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Development of novel and high value products in green algae Edible algae: Production of biologics to improve gut health Efficient production of biologic molecules 1978 1950s Bacteria (e. coli) Swine pancreas Insulin Insulin Use microbial organisms to efficiently and economically produce biologic molecules important to human or animal health How can we harness those advantages to produce new biologic products? Where does Algae have an advantage? Algae biologic molecules? 3 Biologics are expensive with existing systems Expensive Traditional Platforms • Production in traditional protein expression platforms (requiring purification) is too expensive Host Coli Yeast CHO • Algae offer a scalable, low-cost alternative Insect Algae Low cost production No Purification required? Algae are edible • They can serve as a delivery vehicle for biologics in the mammalian gut • Purification cost are eliminated 4 Global health: Triton’s products enable treatment of disease through food products Diarrhea is the largest public health problem in the world • Leading cause of mortality, with 1.5 million deaths annually; primarily in developing countries Botanicals • Botanicals address these diseases • Consumers or animals will eat enhanced foods for nutritional and health benefits Major issue for young children: • • • 33% of children under the age of five experience extreme diarrhea at least once per year 750,000 children die each year from diarrheal disease Especially an issue for women do not breastfeed; infant formula ingredients do not address diarrhea Animal Health • Dependency on antibiotics to reduce diarrhea 5 Products: Triton has a pipeline of products Botanicals Highlights of current products MAA Osteopontin • An active GI biologic, tested and proven in human and animals to prevent GI disease and improve digestive function • • • • A phosphorylated biologic that naturally occurs in mammalian milk Helps to increase bone fracture resistance Prevents calcification Plays a major role in neonatal immune development 6 Why osteopontin as a health and nutrition product? Low levels of OPN in infant formula and bonvine milk Scack et al. 2009 Considerable variation in the concentration of osteopontin in human milk, bovine milk and infant formula Osteopontin (OPN): Phospho-protein with 36 phosphates on human osteopontin and 28 phosphates on bovine osteopontin Chatterton et al. Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns. 2013 Human milk contains significantly more OPN than either bovine milk or infant formula 7 Osteopontin’s role in neonatal gut protection and development Function of Th-1 cells • Enhance macrophage phagocytosis of bacteria and protozoa • Maximize the proliferation of CD8 T-cells - Improve viral clearance Arla Foods 8 Expression and accumulation of osteopontin in chloroplast of green algae 1 2 3 4 5 6 Lane 1: Negative Control Lane 2: Chloroplast ostoepontin 1 Lane 3: Chloroplast osteopontin 2 Lane 4: Chloroplast osteopontin 3 Lane 5: Chloroplast osteopontin 4 Lane 6: Chloroplast osteopontin 5 9 Phosphorylation of osteopontin by chloroplast Mass spec identification of Osteopontin Phosphorylation Sites 10 Algae expressed Osteopontin is biologically active CD4 Th1 cell activation %CD4 T cells in culture Osteopontin mediated Cell Adhesion Algal OPN None nM algal osteopontin 11 Trtion Conclusions • Osteopontin is currently isolated from whey protein (30 metric tons of whey to get 1kg of osteopontin >$24,000) To get 1kg of osteopontin from algae a company would only need to produce 100kg of biomass • Algae biotechnology opens up new opportunities in health and nutrition • Advances made by algae cultivators can be applied to algal strains producing high value biologics • Trition is constantly seeking new partners, collaborators and opportunities 12 Production of advance next generation targeted antibody therapies Chloroplasts as a unique biochemical environment for protein production • Chloroplasts house the photosynthetic apparatus Maul et al. 2002 • Contains a single cup shaped chloroplast that comprises 4060% of the entire cell • Sequenced genomes • Has ribosomes and translation factors that resemble bacteria • Chloroplast are a distinct biochemical compartment Nield et al. 2011 14 Chloroplasts contain protein disulfide isomerases that are necessary for complex protein assembly FD(red) TR(red) SH PSI SH FD(ox) Light Translation-ON cPDI cPABP HS 5’ SH AUG 3’ TR(ox) S cPDI PSII Translation-Off cPABP S S S 5’ AUG Pi 3’ eH2O Kinase [ADP] Adapted from Kim and Mayfield 1997 15 Chloroplasts contain chaperones that are necessary for complex protein assembly Schroda 2004 Photos. Res. • in silico analysis of the C. reinhardtii genome showed that chloroplasts contain a complete set of complex chaperones including: Hsp100, HSp90, Hsp70, HSP40, Cpn60, Cpn10/20, and SHSP • Some chaperones appear to be of eukaryotic origin Hsp90, Hsp70 • Some appear to be of prokaryotic origin but contain distinct domains Cpn60 (GroEL), Cpn10/20 (GroEs) 16 The Unique biochemical environment of chloroplast allow them to produce complex antibody toxin fusions Antibody Domain Toxin Domain Becker and Benhar 2012 • The prokaryotic-like translational apparatus allows them to produce toxins capable of killing eukaryotic cells • Complex chaperones and PDIs allow chloroplast to fold and assemble complex multiple domain proteins 17 Algal chloroplasts can accumulate and assemble antibody toxin fusions Reduced Non-reduced Lane 1: wt total soluble Lane 2: α-CD22scFV-Antibody Lane 3: α-CD22scFV-Exotoxin A Lane 4: α-CD22scFV-HCH23-Exotoxin Tran et al. PNAS 2013 αFlag αFlag • Immunotoxins containing exotoxin A accumulate as soluble molecules in algal chloroplasts 18 Algal chloroplasts produced antibody toxins can specifically target and kill cancer cells Tran et al. PNAS 2013 Only targeted cancer cells are killed while normal healthy cells remained unharmed 19 Algal chloroplasts allow for the rapid production of large numbers of antibody toxin fusions Week 1 Antibody genes designed 2 Antibody genes ordered 3 Antibody genes weaponized 4 Algae transformed 5 Transgeneic strains 6 Expression confirmed 7 Purification 8 Cell Viability Assay 1 2 3 4 5 6 7 8 9 10 11 12 13 Dozens of antibodies can be weaponized in months for cancer therapies vs. a few in years using traditional methods 20 Coming of Age: Algae Chloroplast Expression Platform 1989: First Algae Chloroplast Transformation 1997: Identification of Protein Disulfide Isomerase in Chloroplast 2002: Sequenced Chloroplast Genome 2004: Identification of Prokaryotic like elongation factors in chloroplast 2004: Chloroplast Chaperones Identified 2007: Chloroplast ribosomes resemble those in bacteria 2007: High levels of protein accumulation in chloroplast 2012: Chloroplast produce antibody toxin fusions 2009 Chloroplast Assembly of human antibody 21 Closing Remarks • Chloroplast are capable of producing complex multiple domain proteins • Chloroplast can produce a class of cancer therapies that no other expression platform can • Verdant has produced a large library of antibody toxin fusions • Expression levels are still low (ug/L to mg/L) • Verdant will focus on developing novel methodologies for increasing recombinant protein accumulation and recovery 22 Acknowledgements Management Team Dr. Stephen Mayfield Dr. Jason Pyle Nick Hofmesiter Daniel Sachs Funding Sources NSF NIH California Energy commission Scientific Team & Collaborators Dr. Beth Rasala Mike Mayfield Austin Hallgren Dr. Bill Juilien Dr. Livia seno Ferreira Camargo Dr. John Chang Dr. Soo Ngoi Dr. Jack Bui Dr. Zhouxin Shen Dr. Steve Briggs