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Microbial production of C-4 metabolitesMicrobial production of C-4 metabolites
State Research Institute of Genetics and Selection of Industrial Microorganisms
- Moscow -
State Research Institute of Genetics and Selection of Industrial Microorganisms
- Moscow -
- Tigran Yuzbashev -- Tigran Yuzbashev -
C-4 metabolitesC-4 metabolites
butanolbutanol succinic acidsuccinic acid
(I)
Clostridiumacetobutylicum(I)
Clostridium
acetobutylicum(II)
Escherichia
coli(II)
Escherichia
coli(III)
Yarrowia
lipolytica(III)
Yarrowia
lipolytica
low pHlow pHneutral pHneutral pH
– Section I –– Section I –
Realistic scenario based on current technology and potential in fuels
Realistic scenario based on current technology and potential in fuels
Clostridium acetobutylicumClostridium acetobutylicum
♦
Institution culture collection -
Russian State
Collection of Industrial Microorganisms (VKPM) maintains more than 1000
Clostridium strains
♦♦
Institution culture collection -
Russian State Russian State
Collection of Industrial Collection of Industrial Microorganisms (VKPM)Microorganisms (VKPM) maintains more than 1000
Clostridium strains
♦
The genetic engineering approach is worked out and successfully applied for Clostridium gene knock-out
by group II intron
(TargeTron®)
♦♦
The genetic engineering approach is worked out and successfully applied for Clostridium gene knock-out
by group II intron
(TargeTron®)
♦
The high-throughput automated system is available
for screening of mutant libraries of Clostridium strains
♦♦
The high-throughput automated system is available
for screening of mutant libraries of Clostridium strains
Biochemistry of ABE processBiochemistry of ABE process
Strategy for construction of butanol
producing strainStrategy for construction of butanol
producing strain
The expected result: 23%
→ 32% of mass yieldThe expected result: 23%
→ 32% of mass yield
Best producing clostridial
strain (w.t.)
Breeding of over-producing strain
Knock-out of gene buk
Knock-out of genes
pta; ack; ldhBreeding of strain with lowered activity
of NADH-ferredoxin oxidoreductase
– Section II –– Section II –
OHO
OH
O
OO
O
O NH2
O
NH2
O
NN
NH2
NH2
OHOH
O
N ONH
O
OO
1,4-Butanediol
N-Methyl-pyrrolidone
1,4-Diaminobutane
SuccindiamideDimethylsuccinate
γ-Butyrolactone
Tetrahydrofuran
2-Pyrrolidone
Succinonitrile
Building-block chemicals derived from succinic acidBuilding-block chemicals derived from succinic acid
Escherichia coliEscherichia coli
♦
The
E. coli is excellent objects for molecular
manipulations and microbiological
techniques
♦♦
The
E. coli is excellent objects for molecular
manipulationsand microbiological
techniques
♦
The
E. coli strains are widely used in biotechnology for production of many essential
chemical substances
♦♦
The
E. coli strains are widely used in biotechnology for production of many essential
chemical substances
♦
The highest succinate yield (up to 1.68 mol/mol glucose) was published for engineered E. coli strain
♦♦
The highest succinate yield (up to 1.68 mol/mol glucose) was published for engineered E. coli strain
Production
of succinic acid by E. coli Production
of succinic acid by E. coli
Acet-CoA
PEP
PyrOAA
Fum
Suc
MalFormAcet-P
Acetate Lact
Cit
IsoGlyox
α-KG
Suc-CoA
NADH
CO2
ATP
ATP
CO2
ATP
ATP
CO2
NADPH
CO2
ATP
Gluc (out)
Gluc-6-P (in)
CO2
Acet-CoA
Acet-CoA
NADH
MKH2
½ Glucose
NADH
NADH
NADH
7 Glucose + 6 CO2 = 12 Succinate
Maximal theoretical yield = 1.7 mol/mol
Prospective pathway
with supplementation of
exogenous H2Prospective pathway
with supplementation of
exogenous H2
Fumarate
respiration with
H2
as electron donorFumarate
respiration with
H2
as electron donor
Fumarate + H2 = Succinate + 2/3 ATP
MKH2
MK
2H+
2ē
2ē
2
Suc+ Fum
Out
In
Hyd Frd
2H+
Dcu
Suc Fum
ATP-ase
2H+
2H+2H+
2H+
23
ADP
Strategy for construction of succinate producing E. coli strainStrategy for construction of succinate producing E. coli strain
Some arguments for hydrogen usageSome arguments for hydrogen usage
♦
The yield could be increased up to 2.0 mol/mol
♦
The same (anaerobic) conditions could be applied for both stages (growth and production)
♦
The main accumulated by-product (acetate)
could be diminished
♦
Assimilation of greenhouse gas could be increased twice as much
♦♦
The yield could be increased up to 2.0 mol/mol
♦♦
The same (anaerobic) conditions could be applied for both stages (growth and production)
♦♦
The main accumulated by-product (acetate)could be diminished
♦♦
Assimilation of greenhouse gas could be increased twice as much
– Section III –– Section III –
H2SO4
KOH
H2SO4
KOH
Is it possible
to produce succinic acid under low pH conditions?Is it possible
to produce succinic acid under low pH conditions?
Succinic acid could be produced at low pH if aerobic conditions are used
Succinic acid could be produced at low pH if aerobic conditions are used
–
+
–
+ATP
ATP
pH≈6.8 pH<3.2
glucose
acid
ATP
Yarrowia lipolyticaYarrowia lipolytica
♦
Y. lipolytica is an aerobic yeast
which attracts strong
interest in the field of industrial biotechnology
♦♦
Y. lipolytica is an aerobic yeast
which attracts strong
interest in the fieldof industrial biotechnology
♦
Y. lipolytica is capable to produce large amounts of organic acids, particularly TCA cycle intermediates e.g.,
citric, isocitric and α-ketoglutaric
acids
♦♦
Y. lipolytica is capable to produce large amounts of organic acids, particularly TCA cycle intermediates e.g.,
citric, isocitric and α-ketoglutaric
acids
♦
The
wide spectrum of
tools is available for genetic
engineering
with this organism
♦
The
wide spectrum of
tools is available for genetic
engineering
with this organism
Production
of succinic acid by Y. lipolytica Production
of succinic acid by Y. lipolytica
Production
of succinic acid in fed-batch fermentation by Y. lipolytica Production
of succinic acid in fed-batch fermentation by Y. lipolytica
Y. lipolytica strain was
designed based on deletion of sdh2 gene Y. lipolytica strain was
designed based on deletion of sdh2 gene
The strain produces up to 72 g/l
of succinic acid from glycerol 167 g/l
No neutralizing agent was used (terminal pH < 2.5)
The strain produces up to 72 g/l
of succinic acid from glycerol 167 g/l
No neutralizing agent was used (terminal pH < 2.5)
30°C 4°C
– contacts –Institute: State Research Institute
of Genetics and Selection of Industrial Microorganisms
Address: 1-st Dorozhny pr., 1, Moscow 117545, Russia.
Phone: +7 495 3151210. E-mail: [email protected]
to Tigran Yuzbashev
– contacts –Institute: State Research Institute
of Genetics and Selection of Industrial Microorganisms
Address: 1-st Dorozhny pr., 1, Moscow 117545, Russia.
Phone: +7 495 3151210. E-mail: [email protected]
to to Tigran Tigran YuzbashevYuzbashev
Russian State Collectionof Industrial
Microorganisms
Russian State CollectionRussian State Collectionof Industrial of Industrial
MicroorganismsMicroorganisms
