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

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Jun 04, 2023 265 likes •560 views

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

SLIDE 1

FROM BEER TO BIOFUEL

Milwaukee School of Engineering (MSOE)

Selena Dickinson Jenna Harvestine Rebecca Majewski Matthew Reichartz Patrick Van Handel James Wiatr

Grain Waste Powering Eucalyptol Production

SLIDE 2

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

SLIDE 3

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

SLIDE 4

Mevalonate Pathway

 Non-Mevalonate

Pathway in E. coli

 Natural inhibition

 Mevalonate Pathway

 Yields more product  Eukaryotic  Certain parts of

pathway better from different organisms

SLIDE 5

Gene Pathway

Thiolase HMG-CoA synthase HMG-CoA reductase Mevalonate kinase Phospho- mevalonate kinase

Mevalonate pyrophosphate decarboxylase

IPP isomerase FPP synthase 1,8- cineole synthase

Enterococcus faecalis Streptococcus pnemoniae Streptococcus pnemoniae Arabidopsis thaliana Streptococcus pnemoniae

SLIDE 6

Spent Grain Waste

 Milwaukee’s beer waste  Composed of hemicellulose

 Breaks down further

 Co-utilization of sugars

important for 2nd Generation biofuels

 Diauxie

newyorkcorkreport.com

SLIDE 7

Gene Pathway

Thiolase HMG-CoA synthase HMG-CoA reductase Mevalonate kinase Phospho- mevalonate kinase

Mevalonate pyrophosphate decarboxylase

IPP isomerase FPP synthase 1,8- cineole synthase

Enterococcus faecalis Streptococcus pnemoniae Streptococcus pnemoniae Arabidopsis thaliana Streptococcus pnemoniae

Endoxylanase Xylosidase

Fibrobacter succinogenes Fusarium graminearum

SLIDE 8

Xylose

Initial growth on low glucose concentrations Lag as correct enzymes synthesized

E. coli are able to

grow on xylose

SLIDE 9

Eucalyptol

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

SLIDE 10

Secreting Enzymes to Digest Grain Waste

 University of

Washington 2009 iGEM Team

 First Plasmid

 Gene of interest  Secretion tag

 Second Plasmid

 Pump Protein Protein

SLIDE 11

Final Project Design

Pump Pump Tag Tag xynC-A xyloA

Input Output

Bba_K849000 mvaS mvaK1 mvaK2 mvaD TPS-CIN

1 2 3

SLIDE 12

Treatment of Spent Grains

 Obtained spent grains  Treated with NaOH

 Isolated hemicellulose  Gives higher yield of

hemicellulose

 Vacuum filtration

SLIDE 13

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

SLIDE 14

Assembling Parts

 All original genes

ligated into pSB1C3

 Transformation

produced little to no colonies

 Ligation potentially

problematic

 Must repeat steps to

ensure accuracy

SLIDE 15

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

SLIDE 16

Kinetics

SLIDE 17

How much Eucalyptol would this produce?

Reactor Size Operating Cost Product Cost 10,000L $5 million/yr $2.41/kg Current Market Cost Profit at $5/ kg Payoff time $64/kg $5 million/yr 4 years

Fermentation Purification Extraction

SLIDE 18

Future Directions

 Characterize and

submit original BioBrick parts

 Finish assembly of final

system

 Test final system  Optimize/scale up

final system

SLIDE 19

Community Outreach

 Collaboration with

Wisconsin Lutheran College

 Week long summer

camp for high school students

 Daily labs, discussions,

and activities

SLIDE 20

Acknowledgements

 Baxter  MathWorks  New England BioLabs  Wisconsin Lutheran

College iGEM Team

 Lakefront Brewery  MSOE SuperMilage Team  Katarina Midelfort, Ph.D  Jung Lee, Ph.D  Faisal Shaikh, Ph.D  Gul Afshan, Ph.D  Julie LaRose  Victoria Pink  Lisa Kann

Thank you for your attention!

Milwaukee School of Engineering

SLIDE 21

Supplementary Slides

SLIDE 22

Three E. coli System

 Change hemicellulose

to xylose

 xynC-A and xyloA

 Secretion System

 Tag  Pump

 Mevalonate Pathway

 5 introduced genes  3 enzymes already

present

 1,8-Cineole Synthase

One Output E.coli Two Input E.coli

SLIDE 23

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

SLIDE 24

Output E. coli Design

 1,8 Cineole Synthase  HMG-CoA Reductase  Mevalonate

Pyrophosphate Decarboxylase

 HMG-CoA Synthase  Mevalonate Kinase  Phosphomevalonate

Kinase

pSB3K3 pSB6A1

SLIDE 25

Promoter/Terminator

 T7 Promoter

 Strong  High expression when

T7 polymerase present

 Double Terminator

 Most commonly used  Reliable

SLIDE 26

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

SLIDE 27

Safety Information

 Project completed at

BSL1

 DNA synthesized from

rganisms from BSL2

 Completed training

course in Biosafety

 Obtained project

approval from local Biosafety group

SLIDE 28

Enzyme Kinetics

 Input Assumptions

 Maximum amount of

enzyme pumped into solution

 Steady state  Very low inhibitor

concentration

 Output Assumptions

 Maximum metabolic

utilization of xylose

 Eucalyptol was

properly excreted

 Growth is not inhibited

by eucalyptol