Method to Assemble Biosynthetic Pathways
in Microalgae
Samaneh Noor
Department of Chemical Engineering
The University of Tulsa
IPEC 2010
September 2, 2010
Outline
• Goal of Research
• Introduction and Background
• Assembly of Multi-Gene Biosynthetic Pathways into
Microalgae
• Preliminary Results
• Current Work
• Summary
2
Goal of Research
• Develop a Method to Genetically Engineer
Microalgae
• Multiple Gene Biochemical Pathways
3
Why Microalgae?
•
•
•
•
•
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GRAS
Low Impact
Flexible on Water Quality
High Growth Rate
Low Production Cost
High Levels of Oils
and Hydrocarbons
4
Biology of Algae
• Aquatic, Photosynthetic Organisms
– Microalgae, Macroalgae
• Microalgae
– Chlamydomonas reinhardtii
– Single Cell, Eukaryotic
• Mitochondrial
• Nuclear
• Chloroplast
Various Microalgae and Diatoms
5
Image source: Rosenberg, J.N., Oyler, G.A., Wilkinson, L., Betenbaugh, M.J. A green light for engineered algae: redirecting metabolism to fuel a biotechnology
revolution ,Current Opinion in Biotechnology, 19 (5), pp. 430-436 (2008)
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How to Improve Productivity
• Selection / Screening Techniques
• Cultivation
http://news.cnet.com/i/bto/20080620/Seambiotic_Ponds_540x354.jpg
• Genetic / Metabolic Engineering
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http://www.mvm.uni-karlsruhe.de/img/bio/rohrreaktor_neu.jpg
Genetic and Metabolic Engineering
• Nucleus
• Chloroplast
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Chloroplast of C. reinhardtii
– 40% Cell Volume
– Photosynthetic Apparatus
C. reinhardtii
Chloroplast Genome
203,395 bp
– Metabolic Pathways
– RNA and Protein Synthesizing Systems
• Prokaryotic Organisms
Figure from Maul J.E., Lilly J.W., Cui L., et al. The Chlamydomonas reinhardtii plastid
chromosome:islands of genes in a sea of repeats. Plant Cell 14(11):2659–79 (2002)
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Advantage of Chloroplast
Parameters
Chloroplast
Nucleus
Genome Sequence
Complete
Complete
Number of Genes Expressed
Multiple
Single
Gene Integration
Gene Silencing
Site Specific
Not Probable
Not Specific
Probable
Versatility to Express genes
from other organisms
High
Low
Recombination Machinery
Homologous
Non Homologous
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Gene
Gene Product
aadA
uidA
recA
Rluc/luxCt
aphA6
gfp/GFPct
gds
HSV8-lsc
FMDV.VP1
apc
nifH
m-saa
lacI
hMT-2
Trail
amy
CSFV E2
hGAD65
83K7C
Aminoglycoside adenyl transferase
β-glucoronidase
RecA protein
Luciferase
Aminoglycoside phosphotransferase
Green fluorescent protein
Geranylgeranyl pyrophosphate synthase
Single-chain antibody
Viral protein
Allophycocyanin
Nitrogenase subunit
Bovine serum amyloid
Lac repressor protein
Metallothionein-2
TRAIL protein
α-amylase
E2 protein
Glutamic acid decarboxylase
Monoclonal antibody
reporter protein
pharmaceutical protein
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Goal of Research
• Genetically Engineer Microalgae
• Multiple Gene Biochemical Pathways
– Short Term Goal
» Sugar Utilization
» Hydrocarbon Production
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1. Amplify Cassettes and Gene Assembly in Yeast
Yeast ori
5’ UTR
Gene
3’ UTR
E. coli ori
Yeast Selection
OE-PCR
Chloroplast DNA
Chloroplast DNA
1
W
E
E
K
Expression Cassette
2. Transformation
2
W
E
E
K
S
3. Selecting Primary Transformants and Homoplasmic lines
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Plasmid Assembly and Integration
Vector
Yeast ori
E. coli ori
Yeast Selection
Chloroplast DNA
5’UTR
gene
Chloroplast DNA
3’UTR
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Chloroplast Transformation
Biolostic Particle Gun
Transformed Algae
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TAP+Kan Plates
Homoplasmic Lines
1
2
3
4
6
5
Grow
Homoplasmic
Colony
80 Chloroplasts
Contain Gene
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Gene Verification
1
2
3
4
5
120 V, 0.7% agarose, 30 min
1=Mass Marker
2=cc125-pTJ322-aphA6; aphA6 gene
3= cc125-pTJ322-aphA6-aadA; aphA6 gene
4=cc125-pTJ322-aphA6-aadA; aadA gene
5=Mass Marker
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Growth on Antibiotics
aphA6
• One Gene Successfully and
Functionally Expressed
aadA+aphA6
• Two Genes Successfully and
Functionally Expressed
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Growth Curve
cc125-pTJ322-aphA6
2.5
Engineered
Strain
O.D. 750 nm
2
1.5
1
Wild Type Strain
0.5
0
0
20
40
60
80
100
Time (hr)
120
140
160
180
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Current Work
• Other Multiple Gene Pathway Constructions
• aphA6-phbC-phbA-phbB
• aphA6-Arabinose
• Introduce Assembled Genes into Algae
• Assay for Enzyme Activity
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Summary
• Algae - a Promising Source for Fuel and
Therapeutic Proteins
• Success Assembling Up to 3 Gene Pathways
• Success Integrating 3 Gene Pathways into
Chloroplast Genome of Chlamydomonas
reinhardtii
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Acknowledgments
 Professor Tyler Johannes
 Azadeh M Pourmir
 TU Chemical Engineering Department
 Maddie Laizure
 Kelby Aten
 Chris Dean
 Kayla Kutter
 Anne Campbell
For more information please visit: http://johannes.wik.is/
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