Samaneh NoorDepartment of Chemical Engineering

The University of Tulsa

Method to Assemble Biosynthetic Pathways in Microalgae

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

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Goal of Research

• Develop a Method to Genetically Engineer Microalgae

• Multiple Gene Biochemical Pathways

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Why Microalgae?Why Microalgae?

• GRAS• Low Impact• Flexible on Water Quality• High Growth Rate• Low Production Cost• High Levels of Oils

and Hydrocarbons

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Biology of AlgaeBiology of Algae

• Aquatic, Photosynthetic Organisms– Microalgae, Macroalgae

• Microalgae– Chlamydomonas reinhardtii– Single Cell, Eukaryotic

• Mitochondrial• Nuclear• Chloroplast

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Various Microalgae and

Diatoms

6Image 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)

How to Improve ProductivityHow to Improve Productivity

• Selection / Screening Techniques

• Cultivation

• Genetic / Metabolic Engineering

7http://www.mvm.uni-karlsruhe.de/img/bio/rohrreaktor_neu.jpg

http://news.cnet.com/i/bto/20080620/Seambiotic_Ponds_540x354.jpg

Genetic and Metabolic EngineeringGenetic and Metabolic Engineering

• Nucleus

• Chloroplast

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Chloroplast of C. reinhardtiiChloroplast of C. reinhardtii

– 40% Cell Volume

– Photosynthetic Apparatus

– Metabolic Pathways

– RNA and Protein Synthesizing Systems

• Prokaryotic Organisms

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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)

C. reinhardtiiChloroplast Genome

203,395 bp

Advantage of Chloroplast Advantage of Chloroplast

Parameters Chloroplast NucleusGenome Sequence Complete CompleteNumber of Genes

Expressed Multiple SingleGene Integration Site Specific Not SpecificGene Silencing Not Probable Probable

Versatility to Express genes from other

organisms

High Low

Recombination Machinery

Homologous Non Homologous

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Gene Gene Product

aadA Aminoglycoside adenyl transferaseuidA β-glucoronidaserecA RecA proteinRluc/luxCt LuciferaseaphA6 Aminoglycoside phosphotransferasegfp/GFPct Green fluorescent proteingds Geranylgeranyl pyrophosphate synthaseHSV8-lsc Single-chain antibodyFMDV.VP1 Viral proteinapc AllophycocyaninnifH Nitrogenase subunitm-saa Bovine serum amyloidlacI Lac repressor proteinhMT-2 Metallothionein-2Trail TRAIL proteinamy α-amylaseCSFV E2 E2 proteinhGAD65 Glutamic acid decarboxylase83K7C Monoclonal antibody

reporter protein

pharmaceutical protein

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

1 WEEK

Chloroplast DNA

Yeast ori

E. coli ori Yeast Selection

Chloroplast DNA

2. Transformation

3. Selecting Primary Transformants and Homoplasmic lines

5’ UTR Gene 3’ UTR

OE-PCR

Expression Cassette

2WEEKS

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Plasmid Assembly and Integration

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gene5’UTR 3’UTR

Vector

Chloroplast DNA

Yeast ori

E. coli ori Yeast Selection

Chloroplast DNA

Chloroplast Transformation

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Transformed AlgaeBiolostic Particle Gun

Homoplasmic Lines

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TAP+Kan Plates

Grow Homoplasmic Colony

80 Chloroplasts Contain Gene

1 2 43 5 6

Gene Verification

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1=Mass Marker2=cc125-pTJ322-aphA6; aphA6 gene

3= cc125-pTJ322-aphA6-aadA; aphA6 gene4=cc125-pTJ322-aphA6-aadA; aadA gene

5=Mass Marker

120 V, 0.7% agarose, 30 min

1 3 4 52

Growth on AntibioticsaphA6

aadA+aphA6

• One Gene Successfully and Functionally Expressed

• Two Genes Successfully and Functionally Expressed

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Growth Curvecc125-pTJ322-aphA6

Wild Type Strain

Engineered Strain

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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

22For more information please visit: http://johannes.wik.is/