1. Neal C. Connors, Ph.D. RISE Fellow (nconnors@drew.edu, HS 318, x3794)nconnors@drew.edu  B.S. Biology (chemistry minor) 1984 – Norwich University  Ph.D. Microbiology 1991 – Ohio State University  17 years Bioprocess R&D – Merck Research labs (1991 – 2008)  Currently:  RISE associate/fellow since February 2012.  Owner of Phoenix BioConsulting, LLC since 2009,  Founder/CTO of Kalion, Inc. since 2010 1

2. My Interests (microbial physiology & biochemistry)  Employing microbes as “factories” to produce useful products.  Medical: therapeutic proteins, bioactive natural products, vaccines, bioconversions.  Industrial: renewable fuels and chemicals  Other “cool” things that microbes do (good and bad)  Cave microbiology  Bioremediation (e.g. Deepwater Horizon oil spill, 2010)  Microbes using in mining.  Human (gut) microbiome 2

3. “Those biotech companies like Amgen and Genentech, those are the future IBMs” Prof. Roy Bair, Professor of Biology Norwich University, Spring 1983 3 http://farm6.staticflickr.com/5024/5655076099_b60feca501_z.jpg

4. Products We Use Everyday Come from Chemical Building Blocks 4

5. Crude Oil Traditionally the Source of Chemical Building Blocks 5 http://report.basf.com/2009/en/managementsanalysis/businessdevelopment/globaleconomy/chemicalindustry.html

6. Plentiful Agricultural Biomass Becomes the New “Crude Oil” 6 Biofuels and renewable biochemical building blocks from bio-refinery

7. Composition of Lignocellulosic Materials 7 MaterialCellulose (C-6 sugars) Hemicellulose (C-5 sugars) Lignin Hardwood40-55%25-40%20-25% Softwood45-5025-35 Corn Stover382619 Corn Cobs453515 Wheat Straw305015 Rice Straw322418 Switch Grass453112 Paper85015 Paper Refuse6020 Howard et al 2003; Wyman and Yang 2009 DOE/USDA project 1.3 Billion tons of dry biomass in the USA (= 1.5 billion barrels of petroleum). 600 million tons of hexose (C-6) sugar; 400 million tons of pentose (C-5) sugar.

8. DOE’s Top 12 Building Blocks Chemicals from Biomass 8 Werpy and Petersen,2004

9. Bioactives Phosphate Replacement/ Detergent Builder Fabrics Novel or traditional Nylon 6,6 Biomaterials Monomer for novel SAPS Glucaric Acid: $15-40 Billion Potential Market Biomaterials Hyperbranched polyesters 9

10. Engineered Glucaric Acid Pathway in E. coli BL21(DE3) (Prather Lab – MIT, Kalion Inc.) INO1 S. cerevisiae (yeast), MIOX M. musculus (mouse), Udh P. syringae (bacterium) Moon et al, Appl. Environ. Microbiol. 75(3):589-595 US Patent 8,835,147 10

11. Shake-flask Scale Model for Glucose Fed-batch Process Industrial fermentation processes use a glucose fed-batch process - Includes a glucose tank and pump to control culture glucose levels (not practical in shake flask cultures) Glucose “auto-feed” for shake-flask cultures Soluble Starch = “Glucose Tank” Hydrolytic Enzyme (Amyloglucosidase “AGU”) = “Pump” AGU cleaves glucose units from the non-reducing end of the starch molecule

12. Glucaric Acid Production Using Glucose Auto-feed Cultivation Method Stenger AR, Connors NC. (2015) Glucose Auto-feeding for Glucaric Acid Production by a Recombinant E. coli Strain. Society for Industrial Microbiology and Biotechnology Annual Meeting. Philadelphia, PA (poster). Brockman-Reizman IM, Stenger AR, Reisch CR, Gupta A, Connors NC, Prather KLJ (2015) Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes. Metabolic Engineering Communications. 2:109-116.

13. Expression of MIOX: Different Expression Conditions (e.g. free glucose vs glucose “auto-feed” 13 40 kDa 30 kDa 20 kDa [ Insol. Sol. Total ] Auto-feed Free

14. NMR for Monitoring Glucaric Acid and Glucarolactone Production (collaboration w/ Dr. Evans) E. Coli E. coli + GA plasmids Carbonyl Carbons (acid & lactone)

15. Sugars from Waste Cardboard (Avatar Sustainable Technologies – Fayetville, NY)  AST can chemically convert waste cardboard to glucose (C-6 sugar) and xylose (C-5 sugar).  Crude sugar source with other “nasty stuff”  Can we produce glucaric acid and/or ethanol from this sugar source?  Some E. coli growth toxicity observed; no glucaric acid produced.  Ethanol production using yeast, TBD.  Use “adaptive evolution” to condition the cells to produce product of interest from the sugar in the presence of the “nasty stuff” 15

16. Research Paper Topics  Previous  Are Biofuels the Way of the (Transportation) Future?  Modeling Microbial Fuel Cell Efficiency: A Potential Source for Alternative Energy.  A Review of Current Methods of Generating Electricity and a Potential Energy Source in Microbial Fuel Cells.  The Human Microbiome: A Foray into the Host-Bacteria Relationship  Other Topics  Biorefinery (renewable fuels and chemicals from biomass)  Bioactive natural products (bacteria, fungi, plants, invertebrates)  Phage (virus) therapy for treating bacterial infections.  Applications of microbes. 16