FROM BEER TO BIOFUEL Grain Waste Powering Eucalyptol Production - PowerPoint PPT Presentation

FROM BEER TO BIOFUEL Grain Waste Powering Eucalyptol Production Selena Dickinson Jenna Harvestine Rebecca Majewski Matthew Reichartz Patrick Van Handel James Wiatr Milwaukee School of Engineering (MSOE)

Choosing a Project  Wanted to produce a rare fragrance  Eucalyptus Oil  Derived from boiling bark from Eucalyptus trees  Shortage susceptibility  90% Eucalyptol www.eyeondna.com

Eucalyptol (1,8 Cineole)  Potential to be a fuel additive  8:1 ratio eucalyptol to gasoline  I,8 Cineole monoterpenoid  All terpenoids made from universal precursors  DMAPP and IPP meddic.jp

Mevalonate Pathway  Non-Mevalonate Pathway in E. coli  Natural inhibition  Mevalonate Pathway  Yields more product  Eukaryotic  Certain parts of pathway better from different organisms

Gene Pathway HMG-CoA reductase HMG-CoA Mevalonate synthase kinase Enterococcus Streptococcus faecalis pnemoniae Phospho- Thiolase 1,8- mevalonate cineole kinase Streptococcus synthase pnemoniae Arabidopsis thaliana Mevalonate pyrophosphate FPP decarboxylase synthase Streptococcus IPP pnemoniae isomerase

Spent Grain Waste  Milwaukee’s beer waste  Composed of hemicellulose  Breaks down further  Co-utilization of sugars important for 2 nd Generation biofuels  Diauxie newyorkcorkreport.com

Endoxylanase Xylosidase Gene Pathway Fibrobacter Fusarium succinogenes graminearum HMG-CoA reductase HMG-CoA Mevalonate synthase kinase Enterococcus Streptococcus faecalis pnemoniae Phospho- Thiolase 1,8- mevalonate cineole kinase Streptococcus synthase pnemoniae Arabidopsis thaliana Mevalonate pyrophosphate FPP decarboxylase synthase Streptococcus IPP pnemoniae isomerase

Xylose Initial growth on low glucose Lag as correct enzymes E. coli are able to concentrations synthesized grow on xylose

Eucalyptol Lower eucalyptol concentrations lead As eucalyptol becomes more to higher growth rates concentrated, growth rate slows

Secreting Enzymes to Digest Grain Waste  University of Washington 2009 iGEM Team  First Plasmid  Gene of interest Protein  Secretion tag  Second Plasmid Protein  Pump

Final Project Design Input Output mvaK2 Pump 1 Tag xynC-A mvaS 3 mvaK1 mvaD 2 Bba_K849000 Pump Tag xyloA TPS-CIN

Treatment of Spent Grains  Obtained spent grains  Treated with NaOH  Isolated hemicellulose  Gives higher yield of hemicellulose  Vacuum filtration

Obtaining the Introduced Genes  17 IDT Gene Blocks  7 new genes  Plus 3 already present in E. coli  Gibson Assembly  40 base pair overhang  PCR with designed primers

Assembling Parts  All original genes ligated into pSB1C3  Transformation produced little to no colonies  Ligation potentially problematic  Must repeat steps to ensure accuracy

Characterization  Began assembling genes into pSB1C3  A promoter  A ribosome binding site before the gene  The original gene  A ribosome binding site before GFP  GFP  A terminator  3A assembly proved to be difficult

Kinetics

How much Eucalyptol would this produce? Fermentation Purification Extraction Reactor Size Operating Product Cost Cost 10,000L $5 $2.41/kg million/yr Current Profit at $5/ Payoff time Market Cost kg $64/kg $5 4 years million/yr

Future Directions  Characterize and submit original BioBrick parts  Finish assembly of final system  Test final system  Optimize/scale up final system

Community Outreach  Collaboration with Wisconsin Lutheran College  Week long summer camp for high school students  Daily labs, discussions, and activities

Thank you for your attention! Acknowledgements  Baxter  Katarina Midelfort, Ph.D  MathWorks  Jung Lee, Ph.D  New England BioLabs  Faisal Shaikh, Ph.D  Wisconsin Lutheran  Gul Afshan, Ph.D College iGEM Team  Julie LaRose  Lakefront Brewery  Victoria Pink  MSOE SuperMilage Team  Lisa Kann Milwaukee School of Engineering

Supplementary Slides

Three E. coli System Two Input E.coli One Output E.coli  Change hemicellulose  Mevalonate Pathway to xylose  5 introduced genes  xynC-A and xyloA  3 enzymes already present  Secretion System  1,8-Cineole Synthase  Tag  Pump

Terpenoid Synthesis in E. coli  All terpenoids created from - same precursors  IPP and DMAPP  1,8-cineole is a monoterpenoid  Engineering: building blocks  Adjust for variety of terpenoids

Output E. coli Design pSB3K3 pSB6A1  1,8 Cineole Synthase  HMG-CoA Synthase  HMG-CoA Reductase  Mevalonate Kinase  Mevalonate  Phosphomevalonate Pyrophosphate Kinase Decarboxylase

Promoter/Terminator  T7 Promoter  Strong  High expression when T7 polymerase present  Double Terminator  Most commonly used  Reliable

Primers  Used to add the BioBrick prefix/suffix  Adding restriction sites using PCR  Melting points between 52-58˚C  Ensured a GC Clamp held the primers in place 2008.igem.org

Safety Information  Project completed at BSL1  DNA synthesized from organisms from BSL2  Completed training course in Biosafety  Obtained project approval from local Biosafety group

Enzyme Kinetics  Input Assumptions  Output Assumptions  Maximum amount of  Maximum metabolic enzyme pumped into utilization of xylose solution  Eucalyptol was  Steady state properly excreted  Very low inhibitor  Growth is not inhibited concentration by eucalyptol