Saccharomyces cerevisiae :
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Evoutions in Bio Sciences
•
•
•
•
•
•
Ecology
Quantitative ecology
Physiology,
Quantitative biology
Systemic Biology
Holistic Biology
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Yeast asYeast
cell factory
Semi Anaerobiosis
Anaerobiosis
Aerobiosis
Baker yeast
Yeast extract
Flavouring agents
Metabolites, ex food
additives
Recombinant
yeast

enzyme
pharmacentical
Waste water treatment
Yeast as co productanimal feed
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Ethanol (ETBE)
Ethanol solvant
chemistry
alcoolic beverages
MICROBIAL/BIO REACTOR
ENGINEERING:A BASIC TOOL FOR
KNOWLEDGE IN HOLISTIC BIOLOGY
G.Goma,S Guillouet,C Jouve,J L Uribellarea
Laboratoire d ingenierie des systémes biologique
et des procédés
UMR CNRS,INRA,INSA
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Green
biotechs
White
biotechs
Red biotechs
Agro-food
biotechs
Intersections
on technology
and common
fields
Generic technology
 Synthetics, pathways
 Biocatalysis engineering
 Bioprocessing
KBDiSB/Aix 09-07
Basic knowledges
 Focused on
 life sciences …
 engineerig sciences
 biomathematics
 physics
GG LISBP INSA Toulouse
 Economy, sociology, ...
Microbial Engineering:a part of
biotechnologies
Find and improve the microorganisms for bio
processing
Find the conditions of bio processing where the
microrganism is economicaly performant
A multidisciplinary approach
A contraint : find the bottlenecks,eliminate them
An obligation:need of handling a complete tool
box:from genes to bioproducts and bioprocess
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
What kind of technological
strategy?
• Low tech ?
• High tech ?
• Right tech for the goal
• What are the criteria of production ?
•
–
–
–
–
–
–
Production of « active agents »
Cost ?
Invisible technology
Relatively safe technology
Reproducible protocols simplest as possible
Semi
speciality
« de
novo » technology?
use of existing tools of production?
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
The IB Value Chain
Bulk
Biofuels
H2
Ethanol
Agricultural
(by)products
Biomaterials
Sugars
Physical treatment
and/or enzymes
(Micro-)organisms
biocatalysis
Polylactic acid
1,3 propane diol
PHAs
Biochemicals
Food Ingredients
Pharmaceuticals
Fine Chemicals
Fine
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
The steps
• Factory and his environment
• The reactors ,biorector:biocatalist,srategy
• Raw materials and biocatalist,bioreaction
engineering
• The biocatalist
• Global implementation ;find the differents
bottleneks and solve the problems
• Need a tool box,and combining experimentals
datas(strategy?) and simulations
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Take down,
Feeds,
Substrat(s), air,
regulations and
controls
Si X

products:j
X
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
culture medium,
gaz out,
biomass,
products,,,,,,,,,,
Industrial (White) Biotechnology
Biofuels
Biomaterials
Biochemicals
Sugars
KBDiSB/Aix 09-07
Cell factories
GG LISBP INSA Toulouse
Gaz in
Gaz out:analyse
Tank,mixing,
température control
Measures
Régulations
control
Correction
pH, 09-07
KBDiSB/Aix
Antifoam
GG LISBP INSA Toulouse
Dual use of fermentors
Instrumentation of a
fermentor
Use of fermentors
 RPM
 Starters  milk, silage, …
 Qair
 Baker yeast  bread
 Pressure
 Alcoholic beverages
 CO2, O2 ?
 Lactic acid/organic acids
(citric)
 Gas balance
 Antibiotics
 OD?
 Vaccines
 Ph (controlled)
 Monoclonal antibodies
 Temperature
 Recombinant proteins (or toxin
?)
For this 2 controlled parameters,
the analysis of the « work » of the
control regulator gives
informations
KBDiSB/Aix 09-07
 Waste water treatment
 Bioleaching
GG LISBP INSA Toulouse
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Mixing
Réalisation
FLUIDIC Mixing
Jets
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
AERATION : TECHNOLOGIES d’AERATION
FERMENTEUR type air-lift
ICI, Ltd. factory, Billingham, UK,
(Chem. Eng. News, 18-Sep-78)
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Metabolic descriptor
•
•
•
•
Mass conservation
Elemental biologicals reactions
Macroscopic kinetics
Matrix of reactions combining kinetics and
stoechiometry of elemental reaction of metabolic
pathways
• Combining kinetics observed by on line
measurements by robusts sensors evaluation of
metabolics fluxes « on line » and nutritionals
needs
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
• Identification of some bottlenecks
implementation of microbial
processes
 Phenomenogical models
 Behavioural models
 Structured models and stoechiometric/metabolic descriptors
Experimental strategies
+
2
NADPH,H
CO2
Glucose
ATP
Glucose6-P
Pentose P
Sedoheptulose7 P
Qe
Qresp
%pO2
pH
Temps
Fructose-PNADH,H
ATP
+
ATP
GlycerolP glycerol
TrioseP
O2
CO2
S
P
X
O2
CO2
Erythrose4P
+
NADH,H
ATP
Glycerate3P
NAD
FADH
2
PEP
FAD
ATP
ATP
Pyruvate
HS-CoA
+
NADH,H
CO2
+ ATP CO2
NADPH,H
CO2
CO2
Acetyl CoA
OAA
Qs
FadH
2 Succinate
GG LISBP INSA Toulouse
1/2 O2
3 H+
Acétate
ANABOLISME
Malate
+
NADH,H
Fumarate
1/2 O2
H 02 + 2 +H
HS-CoA ATP
+ ATP
NADPH,H
Citrate
IsoCitrate CO NADH,H
+
2
SH-CoA
aKglu
Suc-CoA
GTP
CO2
SH-CoA
+
NADH,H
KBDiSB/Aix 09-07
+ H 0 + 4 +H
NADH,H
2
+
NADPH,H
CO2
Plate-forme métabolomique, fluxomique
Interface de la cellule et échanges
Vers une biologie des systèmes par la réconciliation
des niveaux métaboliques, génétiques et moléculaires
3
Système
d’échanges
Système
métabolique
2
Système
protéique
Système:
d’adaptation et de
défense
Système
génomique
1
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Prerequisite to “Systemic Biology”
Data base
Analytical methods
in situ
continue
on line
in parallel
micro samples
Sequences Profiles
genes
Kinetics
Flux, Stocks
KBDiSB/Aix
09-07
Technology
proteins
(x2 every 18 months)
Metabolome
Definition
of
functions ,
networks
Metabolic pathways
coupled kinetics
relaxation
time,regulations,
GG LISBP INSA
Toulouse
« OMICS »
Top Down strategy
•
•
•
•
•
•
•
Fit the macroscopic environnment,bioreactor
Find reproducible conditions:signature recognition
Biokinetics
Quantitative physiologie
Metabolic pathways
Proteomic
transcriptomics
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
 Analysis of first fermentation
 Comparison with another fermentation with
better performances
> sequencial feeding glucose
> Titer 50 h = 147 g/L,
viability = 30%
> Viability = 80% at 120g/L
- How osmotic conditions affect response to ethanol?
- Genes and mechanisms involved ?
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Motivation
The physiological state recognition
The cell population expresses stable characteristics within
every physiological state, thus an invariant control strategy
can be effectively applied in each state.
 Normally, we have sensors only for the
environmental variables.
 Physiological states are tracked through
offline measurements and analysis, with an
implied delay.
 The physiological state can be identified by
the fusion of environmental measurements.
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
2
Identification and Classification
of Physiological States
• A bottlenek for « the omics »studies,for
control strategies and « quality »
• Morphometry
• Kinetics and stoechiometrics « parameters »
• Differentiation of biologicals and
environmental effects
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Yeast :Axenic culture gives a population
production
linked
to
some
mechanims
The family growth by
budding
Cycle
G1, G2, G3,G4,…
S1 sugar
S2 oxygene
Myyes/no
job is to
produce
S3
ethanol
cell
biomass
I work
My job is
bioconversi
on
I am injuried
KBDiSB/Aix 09-07
I have a
limitation
I work
GG LISBP INSA Toulouse
I am stressed –
I became a
filament
I am ill
I do
nothing
Finish : End ; cryptic
The Tool box
Bio: the “omics”
+
Traditional technologies
+
mathematical tools
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
“the rule of innovation”
Biocatalysis strategy
Diversity
Natural
Natural of
Diversity
Eco-systems
Eco-systems
Genes*
Genes*
et functions
and
functions
screening
screening
Bioprocess strategy
Raw
premières
materials
 Screening
 Screening

 Engineering
metabolic*

 Building strains

 DNA shuffling
 Global analysis “Omics
and engineering”
Production-formulation
Production-formulation
Bioprocédés »
«« Bioprocesses
»
Strategy on co-products
/bio-products
Needs in size of
market :
animal feed
KBDiSB/Aix 09-07
Co-products
Biomaterial
onplus
energy
value
Increase the value
Biomolecules
Biomolécules
High added value
* e.biotechnology's and engineering
GG LISBP INSA Toulouse
Microbial engineering is multidisciplinary : need
of quantitative and “system” biology
Molecular
physiological engineering
Physiological
engineering
Microbial
processing
Microbial
engineering
KBDiSB/Aix 09-07
Microbial
process analysis
and control
engineering
GG LISBP INSA Toulouse
+ system biology modelling
Dual use of fermentors
What is a fermentor ?
Elemental biokinetics
x Biomass
p Product
s Substrats
t Time
x
p
s
t
x
p
s1
t
ou
x
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
s1
p
s2
t
6000
350
300
(g)
400
5000
200
3000
150
2000
100
1000
50
0
0
0
5
10
15
20
25
Time
KBDiSB/Aix 09-07
30
(h)
GG LISBP INSA Toulouse
35
40
45
50
Biomass
250
4000
Viable Biomass
(g/L)
Ethanol
7000
(g)
(g)
Glucose
Cell and glucose ethanol
concentration vs time (Fed batch
with nutritional strategy)
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Study of a reference fermentation
20
Biomass
18
(g/L)
16
Ethanol 200
Glucose 180
(g/L)
I II
III
IV
VI
1,2
V
Viability
1,0
160
14
140
12
120
10
100
8
80
6
60
4
40
2
20
0,8
0,6
0,4
0,2
0
0,00
0,0
10,00
20,00
Ethanol
30,00
40,00
glucose
50,00
Biomass
 2 phenomena:
- Decoupling
growth-production
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
- Loss of viability
60,00
70,00
viability
80,00
Studying the fast biological
responses ...
Measurement
& rates
/ 20 sec
Gas balances
(Mass spectr.)
out
qO2 , qCO2 , Qresp
Monitoring
Qair
rpm
in
Qair
Controlled
environment
T°
control
pH
pO2
µ
Biomass sensor
Fast sampling :
Q, qH+
Measurement of extracellular
metabolites- direct filtration
through adaptated membrane
Measurement of intracellular
metabolites
Sample quenching in -60°C methanol
Sampling for extraction of RNAs
and proteins
Xestim
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
The hyper yeast
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Data Acquisition : Measures
temp. (°C) RPM pH Pres ion (mb)
Fermentation Parameters
5
2
0
P
a
r
a
m
è
t
r
e
s
d
e
f
e
r
m
e
n
t
a
t
i
o
n
5
1
5
2
D
G
r
a
p
h
3
5
1
0
7
.
0
0
6
.
9
2
4
6
8
1
0
1
2
1
0
1
2
6
.
8
2
D
G
r
a
p
h
2
6
.
7
6
.
6
1
8
0
0
0
1
5
0
0
1
2
0
0
2
4
6
8
p
a
r
a
m
è
t
r
e
s
d
e
f
e
r
m
e
n
t
a
t
i
o
n
9
0
0
6
0
0
3
7
.
4
0
2
4
6
8
1
0
1
2
8
1
0
1
2
3
7
.
2
3
7
.
0
Time(h)
t
e
m
p
s
(
h
)
3
6
.
8
KBDiSB/Aix
09-07
0
2
GG4
LISBP INSA Toulouse
6
Interface de la cellule et échanges
Le biotope du
système microbien
crée un
environnement;
« en soi ,
un système »
Système
d’échange
s
Système
métaboliqu
e
système
protéique
Système
génomique
Système:
d’adaptation
et de défense
Le
microorganisme
est un système
biocatalytique
évoluant dans
KBDiSB/Aix 09-07
un système
« interactions de systèmes et hiérarchies »
Biocatalyse
enzymatique
Biocatalyse
microbienne
GG LISBP INSA Toulouse
« impact
socio-économique »
Cell
RNA control
Mass action law
Modification of enzymatic
pools
Allosteric controls
10-6 10-5 10-4 10-3 10-2 10-1 100 10+1 10+2 10+3 10+4 10+5 10+6
s-
Environment
Gradients due to
mixing
Continuous culture
Batch, Fed-batch
Phenomenological
model
Metabolic model
Behavioural models
KBDiSB/Aix 09-07
Virtual cell
GG LISBP INSA Toulouse
Perspective :
Use of behavioural modelling
Extracellular
components
Segregation (size,
viability, …)
Intracellular components
Descriptor of
physiological
state A
*
Descriptor of
physiological
state B
Analysis of population or « dynamic systems »
KBDiSB/Aix
* Relaxation
time
09-07
GG LISBP INSA Toulouse
What kind of micro
organisms
 Bacteria
 Yeast
 Fungi
What kind of profile
Equations bilan
r .Vliq  Qent O
liq
O2

d (O2gaz .Vgaz )
d (O2dis .Vliq )

dt
dt
gaz
d (CO2 .Vgaz ) d (CO2dis  carbo .Vliq )


dt
dt
1.3
1.2
Coefficient
respiratoire
1.1
1
0.9
0.8
In every case
But, every case have rules of
utilisation with typical profile
 Qsot O
sort
2
liq
rCO
.Vliq  Qent .CO2ent  Qsot .CO2sort
2
 Eucaryotic cells
The basic law of biokinetics and
stoechiometry are the same
ent
2
0.7
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1.4
rO2
rCO2
moles/h
1.2
1
0.8
0.6
0.4
0.2
0
0
0.5
1
1.5
2
2.5
3
3.5
Temps (h)
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
4
4.5
5
CATEGORISATION des SIGNAUX
Item : temps
CLASSES
Item : temps
Temps
Identification de classes de comportement
GG LISBP INSA Toulouse
Mesures pertinentes
/ Comportements physiologiques
KBDiSB/Aix 09-07
implementation of microbial
processes
 Phenomenogical models
 Behavioural models
 Structured models and stoechiometric/metabolic descriptors
Experimental strategies
+
2
NADPH,H
CO2
Glucose
ATP
Glucose6-P
Pentose P
Sedoheptulose7 P
Qe
Qresp
%pO2
pH
Temps
Fructose-PNADH,H
ATP
+
ATP
GlycerolP glycerol
TrioseP
O2
CO2
S
P
X
O2
CO2
Erythrose4P
+
NADH,H
ATP
Glycerate3P
NAD
FADH
2
PEP
FAD
ATP
ATP
Pyruvate
HS-CoA
+
NADH,H
CO2
+ ATP CO2
NADPH,H
CO2
CO2
Acetyl CoA
OAA
Qs
FadH
2 Succinate
GG LISBP INSA Toulouse
1/2 O2
3 H+
Acétate
ANABOLISME
Malate
+
NADH,H
Fumarate
1/2 O2
H 02 + 2 +H
HS-CoA ATP
+ ATP
NADPH,H
Citrate
IsoCitrate CO NADH,H
+
2
SH-CoA
aKglu
Suc-CoA
GTP
CO2
SH-CoA
+
NADH,H
KBDiSB/Aix 09-07
+ H 0 + 4 +H
NADH,H
2
+
NADPH,H
CO2
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Study of a fermentation of
First results:genes over expressed
reference
% des genes significatif dans la famille considerée
16,0
14,0
12,0
III ="60g/L ethanol"
10,0
IV="80g/L ethanol"
8,0
V="90g/L ethanol"
VI="100g/L ethanol"
6,0
X="120g/L ethanol"
4,0
2,0
TRANSPORT
FACILITATION
GG LISBP INSA Toulouse
TRANSCRIPTION
PROTEIN FATE
(folding,
modification,
PROTEIN
SYNTHESIS
REGULATION
OF /
INTERACTION
METABOLISM
KBDiSB/Aix 09-07
ENERGY
CELL RESCUE,
DEFENSE AND
VIRULENCE
CELLULAR
TRANSPORT
AND
CONTROL OF
CELLULAR
ORGANIZATION
CELL FATE
CELL CYCLE
AND DNA
PROCESSING
0,0
0,0
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
TRANSPOSABLE
ELEMENTS,
VIRAL AND
TRANSPORT
FACILITATION
TRANSCRIPTION
REGULATION OF
/ INTERACTION
WITH CELLULAR
PROTEIN
SYNTHESIS
PROTEIN FATE
(folding,
modification,
METABOLISM
ENERGY
CELL RESCUE,
DEFENSE AND
VIRULENCE
CELLULAR
TRANSPORT
AND
CLASSIFICATION
NOT YET CLEARCUT
CELL FATE
CELL CYCLE
AND DNA
PROCESSING
Study of a fermentation of
First results:genes under expressed
reference
% des genes significatif dans la famille considerée
30,0
25,0
20,0
15,0
10,0
60g/L ethanol
80g/L ethanol
90g/L ethanol
100g/L ethanol
120g/L ethanol
5,0
Plate-forme métabolomique, fluxomique
Exploration fonctionnelle des systèmes
métaboliques microbiens
 Analyse des réseaux métaboliques
Environnement
o Reconstruction métabolique
o Analyse topologique
o Modélisation métabolique
 Exploration fonctionnelle
 Analyse in situ: RMN in vivo
o Couplages bioréacteurs / RMN
o Métabolisme énergétique, carboné, etc..
 Métabolomique
o Identification/quantification des métabolites
Génome
Systèmes métaboliques
Métabolisme central E. coli :
89 métabolites, 110 réactions
 Fluxomique
o Quantification des flux métaboliques
o Approches isotopiques (13C)
 Biomathématique/ bioinformatique
KBDiSB/Aix 09-07
o Modélisatio métabolique
GG LISBP INSA Toulouse
o Calculs de flux
o Réconciliation de données
Heterogeneities:gradients(flux,stocks,,,)
:microbe population
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Top Down strategy
•
•
•
•
•
•
•
Fit the macroscopic environnment,bioreactor
Find reproducible conditions:signature recognition
Biokinetics
Quantitative physiologie
Metabolic pathways
Proteomic
transcriptomics
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Synthesis
Engineers Top down strategies
Biologists,bottum up , bioinformatics!!!!!!!!!
Both strategies are necessary
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
Basic concepts
Analysis of information quality
Measures
•3 levels of
multiscale analysis
•Single cell, “statistic”
Classification machine
Fuzzy logic
Hierarchical classification
Biological and engineering
knowledge
Biological modelling
Rules
KBDiSB/Aix 09-07
Programmed by
inductive logic
GG LISBP INSA Toulouse
Hypothesis
or
« Class »
Tackling complexity in
industrial microbiology for
bioprocessing
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse
h
• Biochimie/biologie systémique
• Génie moléculaire enzymatique
• Génie cellulaire des procaryotes et
eucaryotes inférieurs
• Aptitude expérimentation/plate-formes
• Modélisations locales/globales
• Stratégies d’expérimentation/modélisations
• Psychologie d’application
Biocatalyse/enzymologie
Construction/sélection
enzyme
Modélisation
moléculaire PB
• Fluxome, métabolome
• Construction de
souches
• Transcriptome,
régulations
PMM
Biologie intégrative
KBDiSB/Aix 09-07
ascendante
Biologie intégrative
descendante
3
Système
d’échanges
Système
métabolique
2
Système
protéique
• Environnement physicomécanique, physicochimique
•Réponse microbienne
•Modélisation
•Classification
•Corrélation
environnement/mise en
œuvre, réponse
transcriptome
•Dynamique systèmes
•Programmation
expériences
Le
Le biotope
biotope du
du système
système
microbien
crée un
un
microbien crée
environnement;
environnement;
en soi,un
soi,un système
système »»
«« en
PGM
Système
génomique
1
GG LISBP INSA Toulouse
Système:
d’adaptation et de
défense
KBDiSB/Aix 09-07
GG LISBP INSA Toulouse