10/21/2008 Research Overview • Plant biotechnology/bioprocess engineering with applications to – Bioppp(gharmaceutical production (e.g. human α1- antitrypsin) • Ting-Kuo Huang (transgenic tobacco cell suspension cultures in stirred tank bioreactors) • Kittipong Rattanaporn (development of improved viral amplicon expression vectors for transient agroinfiltration) – Biofuels applications (in-planta production of cellulase enzymes) • Ben Lindenmuth (transient production in tobacco leaves) • Sang-KJKyu Jung (trans ien t pro duc tion in sw itc hgrass ) – Biopolymers (human gelatin and collagen) • Corey Dodge (rice cell suspension cultures in bioreactors) • Lucas Arzola (maize cell suspension cultures in bioreactors) Research Goals • Efficient production of recombinant proteins in plants and plant cell cultures Genetic Instructions Host Factors Bioprocess Approaches - Amino acid sequence - Choice of plant - Environmental conditions - Transient vs stable production - Stability - Operational strategies - Inducible promoter - Byproducts - Scale-up - Secretion signal - Optimized genes (codon usage/introns) 1 10/21/2008 Collaborations University: • Abhaya Dandekar, Plant Sciences • Bryce Falk, Plant Pathology • Jean VanderGheynst, Bio & Ag Engr. Industry: • Ventria Biosciences (Applied Phytologics) •FibroGen • Planet Biotechnology • Chevron •BioRad Ben Lindenmuth • PhD candidate in Chemical Engineering with a Designated Emphasis in Biotechnology • B.S. in ChE from Penn State 2 10/21/2008 Transient in planta expression of cellulose-degrading enzymes: Plant tissues as bioreactors Ben Lindenmuth CREATE-IGERT Trainee Dept. of Chemical Engineering & Materials Science University of California - Davis October 16th, 2008 Overview • Why produce cellulose-degrading enzymes? • Why use harvested plant tissue as a bioreactor? • How can plant tissue be used as a bioreactor? • Preliminary data. 3 10/21/2008 Cellulosic Biofuel Production Enzyme Production Cellulose + Enzymes Exposed Cellulose Other Polymers Cellulosic Biomass Disrupted Biomass Sugars Fuel Alcohol: Fermentation Ethanol, Butanol, etc. Distillation Cellulase Enzyme Production • Protein production on an unprecedented scale – Base case: replace 1% of U.S. gasoline consumption with cellulosic ethanol. – Current estimate: requires 31,000 tons of enzymes per year. • State of the art process: fungal cell fermentation – 76 x 100,000L bioreactors running constantly – Capital cost: $800 million – Alterna tive process: • Use bioreactor cultures to produce genes, not proteins • Transfer genes to plant tissue to produce enzymes 4 10/21/2008 Transient In Planta Expression of Enzymes • Agrobacteria known as “nature’s genetic engineers” - can transfer DNA to plant cells. • Plant cell transiently transcribes the transferred genes. • For these experiments: T-DNA replaced with an expression cassette. – Viral promoter (CaMV 35S) – Endoglucanase (thermostable enzyme from A. cellulolyticus) http://openlearn.open.ac.uk/file.php/2808/S250_1_003i.jpg Leaf Tissue Cuticle (Wax) Mesophyll Cells (air-filled extracellular space) Stoma (air exchange w/ atmosphere) http://www.science-art.com/gallery/24/24_120200693546.jpg 5 10/21/2008 Vacuum Infiltration of Leaf Tissue Vacuum PressurePressurePressure Air (14.7(14.7(2 psia) psia) psia) Agro. Suspension Agro. Suspension Agro. Suspension Air Fungal Cell Culture vs. Agroinfiltration • Advantages of using plant tissue – Plants grown outside using energy from sunlight. – Fungal cultures grown in bioreactors require a carbon source for energy. • Advantages of using Agrobacteria – Bacteria grow faster than fungal cells – Less bioreactor volume is needed • Capital cost comparison – Base case: enough enzyme to displace 1% of domestic gasoline consumption – Fungal cell culture bioreactors: $800 million – Agrobacteria bioreactors: $58 million 6 10/21/2008 Agroinfiltration Process + Agrobacteria Suspension N. benthamiana Leaves Vacuum Infiltration CH 3 CH3 E1 cat OH OH OH OH O HO + O HO HO O OH HO O O O O HO O HO O O HO O OH OH OH OH 4-methylumbelliferyl-β-D-cellobioside (MUC) cellobiose 4-methylumbelliferone (MU) Analyze Enzyme Yield Incubate Leaves Enzyme Production in Harvested Leaves Transient expression of Endoglucanase in N. benthamiana 7.0 6.0 resh weight) resh f 5.0 4.0 Day 4 Data Day 6 Data 3.0 2.0 1.0 glucanase (mg enzyme/kg plant plant enzyme/kg (mg glucanase 0.0 Endo Intact Plants Intact Plants Harvested Harvested (Hot/Light) (Cool/Light) Leaves Leaves (Cool/Light) (Cool/Dark) • Harvested leaves have the same enzyme yield as intact plants. • Leaves do not need light to produce high amounts of enzyme. 7 10/21/2008 Concentration of Agrobacteria Suspension In Planta E1 Expression vs. Agro-Suspension OD600 6.5 5.5 Day 4 Data Day 6 Data 4.5 3.5 2.5 Agrobacteria Suspension 1.5 0.5 -0.5 0.0000 0.0020 0.0033 0.0067 0.0167 0.0333 0.3333 1.0000 OD600 (AU) • OD = 0.33 or 1.00, same enzyme yield. • 10X OD increase (0.03 to 0.33), only 2X enzyme yield increase. Additional Chemicals Needed for Activation? Effect of Agrosuspension Conditions on EG1 Yield 5.5 Day 4 Data Day 7 Data 4.5 ) W 95% confident that Day 7 3.5 results are significantly different. 2.5 Agrobacteria Suspension 1.5 Endoglucanase (mg/kg F 0.5 -0.5 Sterile Water Active Suspension • Agrobacteria activated with low pH & phenolic compounds produce 2X more enzyme. • Is the additional yield worth the cost of buffers and phenolic compounds? 8 10/21/2008 Conclusions & Future Work • Cellulose-degrading enzymes can be produced in harvested plant tissue. – Similar yield as in intact plants. – Both show low yield, future work includes using Agrobacteria with a viral replicase system to increase yield. • Concentration of Agrobacteria infiltrated into leaves can be optimized. – Experiment will be repeated with Agrobacteria/viral replicase sys tem. • Agrobacteria do not have to be chemically activated prior to infiltration. – Will factor into cost analysis when higher yields are achieved. Acknowledgments Advisor/CREATE-IGERT Trainer: Prof. Karen McDonald & other students in the McDonald Lab Collaborators: Prof. Abhaya Dandekar - UCD Plant Biology Sandra Uratsu, Ph.D. - UCD Plant Biology Prof. Bryce Falk - UCD Plant Pathology Minsook Hwang, Ph. D. - UCD Plant Pathology Funding: NSF CREATE-IGERT Award #0653984 and Chevron Technology Ventures, L.L.C. (Project #23) 9 10/21/2008 Sources for images: • http://lpmpjogja.diknas.go.id/kc/b/beer/beer-fermenter.jpg • http://upload.wikimedia.org/wikipedia/commons/1/1a/Hay_bale_with_bird.jpg • http://www.eia.doe.gov/kids/energyfacts/sources/non-renewable/images/FCCDistCol.jpg • http://images.google.com/imgres?imgurl=http://genomics.energy.gov/gallery/biomass/originals/558.jpg&img refurl=http://genomics.energy.gov/gallery/biomass/detail.np/detail- 09.html&h=547&w=757&sz=301&hl=en&start=2&um=1&usg=__Pn2sL5vg0a0WbBSfAWSRft5II1E=&tbnid= eALHPrgrrr- oqM:&tbnh=103&tbnw=142&prev=/images%3Fq%3Dbiomass%2Bpretreatment%26um%3D1%26hl%3Den %26safe%3Doff%26sa%3DN • http://www.theautochannel.com/news/2007/02/21/037859.1-lg.jpg • http://images.vertmarkets.com/CRLive/files/images/753892d3-4379-488a-ad68-03a2b6fb26a1/prosuites.jpg 10.