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Seminar in Microbiology and Cell

Developing Aspergillus as a host for heterologous expression David Lubertozzi, Jay D. Keasling Biotechnology Advances 27(1):53-75, 2009

Oct. 04, 2010 Speaker : Ming-Yueh Wu Advisor : Prof. Ching-Tsan Huang

INTRODUCTION Expression System PRODUCT

HOST GENE

TRANSFORMATION

Quality

EXPRESSION

Quantity • Efficiency • Convenience • Cost 2

INTRODUCTION Prokaryotes

Eukaryotes Fungi Yeasts

E.coli Bacillus spp.

Plants

Animals

Filamentous fungi

Tobacco Aspergillus Tomato Rat S. cerevisiae spp. Potato Pig P. pastoris Trichoderma Arabidopsis spp.

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INTRODUCTION • Aspergillus – Protein production – Metabolite production – Bioconversion – Bioremediation – Food, fuel and fiber…

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INTRODUCTION • High environmental adaptability – Temp., pH, osmolarity, Aw

• Convenient ,cheap, and fast cultivation • Large amount extracellular secretion • Simple product recovery – Filamentous morphology

GOOD EXPRESSION HOST 5

CONTENTS TRANSFORMATION

Aspergillus Heterologous Expression

EXPRESSION STRAIN IMPROVEMENT

PRODUCT

HOST GENE

TRANSFORMATION

EXPRESSION

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Aspergillus Heterologous Expression

TRANSFORMATION

HOST GENE

TRANSFORMATION

PRODUCT

EXPRESSION

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TRANSFORMATION PROTOPLAST

PLASMID

TARGET GENE

PEG-MEDIATED

ELECTROPORATION

TRANSFORMATION GENE GUN

AGROBACTERIUM

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TRANSFORMATION PROTOPLAST

PLASMID

TRANSFORMANTS

TARGET GENE

TRANSFORMATION

SPORULATION REGENERATION 9

TRANSFORMATION • Multi-copy transformant – Stable – Homologous Recombination

– Non-Homologous Recombination

TARGET GENE TARGET PRODUCT

HOMOLOGOUS REGION

HOUSEKEEPING GENE

HOUSEKEEPING PRODUCT

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TRANSFORMATION • Homologous recombination improving – Agrobacterium-mediated transformation – Specific strains • Non-homologous end-joining (NHEJ) factors deficiency • High gene-targeting efficiency

1. Less unpredicted gene disruption 2. Gene targeting (knock-out, etc)

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Aspergillus Heterologous Expression

EXPRESSION

PRODUCT

HOST GENE

TRANSFORMATION

EXPRESSION

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EXPRESSION DNA

• Transcription level RNA

• Translation level Protein

• Secretion level • Protein stability 13

EXPRESSION • Transcription level – Copy numbers

DNA

RNA

– Gene loci

TARGET GENE

Low-yield gene

HOMOLOGOUS REGION

Low-yield product

HOUSEKEEPING GENE

High-yield gene

TARGET PRODUCT

High-yield product

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EXPRESSION • Translation level – mRNA stability • Secretion-carrier / 3’ UTR

RNA

Protein

– Translation efficiency • Secretion-carrier/ 3’ UTR/ 5’ UTR/ Codon usage Secretion-carrier

Mignone et al., 2002

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EXPRESSION • Secretion level

– High-branched strain – Gene fusion – ER-targeting sequence promoter

Secretion-carrier

Target gene

terminator

TRANSLATION Secretion-carrier

Protein ER-MODIFICATION

SECRETION 16

EXPRESSION • Protein stability – Protease

Protein

• Protease-deficient strain • Protease inhibiter • Cultivation condition

– Folding • Modification of chaperone expression • Unfolded Protein Response (UPR) induction

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EXPRESSION

M. Schröder, J. S. Chang, and R. J. Kaufman, Genes Dev. 14 (2000) 2962-2975

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Aspergillus Heterologous Expression

STRAIN IMPROVEMENT

PRODUCT

HOST GENE

TRANSFORMATION

EXPRESSION

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STRAIN IMPROVEMENT

• Why? –Quantity –Quality –Purity 20

STRAIN IMPROVEMENT • Better strain screening – Comparing, choosing！ • Transcriptional data

GENE

– DNA microarray

• Metabolite profiling

PRODUCT

– Multi-dimensional chromatographic and mass spectroscopy

• Genome mining – Prediction

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STRAIN IMPROVEMENT • Undesired products reduction – Knock-out • Agrobacterium • Transposon • Restriction enzyme mediated integration (REMI)

– Knock-down

siRNA

• RNAi

– Regulation

Aptamer

• Riboswitch 22

STRAIN IMPROVEMENT • Gene improvement – In vitro revolution • DNA shuffling

– In vivo revolution • Genome shuffling • Repeat-induced point mutation (RIP) – Regulation • Polyketide synthase (PKS) • Non-ribosomal peptide synthase (NRPS) Targe t

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STRAIN IMPROVEMENT • Metabolic engineering

DATABASE PROCESSING

BOTTLENECK

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STRAIN IMPROVEMENT • Inverse metabolic engineering DEFINE 1

PHENOTYPE

2

RELATED FACTORS

HOST

ASSEMBLE

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CONCLUSION • Aspergillus is not only a widely-used microorganism, but also a potential expression host. • Due to Aspergillus’ capabilities as an expression host, researches related to its expression system are still hot and ongoing.

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Any questions?