PRESENTATION BIO WORLD CONGRESS – 19 JUNE 2013
COMPANY SNAPSHOT
DEINOVE is a cleantech company that designs, develops and markets a new
generation of industrial processes based on ancestral bacteria with untapped
potential: the deinococci.
These new systems open the way for manufacturing of rare or technologically inaccessible
products:
• Second-generation biofuels (DEINOL) • High added value biochemicals (DEINOCHEM) • New antibiotics (DEINOBIOTICS) • Enzymes for the remediation of plastics (THANAPLAST™) DEINOVE intends to provide new industrial technology standards that enable it to meet the great
world challenges: preparing for the post-oil era, switching to green chemistry, fighting against
antibiotic resistance and improving the life cycle of plastics.
DEINOVE, THE DEINOCOCCUS WAY
2
DEINOVE
KEY DATES
3
2009 2007 2008
Inception, co-founders Pr Miroslav RADMAN Dr Philippe POULETTY
2013 2006 2010
DEINOL
EC1
2011 2012
OSEO funding (€ 1.57 m)
Jacques BITON CEO
IPO (€ 12 m) Partnership with TEREOS Partnership with VTT OSEO funding (€ 1.4 m)
OSEO funding (€6 m)
Deinobank (6 000 strains) Initiation of DEINOL program
Deinobank (25 strains)
Proprietary technological platform Initiation of DEINOBIOTICS program
Initiation of DEINOCHEM program
DEINOL EC2 (POC) THANAPLAST™ program
DEINOBIOTICS SAS
OSEO funding (€ 1.15 m) Market Cap: € 53 m
Emmanuel PETIOT CEO
2 5 31 Staff
DEINOCHEM
DEINOVE
STRATEGY FROM 2012 to 2013
4
DEINOL…and beyond
Second generation biofuels
Plastics Fine chemicals
Sweeteners Fragrances Flavors
Detergents =
Other programs
DEINOBIOTICS THANAPLAST™
Antibiotics
Plastic remediation
DEINOCOCCUS: A NEW CHASSIS FOR FERMENTATION
5
D.
geothermalis
S.
cerevisiae E. coli
C.
glutamicum
C5 and C6 assimilation
C5 and C6 co-assimilation
amylolytic
cellulolytic
Cello (bi-tri-tetra) ose
Xylanolytic
Resistance to dessication
Resistance to xenobiotics
Rs glucose
Biomass in a mineral
media
Kla
Thermophile
Genetic engineering tools
6
DEINOCOCCUS: A NEW CHASSIS FOR 2nd GENERATION BIOFUELS
A UNIQUE SUM OF BENEFITS
THE GENUS DEINOCOCCUS
A NEW SCIENTIFIC AND TECHNOLOGICAL WAY
7
• The largest collection of Deinococci in the world (6 000 strains)
• A proprietary technological platform (16 patents) with validation through a POC (DEINOL)
Num
ber
of public
ations
200 000
100 000
300 000
Microorganisms
NUMBER OF PUBLICATIONS PER GENUS
Num
ber
of pate
nts
4 000
0
8 000
Microorganisms
NUMBER OF PATENTS PER GENUS
Escherichia coli
Saccharomyces cerevisiae
Deinococcus
Deinococcus geothermalis
The world pioneer of deinotechnologies
OUR PROPRIETARY TECHNOLOGICAL PLATFORM
THE BEST OF GENETIC ENGINEERING AND BACTERIOLOGY
8
ARCHIVING
Library of cryopreserved
strains
Physiology
High-throughput growth assays
Metabolomic
Functional tests Gene expression
measurement
Microbiology
Genomic
PCR Sequencing & Annotation
DEINOBANK (6 000 clones)
Genetic & Metabolic
Engineering
Gene insertion Gene deletion
Sequence optimization
LAB PILOT
INDUSTRIAL PILOT
PRODUCTION
COLLECTION SELECTION OPTIMISATION SCALE UP
LEAD DEINOCOCCUS CANDIDAT
S1 S2 S3 POC
Fermentation engineering
Optimization of culture &
fermentation processes
Industrial engineering
Lab Assays Test &
Integration
Samples collection in
extreme environment
THE DEINOVE TEAM
A YOUNG INNOVATIVE COMPANY
9
A BACTERIAL LIBRARY OF 6 000 STRAINS
Diverse environments have been targeted…
…unusual selection by irradiation
…unusual carbon sources
Deinococcus library (1800 strains) AND diverse library of original strains (4200)
All strains collected according to the international laws (CBD)
Source of original new biocatalysts
MANY UNSUAL STRAINS DUE TO SELECTION BY IRRADIATION
10
INHIBITORS RESISTANCE
11
THE DEINOCOCCUS ADVANTAGES
Metabolization test (MTT)
IC50 (g/L) 5-HMF Furfural Aldehyde from lignin
E. coli 26 3 5
S. cerevisiae 18 1 3
D. radiodurans R1 93 20 14
D. geothermalis MX6-1E 104 42 12
D. geothermalis MX6-1C 112 46 9
D. geothermalis MC2-2A 112 3 13
D. geothermalis M36-7D 97 30 ND
THE DEINOCOCCUS ADVANTAGES
A GENETIC MOSAIC WITH UNUSUAL ROBUSTNESS…
12
2 synthetic operons have been inserted and the bacteria have been diluted without selection pressure. Both are stable after 100 generations even without selection pressure
12
1 2 3 1 2 3
ETOH OPERON
1 Recombinant D. geothermalis 2 Recombinant D. geothermalis after serial dilution 3 Wild type D. geothermalis,
BIOMASS OPERON
Among the most resistant genomes of
the world : • Most of the publications on D. radiodurans
(900) focused on radiation resistance
• Multiple copies of the genome (Battista J,
Annu. Rev. Microbiol. 1997)
• Specific DNA repair machinery (Repar J,
DNA Repair 2010)
THE DEINOCOCCUS ADVANTAGES
… NOW ACCESSIBLE TO DIRECTED EVOLUTION
13
-20
0
20
40
60
80
100
120
0 2 5 10 15 20 25 40
OD
57
0 N
M (
% C
ON
TRO
L)
% ÉTHANOL
E. coli
S. Cerevisiae
D. radiodurans R1
Recombinant D. geothermalis
Evolved recombinant D. geothermalis
0 2 5 10 15 20 25 40
% ETHANOL
DEINOVE’S PLATFORM
14
PLATFORM
STRAIN CONSTRUCTION PLATFORM
15
Library of standard
elements: promotors
RBS…
Omic databases
genomic,
transcriptomic,
proteomic…
A large strain library
6000 strains, 1800
Deinococcus
0
1000
2000
3000
4000
5000
6000
S1 2011 S2 2011 S1 2012 S2 2012
Generated strains A robotic platform
Dedicated software Robotic cloning Deinococcus allows fast
strain construction
Rémi Bernard
Molecular Biology Manager
PLATFORM
FERMENTATION METABOLISM ANALYTIC
16
Fermentation & Metabolism
Multi parallel
fermentors:
1L, 2 L, 5L,
20L.
Enzymology,
fluxomic,
metabolomic
Analytical Platform
MicrOTOF-QII Bruker
(intra and extracellular
metabolites)
Dionex ICS (sugars, acids) Gaz
chromatorgraphy
variant
DEINOCOCCUS ENZYMES, BIOMASS DEGRADATION, SUGAR ASSIMILATION
17
BIOMASS HYDROLYZING ENZYMES
50 STRAINS SEQUENCED, PHYLOGENY OF GLYCOSYL HYDROLASES
18
Mx6
-1B
DR
H-4
6
M1-3
H
M13-1
A
MC
3-9
A
MI3
-1C
M11-1
2B
MI3
_10
M23r-
2A
M23-2
E
M31-8
F
MC
5-1
1A
MX
4-5
F
M31-1
0
M36-8
C
M19-G
G
M26-8
A
MC
3-4
A
MC
3-4
B
MC
3-5
G
MC
2-2
C
MC
2-2
A
M36-7
0
MX
6-1
C
MX
6-1
0
MX
6-1
E
DR
H-3
7
DR
H-3
8
DS
M11300
Maltooligosyl trehalose synthase
Maltooligosyl trehalose trealohydrase
1,4 aglucan branching enzyme
4 aglucanotransferase; amiloa 1,6 glucosidase
Amylosucrase
Isoamylase
aglucosidase
aamylase
4agluconotransferase
aglucosidase; atrehalase Glucoamylase
aamylase
Pellulanase; Isoamylase
bgalactosidase
agalactosidase
bglucosidase
GlycopeptideaN acetylgalactosaminidase
Endo 1,4 bxylanase
Xylane 1,4 bxylosidase
Cellulase
bglucosidase; bNacetylhexosaminidase aLrhamnosidase
XYLANOLYTIC
CELLULOLYTIC
AMYLOLYTIC
5.4.99.15
3.2.1.141
2.4.1.18
2.4.1.25 (33)
2.4.1.4
3.2.1.68
3.2.1.20
3.2.1.1
2.4.1.25
3.2.1.20(28)
3.2.1.3
3.2.1.1
3.2.1.41(68)
3.2.1.23
3.2.1.22
3.2.1.21
23.2.1.97
3.2.1.8
3.2.1.37
3.2.1.4
3.2.1.2152)
3.2.1.40
3.2.1
GH13
GH13
GH13
GH13
GH13
GH13
GH13
GH13
GH77
GH13
GH15
GH13
GH13
GH42
GH36
GH1
GH101
GH8
GH52
GH5
GH3
GH78
GH3
BIOMASS HYDROLYZING ENZYMES
DEINOCOCCUS ENDOCELLULASE
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Closest relative: Clostridium
cellulolyticum GH5 b1,4-glucanase
Clostridium cellulolyticum (65% identity)
1 125 250 375 500 625 750 806
QC
Cellulase
Cellulase superfamily
SH
SpF CBM 1728 CBM 1728
Control (no bacteria)
Deinococcus
AZO-CMC (cellulose 0.5%)
BIOMASS HYDROLYZING ENZYMES
GH10 XYLANASE
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Glycoside hydrolase family 10, closest relative Clostridium thermocellum (45% identity)
1 50 100 150 200 250 300 344
QC
Glyco hydro 10
Glyco hydro 10 superfamily
SH
SpF
AZO-Xylan (0.5%)
DEINOCCOCUS IN THE REAL LIFE
ASSIMILATION OF MIXES OF GLUCOSE & XYLOSE
21
0
20
0.2 0
40
60
80
100
120
0.4 0.6 0.8 1 1.2
% OF RS MAX
Ratio glucose/xylose g/g
Rs up to 4.5 g/l/h in a fermentor (≈ E. coli) and still non optimal
0
2
4
6
8
10
12
0 10 20 30 40 50 60 70 80
Fre
e s
ug
ars
(g
/l)
Times (h)
WHEAT BRAN, shake flask
glucose xylose
22
• Xylan is directly hydrolized by the strains and assimilated
0
20
40
60
80
100
120
0 50 100 150 200
%to
tal
Kinetic of xylose assimilation on birch wood xylan
80% of the total xylan is
consumed after 48h of
cultivation
DEINOCCOCUS IN THE REAL LIFE
ASSIMILATION OF MIXES OF BIRCH WOOD XYLAN
DEINOCCOCUS AS A SECOND GENERATION CHASSIS
CELLULOLYSIS & ASSIMILATION OF SMALL CELLULOSE POLYMERS
23
0
2
10 0
4
6
8
10
12
14 16
20 30 40 50 60 70 80
GLUCOSE
Time(h)
g/l
0
2
10 0
4
6
8
10
12
14 16
20 30 40 50 60 70 80
CELLOBIOSE
Time(h)
g/l
0
2
10 0
4
6
8
10
12
14 16
20 30 40 50 60 70 80
CELLOTRIOSE
Time(h)
g/l
0
2
10 0
4
6
8
10
12
14 16
20 30 40 50 60 70 80
CELLOTETRAOSE
Time(h)
g/l
Activity on watman paper
Inoculated
Control
FROM 1.5 TO 2G BIOETHANOL
24
THE DEINOL PROGRAM
TOWARD 2 G BIOETHANOL
25
PROCESSES IN
DEVELOPMENT
DEINOL
NON FOOD BIOMASS
Distillation
2G bioethanol
Coproducts
Pretreatment
Enzymatic hydrolysis 70°C
Fermentation 37°C
NUTRITIONAL QUALITY ++
LIGHT
YIELD ++
ONE STEP AT 45°C VERY ROBUST NO ADDITIVE
Proof of
concept
DEINOL
AN ACCELERATED DEVELOPMENT
26
2011 PROCESS
PREVALIDATION (EC1)
The optimized strain degrade
more than 80% of a wheat-based biomass into simple sugars
2012 PROOF OF CONCEPT
(EC2)
Target: process validation in large fermenter (300L) and further
optimization
2013 PILOT LAB
(EC3)
Without additive, the optimized
strain transforms the same substrate
into bioethanol with an alcohol content of 3%
2013-2014 INDUSTRIAL PILOT
(EC4)
2014 PRODUCTION
Industrial validation in operating plant
Market production
Soft endosperm Hard endosperm
Germ
Aleurone
Pericarp
Tip cap
1.5 & 2G BIOFUELS
THE WAY TO GREEN CHEMICALS
27
DEINOCHEM
DEINOCHEM program • Resources dedicated to the program (2012
launch / 2013 acceleration)
• Promising early work from our laboratories
• Advanced discussions with many world
leading industrials partners
• Research contracts by the end of 2013
The largest family of natural
substances in the world • More than 22,000 isoprene compounds
• Many industrial applications: creams,
perfumes, detergents, anti-cancer agents…
Looking for new production
standards • Extraction from plants and chemical
synthesis suffer from low yields and high
production costs
• Deinove’s synthetic platform is ready and
increased resources are on the way
THE WAY TO ISOPRENOIDS
28
THE DEINOCOCCUS ADVANTAGES
HIGHER RESISTANCE TO ISOPRENOÏDS
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IC50 (G/L) ISOPRENE GERANIOL LINALOOL b-PINENE
E. coli 7 0,6 4 91,7
S. cerevisiae 8 0,8 4 22,5
D. radiodurans R1 21 No inhibition 25 22,5
D. geothermalis MX6-1E 12 7,8 5 ND
D. geothermalis MX6-1C 14 No inhibition 7 ND
D. geothermalis MC2-2A 14 No inhibition 7 ND
D. geothermalis M36-7D 22 0,6 3 91,7
METABOLIZATION TEST (MTT)
CONTACTS
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Emmanuel PETIOT CEO Tel : + 33 1 42 03 27 37 [email protected]
Jean-Paul LEONETTI VP R&D Tel : + 33 1 42 03 27 37 [email protected]
………………………………………………………………………………………….........
………………………………………………………………………………………….........
DEINOVE, a member of IAR
• 210 members
• 40% SMEs
• 135 R&I projects
• Total R&I budget: > 1,2 bn €
www.iar-pole.com
M a r k e t s
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