Production of renewable materials
through systems and synthetic
biology
Lars Nielsen
Systems & synthetic biology
Model
Manipulate
Mine
Measure
Sucrose for biorefineries
Fuel versus chemicals
Crude
40 GJ
$575
→
Petrol
40 GJ
$600
Sugar
15 GJ
$300
→
EtOH
27 GJ
$600 ($900oe)
In conventional combustion engines,
no premium for higher purity
Chemicals
Ethylene
Propylene
Styrene
Asia spot
$ 825/t
$ 930/t
$ 1010/t
20 Oct 2008
Metabolic engineering
We are studying microbes as "programmable"
manufacturing factories to make chemicals,
monomers and polymers from different nutrient
feedstocks. Current feedstocks for these materials
are petrochemicals from oil. We are programming
microbes to make very sophisticated polymer
building blocks and molecules out of simple,
renewable feedstocks, like glucose and methane.
Chad Holliday, Chairman & CEO – DuPont, Boston Chief Executive Club, Sept 99.
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Classical biotechnology
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Random mutations
Process optimisation
Fixed product range
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Classical biotechnology
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Genetic engineering
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Random mutations
Process optimisation
Fixed product range
New products
Enzyme engineering
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Improved kinetics
New products
PDO
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New products
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Enzyme engineering
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Random mutations
Process optimisation
Fixed product range
Genetic engineering
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Classical biotechnology
Improved kinetics
New products
PDO
Metabolic engineering
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Pathway redesign
Control redesign
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Metabolic engineering
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From retrofitting to green field design
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PDO (2002)
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3 years, 10 staff using systems biology
Amino acids (2007)
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7 years, 15 staff using conventional metabolic
engineering
Succinic acid (2005)
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Genetic engineering → systems & synthetic biology
2-3 years, 3 staff using synthetic biology followed
by systems biology
Novel compounds
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Engineer new pathways for non-biological
compounds
Bio vs inorganic catalysis
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Broad reaction potential
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High catalytic potential
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Effective in water at RT, pH 7
High specificity
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~150 fundamental reaction types (EC Level 3)
Plus polymerisation
Stoichiometric yield over 10-20 catalytic steps
Complex molecules from inexpensive feedstocks
Stereospecificity
Cells = Self-replicating catalytic microreactors
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Low cost and ease of scale-up
In silico strain design
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Is used to evaluate the impact on the product
yield when using:
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This evaluation is based on the organisms
biochemical network and its stoichiometry:
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different organisms,
different culture conditions (e.g. available
substrates, aerobic vs anaerobic)
knockout candidates etc
This means kinetics are neglected
Upper boundaries for the system are calculated that
will not be surpassed in vivo.
Theoretical product carbon yield is used to
evaluate different networks, pathways
In silico E. coli model
Network:
71 reactions
- 21 reversible
55 internal metabolites
13 external metabolites
Captures:
- Substrate uptake
- Glycolysis
- PPP, EDP, TCA-Cycle
- Anaplerosis
- Respiration
- Fermentation
- Biomass formation
- 4 different pathways
for product formation
4 natural pathways for 3HP production (KEGG)
3HP
Effect on product formation (anaerobic,
aerobic)
Left (1)
Right (2)
Center(3)
Centerright (4)
3-HP Yield
(aerobic)
95.8 %
96.6 %
84.2 %
100 % **
3-HP Yield
(anaerobic)
100 %
100 % *
(50%)
85.7 %
100 % **
Yields are given in C-mol / C-mol [%]
In general the yields are higher under anaerobic conditions
The second pathway is strongly dependent on a reversible
acetate kinase.
The third pathway underperforms the other in both
scenarios.
Two unknown enzymes in the fourth pathway
In silico analysis of β-alanine pathway
for the production of 3-HP in E. coli
#product synthesis - left pathway
R54 : OAA + GLU = ASP + 2-OXO
R55 : ASP = bALA + CO2
R56 : bALA + 2-OXO = 3-OXOPRO + GLU
R57 : 3-OXOPRO + NADH = 3-HPA + NAD
3HP
Carbon yield for biomass
Elementary mode analysis
16881 elementary modes
2292 make the desired product
Mode #11048
Max P with X>0
Anaerobic
Does not form acetate
Does not require PEP-carboxylase and GPI
Highly dependent on malic enzyme (NADPH)
Carbon yield for 3-HP
Knock-out of GPI, PEP-C and Acetate kinase
Anaerobic conditions
Carbon yield for biomass
464 elementary modes
126 make the desired product
Carbon yield for 3-HP
Systems biology
Model
Manipulate
Mine
Measure
Flow
Flux
=
=
Capacity
Activity
x Force
x Thermo driving force
2
1
5 mL culture
pH control
2 minutes centrif ugation
S. zoo f ermentation
pellet
4
5
3
SPE column
80 C
ETOH
HPAEC
Sample purif ication
(Jensen et al., 1999)
Cell lysis
We always overestimate the change that will occur in the next
two years and underestimate the change that will occur in the
next ten. Don't let yourself be lulled into inaction.
Bill Gates