Bioengineering ToolboxNow available to industry clients through Bioengineering Solutions

Ideas Robust CommercialProcesses

Idea(molecule)

PathwayPredictor

Gene Pipeline

PathwayPrototyping

HT Strain Engineering

Fermentation Engineering

SeparationsEngineering Product

Technology Platform

BioinformaticsMetagenomicsDirected evolution

TX-TLQSS

HT CloningGenome editing

13C-fluxome

Metabolome

Proteome

Transcriptome

Genome

Genomatica’s Bioengineering

Toolbox

Sector Independent

Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer)

Precision FermentationPathway PredictorOH

HOBDO

Scalable Kilo Piloting

1

SimPhenyTM

Bioengineering SolutionsKey Offerings

Integrated Strain/Process Dev.

Idea(molecule)

PathwayPredictor

Gene Pipeline

PathwayPrototyping

HT Strain Engineering

Fermentation Engineering

SeparationsEngineering Product

BioinformaticsMetagenomicsDirected evolution

TX-TLQSS

HT CloningGenome editing

13C-fluxome

Metabolome

Proteome

Transcriptome

Genome

Genomatica’s Bioengineering

Toolbox

Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer)

Precision FermentationPathway PredictorOH

HOBDO

Scalable Kilo Piloting

FeedstockEvaluationBioprocess Scale-up/Scale down Quantitative Small Scale

Strain Diagnostics Pathway Enzyme EngineeringEvaluation Studies

develop a predictive and quantitative small scale in vivo assay for strain evaluation

enable reliable process scale-up; improve strain and process robustness for successful technology transfer

enable new business opportunities or improve existing fermentation processes

assess new bioprocess ideas using techno-economic analysis and computational biology tools;

develop enzymes for improved in vivoperformance; such as for activity and specificity on non-natural substrates;

provides a ‘view inside the cell’ to gain deep understanding of metabolism using systems-based modeling and omicsProcess Model

TechnicalMetrics(TRY,OUR,impurities,recovery,wastes,etc.)

CostImpact$/ton

Variable 1

Variable 2

2

Bioengineering ToolboxDemonstrated on Numerous Organisms

EukaryotesProkaryotes

MammaliancellsHumancells

Modeling

Systems-based omics

ProveninyeastandGram+andGram- bacteria

Molecular/Micro Biology

EukaryotesProkaryotes

Mammaliancells

QSS

ProveninyeastandGram+andGram- bacteria

Fermentation

Experienceineukaryotesandprokaryotes;yeast,

Streptomyces

GENO Technology Platform

3

SimPhenyTM – High Performance Strains by DesignModeling and Simulation

4 Deepunderstandingofmetabolicnetworkandimpactofgeneticchanges

4 Providesanunderstandingofbyproductsandthecausesfortheirproduction(Forexample,isATPorredoxlimiting?)

4 Prioritizationofpathways,straindesignsandprocessengineeringstrategies

4 Analysisandinterpretationof“-omics”datainglobalnetworkcontext

4

System Biology Tools Enable High Performance Strains

4 RegularOmicsexperiments4 Networkanalysistools4 Statisticaldataanalysis

Iterativemulti-omicsprovidesvaluableinformationaboutcellularenvironment

Data-drivenhypothesisgeneration/decisions

Genomics

Transcriptomics• RNAseq• qPCR

• Productionandevolvedstrains• SNPs,ISelements,deletions,duplications

Proteomics• iTRAQ (global)• MRM(targeted)

Fluxomics• 13C-labeltracing• Computationaltools

Metabolomics

0 20 40 602

4

6

8

10x 105

time [h]

tota

l NAD

(H)

M1M2G1G2

0 20 40 600

2

4

6x 105

time [h]

NADH

0 20 40 601

2

3

4

5x 105

time [h]

NAD

0 20 40 600

1

2

3

time [h]

NADH

/NAD

0 20 40 600

1

2

3

4x 105

time [h]

tota

l NAD

P(H)

0 20 40 600

1

2

3x 105

time [h]

NADP

H

0 20 40 600

1

2

3

4x 104

time [h]

NADP

0 20 40 600

5

10

15

time [h]

NADP

H/NA

DP

• TriplequadLC-MS• Orbitrap - exactmass

5

Provides Key Insights into Metabolism

Systems Biology Example: Eliminating GBL

Two approaches to eliminate GBL by-product:1. Delete genes responsible for GBL

formation, deduced from transcriptomics

2. Identify and introduce new hydrolase

GBL

O Ohydrolase • GBL formed by cyclization of 4HB-CoA (C-yield loss)• Boiling point very close to BDO- expensive separation• GBL formation enzyme induced and spontaneous

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

879 1889 1889+hydrolase

mMBDO mMGBL

BDO

GBLNoGBL

KO hydrolase

4-HB 4-HB-CoA 4-HBal BDO

AldCat2 AdhHO

O

OHHO

O

SCoA HOO

HHO

OHorHOO

OHHO

O

SCoA HOO

HHO

OHor HOO

OHHO

O

SCoA HOO

HHO

OHor

Biological Solution to an Engineering Challenge

8

Ø 321kbregionspansdiversefunctionsincludingessentialgenesandproteasesØ RegionbordershavemobilegeneticelementsØ StrainstabilizedbyremovalofflankingISelementsØ Performancerescued,nogenomicinstabilityobservedinsubsequentlots

Example: Improving Robustness of Commercial Strain

Problem:Ø Anewlotof‘startercultures’forproductionstrainslatedforscale-upperformedpoorly

Approach:Ø WGS(MiSeq)revealedalargeduplicationeventinnewlotrelativetopriorlots

Genetic Instability Identified and Resolved in 5 Weeks

7

Genomatica Enzyme Engineering

Enzyme Structure

HTP in vitro

Screening

In vivo Screening

&Validation

• Quantitative small-scale assay

• Fermentation• Systems/Omics

• Predictive HT assays• 96/384-well• ~ 30,000 clones/day

• DNA synthesis to construct only desired mutations

• Outsource structure determination

• Rosetta enzyme modeling

• Substrate docking

Smart Library Design

Optimizing in vivo Performance of Pathway Enzymes

10

Demonstrated applications: Improve substrate specificity Switch cofactor specificityIncrease specific rate Improve enzyme stabilityLower product inhibition

Example: Improving Downstream Pathway Enzymes

4-HB 4-HB-CoA 4-HBal BDO

AldCat2 AdhHO

O

OHHO

O

SCoA HOO

HHO

OHorHOO

OHHO

O

SCoA HOO

HHO

OHor HOO

OHHO

O

SCoA HOO

HHO

OHor

• ImproveCat2BDOtolerance• Discovery/evolutionapplied• →20Xactivityin1MBDO

• ImproveAld stability• Discovery/evolutionapplied• →10Xactivity,nodegradation

Variant223848

Ald223848FermTime(h):

Ald

85.00

90.00

95.00

100.00

105.00

110.00

115.00

120.00

Parent Cat2* ALD* Cat2*+ALD*

BDO(g/L)

Titer@48hr

EvolvedCat2

EvolvedAld

EvolvedCat2+Ald

Parentenzyme

BDOTiterinFermentations

EvolvedCat2andAld:4Individually– noimprovement4Combined– 15%increaseintiter

Multiplestrainchangesoftenrequiredforimprovedperformance

Improvements in Activity, Tolerance and Stability Accomplished

11

strainscreening&evaluationtopredictlarge-scaleperformance

quantitativemetabolicprofiling,scale-downdiagnostics

systems-levelmetabolicphenotyping(omics)

QSS: Quantitative Small Scale Technology

smartdesign

information

cleanexperiment

Ü V Vgenomaticatechnology

commercialtools

customtools&modeling

Smart Design to Maximize Information at Optimal Throughput and Low Cost

12

0.70.80.91

1.11.21.31.4

BR QSS

StrainAStrainBStrainCStrainD

0.7

0.8

0.9

1

1.1

1.2

BR QSS

StrainAStrainBStrainCStrainD

2 2.5 3 3.5 4 4.5 5 5.50

10

20

30

time(h)

H16P

/H6P

2 2.5 3 3.5 4 4.5 5 5.50

10

20

30

time(h)

Cit/akg

QSS: Proprietary In-house Methods

Predictive

Rankable

SystemsBiologyReady

Relative Product Yield 12

34

12 34

12

34

12 34

Relative Byproduct Yield

01234567

Sp. Product Rate

0

2

4

6

8

10

Sp. Substrate Rate

0.00

0.10

0.20

0.30

0.40

0.50

Product Yield

Metabolomics

QSS

BR

QSS BR

0

1

2

3

4

5

0

30

60

90

120

150

ug/m

g to

tal

Proteomics

Correlation Between Bioreactor and QSS Scale

11

Scalable Bioprocessing: Robust Commercial Process DeliverySmaller, Cheaper, Better, Faster

• Full capability process lab, 6000 ft2

– 36 bioreactors, 2 L, 5 L, 30 L

– Wide range of unit ops for separations and purification

– Integrated LIMS (microbe-to-product)

• Quant small-scale fermentation– 98% carbon closure, 2% CVs

– Mimics large-scale environment

– 200+ process variables monitored

• 30 L piloting suite– Kilogram samples

– 80% of the data in 20% of the time/cost

– Full technology transfer services

Flexible, high-precision fermentation

Kilo-scale integrated piloting

Fermentation

HTST

S/LSeparations

Centrifugation30Lscale

Recovery/Purification

Microfiltration

Nanofiltration

Chromatography

SolventExtraction

Distillation

12

Example: Highly Predictable Scale-up/down

Robust performance at commercial-scale

• Fermentation performance across lab, pilot, and demo scales is highly consistent, enabling rapid lab-to-commercial scale development path • Average commercial-scale performance over ~50 campaign fermentations equivalent to demonstration-scale performance

for same strain (+/-2%)• Low variability in performance across ~50 campaign fermentations, indicates process robustness and predictability

Consistent scale-up to commercial

13,000L

Demonstration Scale

Commercial Scale

~ 50x scale-up

Campaign StrainTiter 98%Rate 104%Yield 100%

Average fermentation performance

( ~50 runs at commercial scale vs. average demo

scale)

0%

20%

40%

60%

80%

100%

120%

0 10 20 30 40 50

Batch No.

Percent average commercial-scale IRR'

0% 20% 40% 60% 80%

100% 120%

0 10 20 30 40 50

Batch No.

Percent average commercial-scale yield

Top 5 Fermentation RunsFermentation Run

Consistent performance across development scales

0%

30%

60%

90%

120%

2L 30L 13kL

BD

O, g

/L

(as

% o

f 2L)

0%

40%

80%

120%

160%

2L 30L 13kL

Rat

e, g

/L.h

r (a

s %

of 2

L)

0%

30%

60%

90%

120%

2L 30L 13kL

Yiel

d, g

/g(a

s %

of 2

L)

13

2013 Kirkpatrick Chemical Engineering Achievement Award

Integrated Solutions Example: Correlation Between O2 Supply and Yield

Ü Diagnostics,omics,strainengineeringrequiredtoincreaseBOTHrateandyieldunderlowO2 conditions

4LoweringO2 decreasesexcessCO2andincreasesyield- good

4LoweringO2 decreasesgrowth,rate,andBDOtiter- bad

BDOtiter(g/L)

BDOprodu

ctivity

(g/L/hr)NormalO2

LowerO2

Biom

ass(g)

BDOYield

%ofG

lctoExcessC

O2

CommercializationYieldTarget

BDO↓Growth↓

Rate↓

CO2 ↓ Yield↑

14

Integrated Approach to Improving Fermentation Performance

Reducing Flux Through CO2 Forming PathwaysGlucose

NADH NADH ATP NAD(P)H NADPH 1,4-BDO

PPpathway

CO2

acetyl-CoA

OAA

aKG

SSA 4HB

CO2

CO2

CO2

CO2

PEP

G6P

isocitrate

succinate

malate

zwf

sucCD

aceBAK

1. pentosephosphatepathway2. completeoxidativeTCAcycle3. glyoxylate shunt

DeletedthreeCO2 formingpathways:

• BDOdecreased• Growthratedecreased• ExcessCO2%increased!!

1. 13Cisotopetracingrevealeda“rogue”flux,convertingsuccinyl-CoAtosuccinate&formingexcessCO2.

2. ExcessCO2 pathwaysmayberequiredtosupplycofactors(ATP,NADPH,NADH)forgrowth&maintenance.

RogueFlux- complementsDsucCD andcompletesTCAcycle

Ü Thecellistellingusitneedsmoreofthesecofactors

Systems Biology Based Diagnostics Results in Key Discovery

Identifying and Reducing the “Rogue Flux (RF)”

4Potentialsuccinyl-CoAhydrolasecandidatesprioritizedfordeletionusingbioinformatics &omics dataOAA

aKG

CO2

CO2

isocitrate

malate

DsucCD

Rogueflux

fumarate

citrate

→InsufficientATP?NAD(P)H?=>Bothneeded

LowBDO(10g/L) Slowgrowth

TCA-impairedstrainsperformpoorly

4TCA-impairedstrains– poorBDOproductionandgrowth

X BDO↑

cyd+

Growth↑

Dcyd,pntAB OE

16

Critical solution identified based on systems-based understanding of metabolism

140 g/L

2012/13 (650 m3)Commercial scale

2011Shipping tons at a time

2011 (13 m3)Integrated demo plant

2010 (3 m3)Piloting

2009 (30 L) Purified Bio-BDO

2008 (2 L)Bio-BDO producing organisms

GENO BDOTM Process

5 mg/L

50 g/L

80 g/L

Genomatica’s Capabilities: Geno BDO™ CommercializationProven in Practice: 5 years from Concept to Commercialization

20 g/L

100 g/L

17