1. Glycos Biotechnologies
Summer Internship 2014

2. Presentation Outline
Nothing in this presentation is confidential, all has been cleared as public information
• GlycosBio
Purpose of the company
What they produce and why
Company components
• Project – controlling cell growth by modifying theTCA Cycle
Importance of gltA deletion
Methods of gltA deletion
Fermentation testing of deletion

3. Company Mission
Create high value biochemicals through microbial
fermentation using sustainable, renewable, non-food based
feed stocks

4. BIO-SIMTM
Isoprene
GlycosBio is creating an isoprene polymer as a “Drop-in”
chemical for the rubber industry
Glycos Biotechnologies 2007-2014

5. Glycerol and PFAD Feedstock
Crude glycerol from
biodiesel production
Palm fatty acid distillate(PFAD) from refining palm oil
Glycos Biotechnologies 2007-2014

6. GlycosBio uses glycerol and fatty acids to produce isoprene
…through microbial fermentation
Isoprene
Glycos Biotechnologies 2007-2014Genetically modified E.coli

7. Poly-Isoprene
PVC
Biosynthesis
Chemical Synthesis
Feedstock
C2
Intermediate
Monomer Polymer
glycerol
fatty acids
ethane
propane
butane
GlycosBio Microbe
Steam Cracker + Reactor
Ethylene VCM
Acetyl-CoA Isoprene
Traditional Process
GlycosBio Process
Traditional Monomer Production vs. Bio-based Method
Glycos Biotechnologies 2007-2014

8. Steam/Electricity
Nutrients
Carbon4Source
pH4Control
Water
BiomassWater4Recycle
CO2/Off=Gases
Finished4Product
Commercial Production
Glycos Biotechnologies 2007-2014

9. Richard Cilento
Chief Executive
Officer
Walter Burnap
President, Chief
Financial Officer
Kevin Mitchell
VP Finance
Diane Muniz-
Chong Exec.
Assistant
Janel Chitty
Exec. Assistant
Accounting
Donna Muniz
Accountant
Paul
Campbell
Co-Founder,
Chief Science
Officer
Werner
Bussmann
VP Project
Management
David Gaskin
Project
Manager
Shadab
Mohommed
Process Engineer
Alex Reis
Engineering
Intern
Dan Monticello
VP Research &
Development
Matt
Wong
RS
Munira
Momin
RA
Robert
West
RS
Sailandra
Paude
RA
Kristian
Odfalk
Intern
Mai Li
RS
Kimberly
Marroquin
Intern
Stephanie
Doneske
RS
Cindy
Austin
RA
Josh
Munnerlyn
RA
Huajin
Zhou
RS
Allana
Robertson
Intern
Sebastian
Bedrow
RS
Erin Burke
RS
Katherine
Walton
RA
Ivy
Martinez
RA
Ryan Black
Manager of
Process
Economics
Solutions Support
Team
AnalyticalMolecular
Genetics
FermentationEngineeringHuman
Resources
Business
Development

10. Engineering
Business
Development
Research
&
Development
Engineering

11. Business Development
▪ CEO-heads effort but is assisted by theVP Finance; CFO; and Chief Science
Officer.
▪ Together they make up the face of the company
▪ Three main goals are:
1.CreateValue
– Find your market, find you niche within market
2. Raise Funds/ Inv.
– Raises money through venture capitalism; angel investments
– Investment firms; Individual investors
3. Build Partnerships
– Feedstock Suppliers
– Buyers of Final Product

12. Engineering
Research
&
Development
Engineering
Business
Development

13. Research & Development
Strain Development
Fermentation Development
Product Capture & Analysis
▪ DevelopsTechnology
– Makes up 80% of current staff
– Headed by Chief Science Officer
andVice President of Research &
Development

14. Engineering
Research
&
Development
Engineering
Business
Development

15. Engineering
Responsible for:
▪ Scale Up Process
– Run experiments to determine strain constraints i.e. growth rates, flow rates, oxygen
demand, and product per liter of media
– Determine optimum media mixing and feeding process according to calculated demands
(Take feed stock and turn it into a usable form)
▪ Product Capture
– Determine process to turn gas phase isoprene into a liquid (transportable form)
▪ Plant Design
– Build and design production blueprint for collecting isoprene
– Determine optimum equipment sizing according to bacterial and material limitations
– Find businesses equipped to build system components according to specifications. (Process
requires pharmaceutical, fermentation, and chemical industry components)
– Recycle waste water from production process to reduce plant foot print
– Find the most economical way to produce isoprene and maximize returns

16. Attenuated TCA Cycle Project
Problem:We need a method to control cell growth through nutrient
limitation
Hypothesis: Would deleting gltA shut down the movement of carbon into the
TCA cycle, and eliminate cell growth, and if so could carbon source
supplementation restore cell growth?
▪ Molecular
– Why deleted - metabolic diagram
– How deleted- phage/suicide vector
▪ Fermentation
– Purpose-validate deletion
– Verify phenotype

17. Isoprene
Glycolysis
Citrate
Acetyl CoA
oxaloacetate
Lipid Synthesis
PDHc
No O2 Only
Glycerol
Glycerol kinase
Glycerol-3-phosphate dehydrogenase
Triosephosphate isomerase
Glutamine
Glutamate
Proline
Alanine
Mevalonate

18. Beta OxidationTriacylglycerols
Lipase
IsopreneMevalonate

19. Upper/Lower Mevalonate PathwayAcetyl CoA
Acetyl CoA
AcetoAcetyl CoA
3-hydroxyl-3-methylglutaryl-CoA
Acetyl CoA
atoB
HMGS
Mevalonate
2 NADH
2 NAD+
Mevalonate-P
ATP ADP
MEK PMK
Mevalonate-PP
ATP ADP
MPD
Isopentenyl-PP DMAPP
OHLDI
Isoprene
Upper
MEV
Lower
MEV

20. Deletion Methods
Deletion
Method
Time
Range
Chances
of
Success
Special
Construct
Accuracy Scar
Risk of Causing
Contamination
Cause of
Failure
Phage
Method
1-2 weeks
or longer
High No
(donor
strain
required)
Ok
(may cause
other
changes to
chromosome)
Yes Very High Strain gains
resistance to
P1 phage
Suicide
Vector
2 weeks or
longer
Good
(may be
hard to
resolve
backbone)
Yes
(design a
plasmid
that can
not be
replicated)
High No Low Tet resistant
mutation
makesTSS
counter
selection not
effective

21. Fig1
Streips U. N.,Yasbin R. E., Transduction in Gram-Negative Bacteria. 2002. Modern MicrobialGenetics, (2) 561-564.

22. Metcalf W., Jiang W., Daniels L. L., Kim S. K., Haldimann A., and Wanner B. L., Conditionally Replicative and Conjugative
Plasmids Carrying lacZα for Cloning, Mutagenesis, and Allele Replacement in Bacteria. 1995. Academic Press, Inc. 35: 1-13.
Kan

23. 5’ 3’ R6Kyori Kanr Tet RA 5’ gltA 3’
5’ gltA 3’ R6Kyori Kanr Tet RA 5’ 3’
Insert Orientation
5’ Integration
3’ Integration
∆gltA
Doneske S., Suicide Vector Diagram. 2007. Glycos Biotechnologies

24. Proof of Concept-Micro Titer Experiment
0
0.1
0.2
0.3
0.4
0.5
0.6
0 2 4 6 8 10
CellGrowth,OD600 Hours
Casein Amino Acids
MG1655 20g/l Glucose
(Positive Control)
MG1655 No Glucose
(Negative Control)
gltA- 20g/l Glucose;
200mg/l Casein Amino
Acids
gltA- 20g/l Glucose;
400mg/l Casein Amino
Acids
gltA- 20g/l Glucose;
800mg/l Casein Amino
Acids
gltA- 20g/l Glucose;
1600mg/l Casein Amino
Acids
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 2 4 6 8 10
CellGrowth,OD600
Hours
Glutamine
Blank
MG1655 20g/l
Glucose (Positive
Control)
MG1655 No
Glucose (Negative
Control)
gltA- 20g/l
Glucose; 2mM
Glutamine

25. Proof of Concept-Micro Titer Experiment
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 2 4 6 8 10
CellGrowth,OD600
Hours
Citric Acid
Blank
BL21 100mM Citric Acid
MG1655 100mM Citric
Acid
MG1655 20g/l Glucose
(Positive Control)
MG1655 No Glucose
(Negative Control)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 2 4 6 8 10
CellGrowth,OD600
Hours
Glutamate
MG1655 20g/l
Glucose (Positive
Control)
MG1655 No
Glucose (Negative
Control)
gltA- 20g/l
Glucose; 2mM
Glutamate
gltA- 20g/l
Glucose; 4mM
Glutamate
gltA- 20g/l
Glucose; 8mM
Glutamate

26. Isoprene
Glycolysis
Citrate
Acetyl CoA
oxaloacetate
Lipid Synthesis
MalateEnzyme
PCK
PPC
PDHc
No O2 Only
Glycerol
Glycerol kinase
Glycerol-3-phosphate dehydrogenase
Triosephosphate isomerase
Glutamine
Glutamate
Proline
Alanine
Mevalonate

27. 0
5
10
15
20
25
30
0 5 10 15 20 25 30
OD600
Hours
Glutamic Acid
glu 1g/l
glu 2g/l
glu 4g/l
glu 8g/l
0
5
10
15
20
25
30
0 5 10 15 20 25 30
OD600
Hours
CasAA
Negative Control
CasAA 1g/l
CasAA 2g/l
CasAA 4g/l
CasAA 8g/l
gltA Keio Collection-Flask Fermentation

28. gltA Keio Collection-Flask Fermentation
0
5
10
15
20
25
30
0 5 10 15 20 25 30
OD600
Hours
YE
Negative Control
YE 1g/l
YE 2g/l
YE 4g/l
YE 8g/l
YE 16g/l

29. gltA Keio Collection-Flask Fermentation
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14 16 18
MaxOD600
Supplement Conc. (g/l)
Max Cell Growth vs. Supplement Conc.
Glu
YE
CasAA

30. Validating gltA Deletion-Flask Fermentation
0
1
2
3
gly YE glu gly YE glu gly YE glu
gltA- Keio GB130.079 GB130.080
Final OD600

31. Validating gltA Deletion-Flask Fermentation
0
1
2
3
4
5
6
gly YE glu gly YE glu gly YE glu
gltA- Keio GB130.079 GB130.080
# of Doublings

32. gltA Reactor Experiment
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40 45 50
OD600
TIME (H)
CELL GROWTH ACCORDING TO SUBSTRATE
40 g/L YE
40 g/L Casamino acids
5 g/L MSG

33. Flask 1
Flask 2
Flask 1A
Flask 1B
Flask 2A
Flask 2B
1.3L Bench Top Reactor
7L Bench Top Reactor
40ml LB, etc.
40ml LB, etc.
40ml MM34, etc. 40ml MM34, etc.
40ml MM34, etc.40ml
MM34
600mL MM33, etc.
3500mL MM33, etc.
2mL
40mL #2
30mL #3
175mL

34. Xo=0.39g/L
Xf=0.39g/L
So=61.41 g/L
Sf= 31.3 g/L
0
2
4
6
8
10
12
14
16
18
112 114 116 118 120 122 124 126 128 130 132 134
CellConcentration(g/L)
Time (hrs)
Cell Concentration Over Time
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
112 114 116 118 120 122 124 126
µ(hr^-1)
Time (hrs)
Growth Rate Over Time
µmax= 1.44 hr -1
Gas Chromatography
Product Capture= 0.05g/L
Total Time=17.9 hrs
Yx/s= 3.3 (g/L)/(g/L) Wild Type E.coli
K-12 MG1655
Carbon source: Glycerol
µmax=0.040 – 0.003 h -1
Murarka A., Dharmadi Y., Yazdani S. S. and Gonzalez R. 2008. Fermentative Utilization of Glycerol by Escherichia coli and Its
Implication for the Production of Fuels and Chemicals. Appl. Environ. Microbiol. 74(4): 1124-1135.
3.5L Batch Reaction [Glycerol to Isoprene]
Growth Rate Determination via Mid Point Slope Graph Differentiation

40. Thank you