Glycos Biotechnologies

Summer Internship 2014

Presentation Outline

Nothing in this presentation is confidential, all has been cleared as public information

• GlycosBioPurpose of the companyWhat they produce and whyCompany components

• Project – controlling cell growth by modifying the TCA Cycle

Importance of gltA deletionMethods of gltA deletionFermentation testing of deletion

Company Mission

Create high value biochemicals through microbial fermentation using sustainable, renewable, non-

food based feed stocks

BIO-SIMTM

Isoprene

GlycosBio is creating an isoprene polymer as a “Drop-in” chemical for the rubber industry

Glycos Biotechnologies 2007-2014

Glycerol and PFAD Feedstock

Crude glycerol from biodiesel production

Palm fatty acid distillate(PFAD) from refining palm oil

Glycos Biotechnologies 2007-2014

GlycosBio uses glycerol and fatty acids to produce isoprene

…through microbial fermentation

Isoprene

Glycos Biotechnologies 2007-2014

Genetically modified E.coli

Poly-Isoprene

PVCTraditional Process

GlycosBio Process

Traditional Monomer Production vs. Bio-based Method

Glycos Biotechnologies 2007-2014

Commercial Production

Glycos Biotechnologies 2007-2014

Richard Cilento Chief

Executive Officer

Walter Burnap

President, Chief

Financial Officer

Kevin Mitchell

VP Finance

Diane Muniz-Chong Exec.

AssistantJanel 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 Marroqui

n Intern

Stephanie

Doneske RS

Cindy Austin

RA

Josh Munnerlyn RA

Huajin Zhou RS

Allana Roberts

on Intern

Sebastian

Bedrow RS

Erin Burke RS

Katherine Walton

RA

Ivy Martinez

RA

Ryan BlackManager

of Process Economics

Solutions Support Team

Analytical

Molecular

Genetics

Fermentation

EngineeringHuman Resourc

es

Business Developmen

t

Engineering

Business Developmen

t

Research &

DevelopmentEngineering

Business Development

▪ CEO-heads effort but is assisted by the VP Finance; CFO; and Chief Science Officer.

▪ Together they make up the face of the company

▪ Three main goals are:

1.Create Value – 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

Engineering

Research &

DevelopmentEngineering

Business Developmen

t

Research & Development

Strain Development

Fermentation Development

Product Capture & Analysis

▪ Develops Technology– Makes up 80% of current

staff – Headed by Chief Science

Officer and Vice President of Research & Development

EngineeringResearch

& DevelopmentEngineering

Business Developmen

t

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

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

Isoprene

Glycolysis

Citrate

Acetyl CoA

oxaloacetate

Lipid Synthesis

ACS

Pox

B

PD

Hc

No O2 Only

GlycerolGlycerol kinaseGlycerol-3-phosphate dehydrogenaseTriosephosphate isomerase

GlutamineGlutamateProlineAlanine

Mevalonate

Beta Oxidation Triacylglycerols

Lipase

IsopreneMevalonate

Upper/Lower Mevalonate Pathway

Acetyl CoA

Acetyl CoA

AcetoAcetyl CoA

3-hydroxyl-3-methylglutaryl-CoA

Acetyl CoA

atoB

HMGS

Mevalonate

2 NADH2 NAD+ Mevalonate

-PATP ADP

MEK PMK Mevalonate-PP

ATP ADP

MPD Isopentenyl-PP

DMAPP

OHLDI

Isoprene

Upper MEV

Lower MEV

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 makes TSS counter selection not effective

Fig1Streips U. N., Yasbin R. E., Transduction in Gram-Negative Bacteria. 2002. Modern Microbial Genetics, (2) 561-564.

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

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

Proof of Concept-Micro Titer Experiment

0 2 4 6 8 100

0.1

0.2

0.3

0.4

0.5

0.6

Casein Amino AcidsMG1655 20g/l Glu-cose (Positive Con-trol)MG1655 No Glucose (Nega-tive Control)gltA- 20g/l Glucose; 200mg/l Casein Amino AcidsgltA- 20g/l Glucose; 400mg/l Casein Amino AcidsgltA- 20g/l Glucose; 800mg/l Casein Amino AcidsgltA- 20g/l Glucose; 1600mg/l Casein Amino Acids

HoursC

ell G

row

th,

OD

60

0

0 2 4 6 8 100

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Glutamine

Blank

MG1655 20g/l Glu-cose (Posi-tive Control)

MG1655 No Glucose (Negative Control)

gltA- 20g/l Glucose; 2mM Glu-tamine

Hours

Cell G

row

th,

OD

60

0

Proof of Concept-Micro Titer Experiment

0 2 4 6 8 100

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Citric Acid

BlankBL21 100mM Citric AcidMG1655 100mM Citric AcidMG1655 20g/l Glucose (Positive Control)MG1655 No Glucose (Negative Control)

Hours

Cell G

row

th,

OD

600

0 2 4 6 8 100

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

GlutamateMG1655 20g/l Glucose (Positive Control)

MG1655 No Glucose (Negative Control)

gltA- 20g/l Glucose; 2mM Glu-tamate

gltA- 20g/l Glucose; 4mM Glu-tamate

Hours

Cell G

row

th, O

D600

Isoprene

Glycolysis

Citrate

Acetyl CoA

oxaloacetate

Lipid Synthesis

Mala

te E

nzy

me

PC

K

PPC

ACS

Pox

B

PD

Hc

No O2 Only

GlycerolGlycerol kinaseGlycerol-3-phosphate dehydrogenaseTriosephosphate isomerase

GlutamineGlutamateProlineAlanine

Mevalonate

0 5 10 15 20 25 300

5

10

15

20

25

30

Glutamic Acid

glu 1g/lglu 2g/lglu 4g/lglu 8g/l

Hours

OD

60

0

0 5 10 15 20 25 300

5

10

15

20

25

30

CasAA

Negative ControlCasAA 1g/lCasAA 2g/lCasAA 4g/lCasAA 8g/l

HoursO

D6

00

gltA Keio Collection-Flask Fermentation

gltA Keio Collection-Flask Fermentation

0 5 10 15 20 25 300

5

10

15

20

25

30YE

Negative ControlYE 1g/lYE 2g/lYE 4g/lYE 8g/lYE 16g/l

Hours

OD

60

0

gltA Keio Collection-Flask Fermentation

0 2 4 6 8 10 12 14 16 180

5

10

15

20

25

30

Max Cell Growth vs. Supplement Conc.

GluYECasAA

Supplement Conc. (g/l)

Max O

D6

00

Validating gltA Deletion-Flask Fermentation

gly YE glu gly YE glu gly YE glugltA- Keio GB130.079 GB130.080

0

1

2

3

Final OD600

Validating gltA Deletion-Flask Fermentation

gly YE glu gly YE glu gly YE glugltA- Keio GB130.079 GB130.080

0

1

2

3

4

5

6

# of Doublings

gltA Reactor Experiment

0 5 10 15 20 25 30 35 40 45 500

5

10

15

20

25

CELL GROWTH ACCORDING TO SUBSTRATE

40 g/L YE

40 g/L Casamino acids

5 g/L MSG

Time (h)

OD

600

Flask 1Flask 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.

2mL40mL #230mL #3

175mL

Xo=0.39g/LXf=0.39g/L

So=61.41 g/LSf= 31.3 g/L

110 115 120 125 130 1350

2

4

6

8

10

12

14

16

18

Cell Concentration Over Time

Time (hrs)

Cell

Conc

entr

ation

(g/

L)

110 115 120 125 130 1350

2

4

6

8

10

12

Growth Rate Over Time

Time (hrs)

µ (h

r^-1

)

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 ItsImplication 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

Thank you