Handai Cyber University
Biotechnology Basic
Microbial Metabolic Regulation
Ken-ichi Suga
Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka University
1
International Center for Biotechnology, Osaka University
Industrial microbial production and
their relation to metabolic regulation
I. Regulation of primary
metabolite fermentations
II. Overproduction of microbial
products
2
I. Regulation of primary
metabolite fermentations
1. Mechanizm of feedback
repression and feedback
inhibition.
2. Simple metabolic pathway.
3.Branched metabolic pathway.
3
Technical Term
• Technical term should have ONLY one
meaning.
– Ex) Repression, Inhibition
• Usual words may have several meaning.
– Ex) Regulation
4
1.1 Feedback repressible enzyme system
1) co-repressor present
P
R
O
DNA
S
aporepressor
RNA
polymerase
repressor
co-repressor
(end product)
2) co-repressor absent
R
P
O
S
DNA
mRNA
5
enzyme
2. 1 Mechanism of end product inhibition
Enzyme
End product Enzyme
inhibitor
Substrate
Enzyme-substrate
complex
Substrate
Inactive Enzyme
Feedback inhibition is the phenomenon by which the end product inhibits
the early enzyme of pathway. When the inhibitor occupies the regulatory site,
the substrate can not bind to the enzyme at the substrate site.
6
1.2 Feedback regulation in simple
pathway
Enzyme 1
A
Enzyme 2
B
Enzyme 3
C
D
7
1.3 Feedback regulation in
branched pathways
E
D
A
B
F
C
G
H
8
1.3 Feedback repression in
branched pathways
a) Isozymes
E
D
A
B
F
C
G
H
Multiple enzyme are made; each carries out the same reaction
but is regulated by a different end product. This mechanism is
used in both feedback repression and feedback inhibition.
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Isoenzyme in E.coli
Aspartate
Aspartokinase
Aspartyl phosphate
Aspartate semi-aldehyde
Homoserine dehydrogenase
Homoserine
Threonine
Methionine
Lysine
Three aspartokinases are regulated lysine, threonine
and methionine, respectively in E.coli.
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b) Concerted feedback repression
E
D
A
B
C
F
G
H
More than one end product or all end products must
be present in excess to repress the first enzyme.
11
Concerted inhibition in Corynebacterium
Aspartate
Aspartokinase
Aspartyl phosphate
Aspartate semi-aldehyde
Homoserine dehydrogenase
Homoserine
Threonine
Methionine
Lysine
Lysine plus threonine cause concerted feedback inhibition of
aspartokinase
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c) Cumulative inhibition
E
D
A
B
C
F
G
H
Each end product causes a partial inhibition when
present in excess alone, but all end products must
be present to inhibit to a significant degree.
13
Inosine 5-mono-phosphate (IMP) production
Phosphoribosyl pyrophosphate (PRPP)
PRPP amidotransferase
5-phosphoribosyl 1-amine
Adenylosuccinate
synthetase
AMP
IMP
IMP dehydrogenase
Guanosine 5-monophosphate
(GMP)
AMP and GMP cause cumulative feedback inhibition of
PRPP amidotransferase. GMP feedback-inhibits and
feedback-represses IMP dehydrogenase.
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II. Overproduction of
microbial products
1. Decrease in concentration of end
product
2. Mutation to feedback resistance
15
1.1 Overproduction of an intermediate
in unifunctional pathway by auxotrophic
mutant
Enzyme 2
Enzyme 1
A
B
Enzyme 3
C
D
C : Objective product
In parent organism, end product D feedback-regulates
the Enzyme 1. A mutant is obtained which lacks
Enzyme 3. D now must be supplied in the medium.
Because D is generally an essential product for growth.
If D is added at a growth-limiting concentration,
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feedback regulation is broken and C is overproduced.
L-Ornithine production
Glutamate
N-Acetylglutamate
N-Acetylglutamate
syuthetase-
Ornithine
N-acetylglutamyl
N-acetylglutamatekinase phosphate
N-acetylornithine
N-Acetylglutamate
semialdehyde
Ornithine carbamoyl
transferase
Citrullin
Argininosuccinate
Arginine
Feedback inhibition
Feedback repression by arginine
L-Ornithine is produced with a citrulline auxotroph. L-Ornithine
and L-citrulline are intermediates in L-arginine biosynthesis. In
Corynebacterium glutamicum, the first and second enzymes in the
biosynthesis are inhibited by L-arginine. A citrulline auxotroph
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arginine formation and accumulates L-ornithine in large amount.
1.2 Overproduction of an intermediate in a
branched pathway by auxotrophic mutant
Enzyme 3
Enzyme 2
Enzyme 1
A
B
D
C
E
C : Objective product
In parent organism, the Enzyme 1 is subjected to a
cumulative feedback regulation by end products D
plus E. A mutant is obtained which lacks Enzyme
3. D now must be supplied in the medium. If D is
added at a growth-limiting concentration,
cumulative feedback regulation is broken and C is
overproduced.
18
Inosine 5-mono-phosphate (IMP) production
Phosphoribosyl pyrophosphate (PRPP)
PRPP amidotransferase
5-phosphoribosyl 1-amine
Adenylosuccinate
synthetase
AMP
IMP
IMP dehydrogenase
Guanosine 5-monophosphate
(GMP)
AMP and GMP cause cumulative feedback inhibition of
PRPP amidotransferase. GMP feedback-inhibits and
represses IMP dehydrogenase
A mutant is obtained which lacks adenylosuccinate synthetase.
AMP must be supplied in the medium. If AMP is added at
a growth-limiting concentration, feedback regulation by AMP is
broken and IMP is overproduced.
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1.3 Overproduction of one end product
in a branched pathway by auxotrophic
mutant
Enzyme 3
Enzyme 2
Enzyme 1
A
B
D
C
E
E : Objective product
In parent organism, the Enzyme 1 is subjected to a
concerted feedback regulation by end products D plus E.
A mutant is obtained which lacks Enzyme 3. D now
must be supplied in the medium. If D is added at a
growth-limiting concentration, concerted feedback
20
regulation is broken and E is overproduced.
Lysine production by Corynebacerium glutamicum.
Aspartate
Aspartokinase
Aspartyl phosphate
Aspartate semi-aldehyde
Homoserine dehydrogenase
Homoserine
Threonine
Methionine
Lysine
Lysine plus threonine cause concerted feedback inhibition of
aspartokinase
A mutant is obtained which lacks homoserine dehydrogenase.
Threonine plus methionine must be supplied in the medium.
If threonine is added at a growth –limiting concentration, concerted
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inhibition is broken and lysine is overproduced.
Feedback inhibition in simple pathway
Enzyme 1
A
B
C
D
Objective product
End product of a pathway inhibits the action of
an first enzyme of the pathway.
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2.1 Selection of feedback resistant
mutants using toxic analogs
( antimetabolites)
• Toxic analog (antimetabolite)
The toxic analogs are structurally similar to naturally
occurring metabolites and have inhitory effects on the
growth of certain microorganisms.
NH2
CH2
CH2
CH2
CH2
H-C-COOH
NH2
L-Lysine
NH2
CH2
CH2
S
CH2
H-C-COOH
NH2
AEC
CH3
OH-CH
HC-COOH
NH2
L-Threonine
CH3
CH2
OH-CH
HC-COOH
NH2
AVH
(S-2-aminoetyl-L-cysteine)
(α−amino-β−hydroxyvalerate)
Lysine analog
Threonine analog
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Isolation of mutant insensitive to analog
• Wild type
Feedback regulation by end product
Enzyme 1
A
B
C
D
Feedback regulation by D-analog
Enzyme 1
A
B
D-analog
C
D
Starvation
of D
Die
24
Isolation of mutant insensitive to analog
• Mutant type
Insensitive to D-analog
Grow into colony
Enzyme 1
A
B
C
D
Inhibition resistant
Repression resistant
D-analog
Insensitive to end product D
Enzyme 1
A
B
C
D
Overproduce D
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Lysine production by mutant resistant to AEC
in parent cell of Brevibacterium flavum
Aspartate
Aspartokinase
Aspartyl phosphate
Aspartate semi-aldehyde
Homoserine
Threonine
Methionine
Lysine
Lysine plus threonine cause concerted feedback inhibition of
aspartokinase
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Effect of lysine and threonine on the aspartokinase of wild type cell
Supplements
None
L-lysine ( 1 mM )
L-lysine ( 5 mM )
L-threonine ( 1 mM )
L-threonine ( 5 mM )
L-lysine + L-threonine ( 1 mM each )
L-lysine + L-threonine ( 5 mM each )
Relative activity
100
80
45
88
65
6
1
Aspartokinase is subjected to a concerted feedback inhibition
by L-lysine plus L-threonine.
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Growth inhibition by AEC or AEC plus
threonine, and recovery of growth by addition
of lysine
Relative growth
1.0
1 mg/ml each of
AEC + L-Thr
AEC
0.5
AEC + L-Thr
0
0
0.3
1.0
AEC and L-Thr mg/l
3.0
0 0.1 0.3
L-Lys mg/l
1.0
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Isolation of AEC-resistant mutant
Exponentially growing parent cells
are treated with a mutagen, such as
nitrosoguanidine
Plate out and incubate
Colonies appearing on the surface of
Agar are picked up as AEC-resistants
Production of lysine by AEC-resistant
mutants are examined.
Minimal medium supplemented
with AEC (and L-threonine)
29
Primary metabolite fermentation by
auxotrophic mutants and analog-resistant
mutant
Type of mutant Organism Genetic markers Product accumulation Reference
Auxotrophic
Brevibacterium Thrflavum
Thr-, Met-
Lysine
26 (g/l)
Lysine
34
AECr , LHXr
Lysine
Lysine
16
42
AHVr , DPS-
Threonine 16
Shiio et al
(1967)
Shiio et al
(1969)
Analog-resistant
B. lactofermentum AECr
Corynebacterium
glutamicum
B. flavum
LHXr resistance to l-lysine hydroxamate
AHVr resistance to a-amino-b-hydroxyvaleric acid
DPS- dihydro dipicolinate synthase defect
Sano et al (1970)
Smekal et al (1985)
Shiio et al (1990)
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Quiz 1
Q1 What is an auxotroph ?
1. Mutant cells that have lost their ability to
synthesize essential metabolites.
2. Microbial cells that can grow by utilizing only
inorganic compounds.
3. Wild type cells lacking the growth
requirements.
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Quiz 2
Q1 What kind of mutant should be isolated, when
you want to overproduce L-threonine ?
1. Lysine analog resistant mutant.
2. Lysine- and Metionine- auxotroph
3. Threonine- auxotroph.
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Handai Cyber University
Biotechnology Advanced
Microbial Metabolic Regulation
END
Graduate School of Engineering, Osaka University
Graduate School of Information Science, Osaka University
33
International Center for Biotechnology, Osaka University