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
meddic.jp
and IPP
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
synthase
HMG-CoA
reductase
Enterococcus
faecalis
Thiolase
Mevalonate
kinase
Streptococcus
pnemoniae
1,8cineole
synthase
Arabidopsis
thaliana
Phosphomevalonate
kinase
Streptococcus
pnemoniae
Mevalonate
pyrophosphate
decarboxylase
FPP
synthase
IPP
isomerase
Streptococcus
pnemoniae
Spent Grain Waste
 
Milwaukee’s beer waste
 
Composed of hemicellulose
  Breaks
 
 
newyorkcorkreport.com
down further
Co-utilization of sugars
important for 2nd
Generation biofuels
Diauxie
Endoxylanase
Gene Pathway
HMG-CoA
synthase
HMG-CoA
reductase
Enterococcus
faecalis
Thiolase
Fibrobacter
succinogenes
Xylosidase
Fusarium
graminearum
Mevalonate
kinase
Streptococcus
pnemoniae
1,8cineole
synthase
Arabidopsis
thaliana
Phosphomevalonate
kinase
Streptococcus
pnemoniae
Mevalonate
pyrophosphate
decarboxylase
FPP
synthase
IPP
isomerase
Streptococcus
pnemoniae
Xylose
Initial growth on low glucose
concentrations
Lag as correct enzymes
synthesized
E. coli are able to
grow on xylose
Eucalyptol
Lower eucalyptol concentrations lead
to higher growth rates
As eucalyptol becomes more
concentrated, growth rate slows
Secreting Enzymes to Digest Grain Waste
 
 
First Plasmid
  Gene
of interest
  Secretion tag
Protein
 
Protein
University of
Washington 2009
iGEM Team
Second Plasmid
  Pump
Final Project Design
Output
Input
Tag
mvaK2
Pump
1
xynC-A
mvaS
mvaK1
Tag
2
xyloA
Pump
3
mvaD
Bba_K849000
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
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
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
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
Milwaukee School of Engineering
Supplementary Slides
Three E. coli System
Two Input E.coli
 
Change hemicellulose
to xylose
  xynC-A
 
One Output E.coli
 
Mevalonate Pathway
  5
introduced genes
  3 enzymes already
present
and xyloA
Secretion System
  Tag
  Pump
 
1,8-Cineole Synthase
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
1,8 Cineole Synthase
  HMG-CoA Reductase
  Mevalonate
Pyrophosphate
Decarboxylase
 
pSB6A1
HMG-CoA Synthase
  Mevalonate Kinase
  Phosphomevalonate
Kinase
 
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
 
 
2008.igem.org
Melting points between
52-58˚C
Ensured a GC Clamp
held the primers in place
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
  Maximum
amount of
enzyme pumped into
solution
 
Output Assumptions
  Maximum
metabolic
utilization of xylose
  Eucalyptol
  Steady
  Very
state
low inhibitor
concentration
was
properly excreted
  Growth
is not inhibited
by eucalyptol