ser20) Cys25 S (Thr24-Asp35) 

3 Cys50 S (Thr48-Asp52) Cys71 S (Asn70-Asp74) 

4 Cys61 L (Ser58-Asp63) Cys67 H (Asn64-Lys69) 

5 Cys138 L (Val133-Leu140) Cys397 L (Gly395-Val403) 

6 Cys172 L (Met149-Leu180) Cys210 Cys210 

7 Cys176 L (Met149-Leu180) Cys209 S (Gly205-Cys209) 

8 Cys230 S (Ser222-Thr231) Cys256 S (Thr255-Asp257) 

9 Cys 238 S (Glu236-Gly240) Cys243 L (Asp241-Gly254) 

10 Cys261 S (Cys261-Trp263) Cys331 H (Asp328-Phe338) 


10


The expression vector 

cbh1 promoter (red) 

signal sequence (black) 

DNA encoding CBHI core region (blue) 

Venus gene (yellow) 

cbh1 transcription termination region (ttm: truncated terminator; ftm: full 

terminator; grey) 

hygromycin selection marker gene under the pki promoter (green) 


11


PCR amplification of a mutant cbh1 gene (p4) 

Mutant genes transformed into T. reesei conidia by biolistic 

bombardment > strains with one copy of the gene in the cbh1 

locus 


12


Expression of a misfolded CBHI 

causes physiological stress 

Mutations also affected the profiles of secreted proteins 

and cellulase activity 


13


Genome-wide effects of the expression of a 

mutant CBHI 

Custom ArrayTM 12K slides 

Genome wide gene-specific 

microarray: 30-45 mer oligonucleotide 

probes representing 

the 9129 gene open reading 

frames (ORFs) derived from the 

Trichoderma reesei sequencing 

project (http://genome.jgipsf.org/Trire2/ 

Trire2.home.html) 

CVt: Cy3-ULS; 

others: Cy5-ULS 

Set of UPR/ERAD related 

genes 


14


jgi Trire Gene name U/E jgi Trire Gene name U/E jgi Trire Gene name U/E 

120153 19S RPN1 E 120650 20S ALPHA6 E 122396 NPL4 E 

78423 19S RPN2 E 76010 20S ALPHA7 E 131033 PEX4 E 

77591 19S RPN3 E 78882 20S BETA1 E 50647 HRD1 U 

82512 19S RPN4 E 53446 20S BETA2 E 64023 HRD3 U 

68304 19S RPN5 E 58125 20S BETA3 E 121977 PPI U 

54454 19S RPN6 E 78925 20S BETA4 E 52050 CPR3(CYPA) U 

49923 19S RPN7 E 121009 20S BETA5 E 122920 BIP U 

80843 19S RPN8 E 66707 20S BETA6 E 73678 CAL U 

77330 19S RPN9 E 105189 20S BETA7 E 119903 DER1 U 

66591 19S RPN10 E 22994 CDC48 E 35465 LHS1 U 

12189 19S RPN11 E 121397 SEC61 E 122415 PDI U 

48366 19S RPN12 E 80400 UCH1 E 28928 PRPA U 

73574 19S RPT1 E 21246 UFD1 E 119890 TIGA U 

77587 19S RPT3 E 72606 UBA1 E 64285 EDEM U 

63751 19S RPT4 E 123753 RUB1 E 119664 DOA4 U 

23206 19S RPT5 E 47635 SKN7 E 46902 HAC U 

78817 19S RPT6 E 123493 SSM4(DOA4) E 45242 IRE U 

121343 20S ALPHA1 E 123773 UBC1 E 81164 PTC U 

79825 20S ALPHA2 E 123559 UBC12 E 55362 HSP70 U 

73564 20S ALPHA3 E 77732 UBC6 E 119731 HSP60 U 

124031 20S ALPHA4 E 59987 UBC7 E 44504 ACT1 HK 

55644 20S ALPHA5 E 55788 UBC7homolog E 119735 GAPDH HK 

E: ERAD U: UPR HK: House keeping 


15


An overview of gene expression 

Up- ( 1.5 fold) or down-regulated ( 1.5 fold) genes from 

the T. reesei at three time points: A. 12 h; B. 24 h and C. 

48 h 

Most of the differentially expressed genes belonged to 

the class ‘metabolic pathways’ 


16


Up- and down-regulated genes assigned to their functional 

classes in strain CVt4. Up-regulated ; down-regulated . 


17


Expression changes in UPR/ERAD related genes 

RutC30 CVt2 CVt4 CVt5 RutC30 CVt2 CVt4 CVt5 RutC30 CVt2 CVt4 CVt5 

TR2 

code 

Gene Function 12h 12h 12h 12h 24h 24h 24h 24h 48h 48h 48h 48h 

122920 BIP UPR (3.669, 

0.543) 

(3.565, 

0.509) 

(4.4, 

0.505) 

(3.968, 

0.46) 

(3.667, 

0.644) 

(4.54, 

0.621) 

(4.257, 

0.664) 

(0.596, 

4.462) 

(0.652, 

2.931) 

(0.613, 

2.302) 

(0.574, 

2.814) 

(0.451, 

12.325) 

(0.368, 

7.251) 

(0.386, 

9.595) 

(0.407, 

8.18) 

122415 PDI UPR (5.237, 

0.395) 

(0.427, 

26.836) 

73678 CAL UPR (2.022, 

0.619) 

(1.603, 

0.631) 

(2.066, 

(0.612, 

1.998) 

(0.65, 

7.332) 

(0.541, 

3.923) 

0.669) 

(1.704, 

0.644) 

82512 19S RPN4 ERAD (11.622, 

0.445) 

(6.388, 

0.359 ) 

68304 19S RPN5 ERAD (0.655, 

2.512) 

(0.667, 

2.081) 

80843 19S RPN8 ERAD (0.606, 

2.393) 

48366 19S RPN12 ERAD (0.569, 

1.876) 

76010 20S ALFA7 ERAD (0.602, 

2.412) 

(0.619, 

2.232) 

66707 20S BETA6 ERAD (0.578, 

1.539) 

121397 SEC61 ERAD (3.315, 

0.506) 

(0.612, 

5.107) 

(3.52, 

0.571) 

(3.792, 

0.533) 

(0.664, 

1.904) 

(0.628, 

4.888) 

(0.651, 

4.992) 

72606 UBA1 ERAD (0.577, 


2.689) 

(1.816, 

0.665) 

18


Summary 

Amongst the three CBHI mutant strains examined, 

CVt2 did not show major expression changes in the 

genes involved in the UPR and ERAD pathways 

CVt4 was showing a stress response at 48 h and 

CVt5 was already showing signs of stress at 12 h 

Only 2.5 to 6.3 % of the differentially expressed genes 

were common in each strain either at two time points or 

all three time points 

All mutant strains exhibited stress related physiological 

changes such as low biomass synthesis and thinner 

hyphae during cultivation when compared to the nonmutant 

CVt 


19


Visualisation of the Trichoderma reesei proteasome 

A 

B 

C 

T. reesei hyphae immunolabelled with a polyclonal anti-yeast 20S antibody 

detected by Alexa Fluor®488 (green). ToPro3 used for nuclear staining (blue). 

A. 2 d old hyphae showing the 20S proteasome inside the hyphae 

B. Proteasomes in the conidia of 19 h old hyphae 

C. Proteasomes at the actively growing hyphal tip 

20 

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Co-localisation of CBHI to the proteasome 

A. B 

. 

C 

. 

White-resin embedded hyphae of T. reesei 

labelled with monoclonal anti-CBHI (green) and 

polyclonal 20S antiserum (red) 

T. reesei Rut-C30: A. CBHI 

B. 20S proteasome 

C. Merged images 

A 

. 

B 

C 

T. reesei CVt: A. CBHI 


C. Co-localisation (yellow) 

A 

B 

. 

C 

. 

T. reesei CVt4: A. CBHI 


C. Co-localisation 


21


Quantification of gold particles in thin sections with TEM 

5 nm (proteasome); 10 nm (CBHI) 

a, p


Summary 

Co-localisation of mutant CBHI and 20S proteasome 

particles was most obvious in strain CVt4, where they 

seemed to aggregate on the ER membrane (CLSM) 

Distribution of the gold particles in the indirect 

immunolabelling of CBHI and the 20S proteasome was 

different in the mutant strain CVt4 compared to the nonmutant 

CVt and the non-transformant Rut-C30 (TEM) 

These findings would indirectly point to degradation of the 

mutant CBHI by the proteasome 


23


Conclusions 

The T. reesei proteasome seems to have a very similar 

structure to proteasomes isolated from other eukaryotic 

cells 

The use of mutant (misfolded) forms of a native 

highly secreted protein to study cellular protein 

quality control is a new approach 

The heterologous protein Venus did not cause any 

stress to the strain in the culture conditions used when 

compared to the non-transformant Rut-C30 


24


Conclusions cont… 

Of all the differentially expressed genes identified, 23-50% 

had no match to known genes or proteins, or hypothetical 

genes or proteins 

Expression of a mutant CBHI decreased transcription 

and CBHI activity 

The known ER-stress-induced chaperones, BiP, PDI 

and PPI were found to be affected in strain CVt4 (48 h) 

CVt5 responded by mainly up-regulating the genes 

involved in the ERAD pathway 


25


Acknowledgements 

Australian Proteome Analysis 

Facility 

Liisa Kautto 

Jasmine Grinyer 

Junior Te’o 

Peter Bergquist 

Debra Birch 

MichaGodlewski 

Helsingin Sanomat 

Foundation 

Seppo Säynäjäkangas 

Foundation 

Australian Research 

Council 


26


Macquarie University in a nutshell 

• Established in 1969 

• Located about 18 km Northwest from Sydney 

CBD 

• Good public transport (train, buses) 

• About 33,000 students, 30% international 

• 24 h campus security 

http://www.mq.edu.au/ 

27 



Faculty of Science 

• 500 Staff (300 Academic, 100 Admin and 100 

Technical) 

• 500 HDR students 

• 3,500 Coursework students 

• Occupy 12 major buildings on campus 

• 10 National research centres 

• 7 Macquarie University research centres 

• More than 230 Labs 

28 



Department of Chemistry and Biomolecular 

Sciences (CBMS) 

20 academic staff and 60 higher degree research 

students 

• Teachers are active researchers and supervisors 

• State-of-the-art laboratories and facilities 

• Personalised instruction: students receive individual 

attention and advice 

29 



Analytical chemistry: advanced chemical and physical technologies 

Bio-organic and medicinal chemistry: drug discovery and design 

Chemical biology: application of structural chemistry to biomedical or 

biological problems 

Structural genomics: analyses of the 3-D structure and dynamics of 

proteins 

Proteomics: study of the thousands of types of proteins found in all life 

forms 

Functional glycomics: role of sugars in disease and infections 

Agricultural and plant cell biochemistry and biotechnology: metabolic 

processes in crop plants to help select new plant varieties 

Microbial genomics: vaccine and drug discovery, diagnostics, 

bioremediation 

Biotechnology: developing micro-organisms for production of 

commercially relevant proteins for biopharma and industry 


30


Highly specialised instrumentation 

including: 

Fermentors for making gene products 

Flow cytometers for fluorescence based cell 

sorting 

NMR (Nucleic Magnetic Resonance) instruments 

for the studies of chemical and protein structures 

A range of mass spectrometers for protein and 

biomolecular identification and characterisation 


31


Fermentors for making bioproducts 

www.oardc.ohio-state.edu/bioenergy 


32


FACSAria cell sorting 

flow cytometer 


33


A 600 MHerz 

NMR machine 

http://www.pharmacy.arizona.edu/faculty/yanglab/images/NMRFacility/NMR.JPG) 


34


CBMS Centers 

• Australian Proteome Analysis Facility 

• Biomolecular Frontiers Research Centre 

• Macquarie University Centre for Analytical 

Biotechnology 

• Environmental Biotechnology Co-operative Research 

Centre 

• Grain Foods CRC 

• Macquarie NMR Facility 

• Macquarie University VisLab 


35


Biomolecular Frontiers Research Centre 

• Mission: conduct world-class research into proteomics, glycomics 

and genomics and to enable a holistic understanding of organisms 

(systems biology) 

• Specific areas of research: 

• Cell biology (human, animal, plant and microbe) 

• Cellular interactions 

• Human disease biomarker discovery 

• Agri-food quality trait discovery 

• Protein post-translational modification and expression 

• Microbial physiology and pathogenicity 

• Lateral gene transfer and evolution 

• Bioinformatics 


36


Proteomics to identify what membrane 

proteins socialise in cancer metastasis 

(identify binding partners of uPAR) 


1. Dysregulation of proteolytic machinery 

at invasive edge of tumours (uPAR) 

2. Changes in cell adhesive properties as 

cancer becomes malignant (integrins) 

3. Differential responses to existent 

growth signalling pathways changes in 

malignancy (TGFb etc) 

uPAR 

37 

Saldanha et al., J Proteome Res. 2007 6: 1016-1028.


“Glycobiology is one of the 10 emerging technologies 

that will change the world." MIT 2003 

Glycomics @ MQ 

Cancer glycomics 

Profiling the oligosaccharide changes that 

occur on membrane proteins in cancer 

metastasis and drug resistance 

Innate immune system 

Comparison of the glycoproteins of human 

breast milk with cow’s milk – important 

antipathogenic factors 

Technology development 

Specific separation technologies for the 

analysis of recombinant glycoprotein drugs 

(e.g. erythropoetin) (in collaboration with SGE 

Analytical, Melborne and Bruker Daltonics, 

Bremen, Germany) 


38


Mobile DNA and gene diversity 

Understanding the impact of horizontal gene transfer 

on bacterial evolution and antibiotic resistant 

pathogens 

Environmental genomics 

Bioprospecting for genetic and microbial diversity 

G 

H 

I 

IS 

DNA helicase II 

integrase 

dnaK 

orf 

33 

9 

orf 

18 

6 

cox 

transposase 

IS IS IS 

gyrB 

pol 

B 

J 

gyr 

B 

pol 

B 

dn 

aA 

94% 

integrase 

orf 

25 

3 

orf 

11 

4 

orf 

85 

orf 

16 

9 

orf 

15 

2 

xre 

lysR 

IS 

IS 

IS 


39


High throughput genomic approaches to understanding microbial 

systems 

q Microarrays, phenotype arrays 

Microbial genomics 

q Genome sequencing, metagenomics 

q Bioinformatics 

Applications in vaccine and drug discovery, diagnostics, 

bioremediation, carbon sequestration and microbial evolution 


40


Development of fungi as cell factories for the 

production of enzymes and biopharmaceuticals 

Where genomics meets proteomics 

Pulp bleaching, detergents, 

biofuels… 

Antibodies, hormones… 

Directed evolution of 

biocatalysts for 

enhanced performance 


41


Australian Proteome Analysis Facility 

http://www.proteome.org.au/ 

Leading proteomics service provider, nationally and internationally, 

to both academia and industry 

Developer of new technologies for protein analysis, continually 

forming new collaborations with Australian and international 

biotechnology communities 

Aus $16 million investment from National Collaborative 

Infrastructure Strategy (NCRIS) 


42


Selection of mass spectrometers 


43


Selection of mass spectrometers 


44


Masters programs available 

• Master of Biotechnology (12 months coursework) 

• Master of Biotechnology (12 months coursework and 

research) 

• Master of Biotechnology with Master of Commerce (18 

months) 

• Master of Laboratory Quality Management (12 months) 

• Master of Radiopharmaceutical Sciences (18 months, 

available in 2011) 


45

T. reesei - TNAU Genomics

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