Transcript In silico aided metaoblic engineering of Saccharomyces

In silico aided metaoblic engineering of
Saccharomyces cerevisiae for improved
bioethanol production
Christoffer Bro et al. 2005
The problem
• Under anaerobic conditions, S.
cerevisiae produces only four major
products from glucose:
• CO2, ethanol, biomass and glycerol
• To increase the ethanol yield, the
flow of carbon going to biomass or
glycerol should be redirected
towards ethanol.
Overview of main fluxes
Previous work
• Some of the carbon flowing to biomass
can be redirected towards ethanol by
increasing the consumption of ATP for
biomass production or reducing the
amount of ATP formed in association with
ethanol production. (Nissen et al. 2000)
• Deletion of the structural genes in glycerol
biosynthesis is not a successful strategy.
• The maximum specific growth rate is severely
lowered in such strains
• Formation of glycerol is necessary for
maintaining the redox balance by oxidizing
NADH
Strategy #1
• Substitution of NADPH-oxidizing
reactions in biomass formation with
NADH-oxidizing reactions
Strategy #2
• Substitution of NAD+-reducing
reactions in biomass formation by
NADP+-reducing reactions.
Strategy #3
• Introduction of a reaction which
either directly or via a cycle
converts NADH into NADPH.
Strategy #4
• Substitution of the glycerol production
with production of ethanol, which has a
net oxidation of NADH.
In silico model
• iFF708 (Forster et al., 2003)
• 708 genes
• 584 metabolites
• 1175 reactions
Method
• A database of 3800 biochemical reactions is
derived from the LIGAND database of KEGG.
• Each gene (corresponding to a specific
biochemical reaction) was inserted one at a time
into the genome-scale metabolic model, and the
performance of the engineered strain was
evaluated.
• Two other engineered strains:
• Heterologous expression of a non-phosphorylating,
NADP+-dependent D-GAPN
• Deletion of GDH1 combined with simultaneous
overexpression of GDH2 or GLN1 and GLT1.
•
•
•
•
GDH1: AKG + NH3 + NADPH -> GLU + NADP
GDH2: GLU + NAD -> AKG + NH3 + NADH
GLN1: GLU + NH3 + ATP -> GLN + ADP + PI
GLT1: AKG + GLN + NADH -> NAD + 2 GLU
Eight best strains predicted
The best strategy
In vivo testing of the best strategy
• Ethanol production increased by 3%
• Reasons for disagreement between
experiment and model:
• Limited GAPN activity in vivo
• Low intracellular NADP+ concentrations
compared with NADPH
Discussion
• “The success of the strategies is due
to the tight linking of the different
parts of the metabolic network
through the common usage of cofactors like NADH, NADPH and ATP,
and the genome-scale metabolic
model represents a valuable tool for
studying how these co-factors link
the different parts of the metabolism
in a quantitative fashion.”
Efficiency of amino acid production in
Escherichia coli
Anthony Burgard & Costas Maranas, 2001
iJR904
20
8.2609
15.68
18.627
9.4501
11.322
11.81
26.122
7.8728
7.4219
7.5
7.8512
5.7951
5.4913
10.074
20
12.601
4.4907
5.6886
10
Universal model
• The universal model is constructed
by incorporating 3400 cellular
reactions from the KEGG into the
modified Keasling stoichiometric
model.
Arginine production