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2008 NAIST-UM (BTI) Synmposium
Metabolic regulation of cysteine in bacteria
and its application to cysteine production
September 22, 2008
Hiroshi Takagi, Ph.D.
Lab. of Cell Biotechnology
Graduate School of Biological Sciences
Nara Institute of Science and Technology
Microbial production of amino acids
Amino acid
Microorganism
-Glutamate
L-Lysine
L-Phenylalanine
L-Threonine
L-Glutamine
L-Arginine
C. glutamicum
C. glutamicum
C. glutamicum / E. coli
E. coli
C. glutamicum
C. glutamicum
L
(L-Cysteine)
(DL-Methionine)
Market (tons/y)
1,000,000
250,000
8,000
4,000
1,300
1,200
(1,500)
(350,000)
No direct-fermentation process for sulfur-containing
amino acids (Cys, Met) has yet been achieved.
Industrial use and production methods of Cys
・Food
・Pharmaceutical
・Cosmetic
・New material
・Hydrolysis of human hairs
・Asymmetrical hydrolysis of ATC
A world market of 4,000 tons a year
A variety of applications
Environmental issues
Increase of demand
Direct fermentation of glucose
Pseudomonas thiazoliniphilum：
DL-ATC (2-aminothiazoline-4-carboxylic acid)
→ → L-Cysteine
Cysteine desulfhydrase：
L-ClCH2CHNH2COOH + Na2S + H2O → L-Cysteine + NaCl + NaOH
Metabolism and its regulation of Cys in E. coli
2-
SO4 ( external )
L-Serine + Acetyl-CoA
SO42APS
SAT
PAPS
Feedback
inhibition
O-Acetyl-L-serine
SO32-
S2-
OASS
COOH
H2N-C-H
CH2
SH
L-Cysteine
(Cys)
・Feedback inhibition of SAT by Cys
・Cys degradation catalyzed by CD
L-Cysteine
CD
Glutathione
degradation
( Pyruvate, NH3, H2S )
L-Methionine
In E. coli cells…
・No oversynthesis
・No accumulation
Direct fermentation of Cys from glucose
Methionine
Acetyl-CoA
Glucose
Serine
H2S
O-Acetylserine
Serine acetyltransferase
(SAT)
Cysteine
Cysteine desulfhydrase
(CD)
Degradation
1) Enhancing the biosynthetic activity
Functional improvement of serine acetyltransferase (SAT)
2) Weakening the degradation pathway
Identification and the gene disruption of cysteine desulfhydrase (CD)
Serine acetyltransferase (SAT) of E. coli
Denk et al. (J. Gen. Microbiol., 133, 515, 1987)
・Isolation of a Cys+ revertant from a Cys- auxotroph (Cys: 30 mg/L)
・Gene cloning and its deduced amino acid sequence
・Identification of the Met256Ile mutation
＜Feedback inhibition of SAT activity by Cys＞
Relative activity (%)
Enzyme
Substrate
100
Active
＋
site
Allosteric
site
75
50
ES-complex
Ser
Acetyl-CoA
SAT
Ser
25
0
Cysteine
(endproduct)
25
50
75
L-Cysteine conc. (M)
Acetyl-CoA
100
SAT
(inactive)
＜site-directed mutagenesis by PCR＞
＜mixed primers＞
Amino acid substitution of
Met256 of the E. coli SAT
5’-AATGGAT GGG GACCAGC-3’
CCC
AAA
TTT
Analysis of feedback inhibition
and Cys productivity
256All-
<1st PCR>
A
B
256All +
A
Wild-type
Met256
N
B
C
Altered
Met256X
N
C
Ligation
Truncated
N
<2nd PCR>
C
Production of cysteine plus cystine
＜Strain＞
E. coli JM39-8 (SAT-deficient and Cys non-utilizing)
＜Medium＞ Cys production medium (1L) (pH 7.0)
Glucose
30 g
Na2S2O3
15 g
NH4Cl
10 g
KH2PO4
2g
MgSO4・ 7H2O
1g
FeSO4・ 7H2O
0.01 g
MnCl2・ 4H2O
0.01 g
Gly, L-Ile, L-Leu, L-Met 0.1 g each
CaCO3
20 g
＜Cultivation＞ 30℃, Sakaguchi-flask, shaking
＜Determination of cysteine + cystine＞
Bioasay (Pediococcus acidilactici IFO3076)
Cys production by expression of the mutant SATs
Plasmid
Amino acid
residue at
position 256
Activity remaining
in the presence of
100 M cysteine (%)
CySH + Cys
(mg/L)
0.5
ND
pCE
Met (Wild-type)
M256A
Ala
24.1
790 ± 380
M256R
Arg
32.1
600 ± 80
M256D
Asp
24.2
580 ± 50
M256E
Glu
17.3
710 ± 270
M256S
Ser
27.0
610 ± 40
M256W
Trp
18.6
610 ± 70
M256V
Val
25.6
560 ± 70
-*
31.3
730 ± 110
M256Stop
*, termination codon at position 256
Nakamori et al., Appl. Environ. Microbiol., 64, 1607-1611 (1998)
Cys overproduction was achieved by expressing the mutant SAT.
Error-prone PCR random mutagenesis into E. coli SAT
E. coli wild-type SAT gene (cysE)
pCE
Error-prone PCR
EcoRI
XbaI
EcoRI
XbaI
pUC19
EcoRI
＜Reaction mixture＞
10 mM Tris-HCl ( pH 8.3 )
50 mM KCl
1.5 mM MgCl2
0.01 M  -mercaptoethanol
10% DMSO
0.5 mM MnCl2
0.5 M forward primer
0.5 M reverse primer
0.2mM dATP
1mM dGTP, dCTP, dTTP each
1U Taq DNA polymerase
XbaI
pHC
Transformation of E. coli JM39-8
Characteristics of the E. coli mutant SATs
Plasmid
Activity remaining
CySH + Cys
in the presence of
(mg/L)
100 M cysteine (%)
Amino acid
substitution
0.9
ND
pCE M256A
24.1
790
pHC 6
51.9
210 ±170
N51K, R91H , H 233Y
pHC 7
78.2
330 ±70
E166G, M201V
pHC 8
37.1
260 ±50
T167K
pHC 10
20.9
990 ±200
M201R
pHC 11
16.3
740 ±120
M201T
pHC 12
33.2
50 ±20
P252R
pHC 13
28.9
960 ±460
S253L
pCE
M256I
Takagi et al., FEBS Lett., 452, 323-327 (1999)
Several amino acid residues other than Met256 are responsible for
the feedback inhibition by Cys and the overproduction of Cys.
SATs of Arabidopsis thaliana
(Noji et al., J. Biol. Chem., 273, 32739-32745, 1998)
Localization
Feedback inhibition
SAT-m
SAT-p
Mitochondria
Chloroplast
Insensitive
Insensitive
SAT-c
Cytoplasm
Sensitive
SAT
＜Expression plasmids for the SAT cDNA＞
＜Western analysis for the SAT expression＞
cysEp
A. thaliana
Ampr
pEAS-m, pEAS-p
SAT-m
SAT-p
E. coli
wild-type SAT
The A. thaliana SAT-m or SAT-p gene
The E. coli cysE promoter
The A. thaliana SATs are expressed in E. coli cells.
Comparison of catalytic properties of recombinant SATs
plasmid
pEAS-m
pEAS-p
pCEM256I
pCE
SAT
A. thaliana
SAT-m
A. thaliana
SAT-p
E. coli
Met256Ile
E. coli
wild-type
SAT activity
(mU/min/mg)
27.9
21.3
88.0
2,273
100
100
100
100
100
100
100
88
24
100
1.5
Relative activity (%) for
L-cysteine added (M)
0
10
100
ND
ND : Not detected.
The A. thaliana SATs were insensitive to feedback inhibition.
Cys production by recombinant strains
SAT
SAT-m
SAT-p
E. coli Met256Ile
( A ) Growth (OD562)
0.91 ± 0.02
0.77 ± 0.10
0.64 ± 0.12
( B ) L-Cysteine produced
(mg/L)
1,580 ± 100
1,660 ± 200
870 ± 160
(B)/(A)
1,750 ± 100
2,140 ± 200
1,360 ± 70
Takagi et al., FEMS Microbiol. Lett., 179, 453-459 (1999)
Expression of two cDNAs encoding SAT-m and SAT-p in E.
coli cells significantly increased the Cys productivity.
Enhancement of Cys biosynthetic activity
1) Functional improvement of the E. coli SAT
(1) Site-directed mutagenesis into Met256
・Desensitization to feedback inhibition by replacing Met with other residues
→ Met at position 256 is important for feedback inhibition by Cys
・Cys overproduction (ca. 800 mg/L)
(2) PCR-random mutagenesis into cysE
・Identification of several residues other than Met256 involved in
desensitization to feedback inhibition and Cys production
2) Use of the A. thaliana SATs
(1) Expression of the A. thaliana feedback-insensitive SATs in E. coli cells
(2) Improvement of Cys productivity (1,600 - 1,700 mg/L)
Ser
Arg89-Asp96
Kai et al., Prot. Eng. Des. Sel., 19, 163-167 (2006)
Direct fermentation of Cys from glucose
Methionine
Acetyl-CoA
Glucose
Serine
H2S
O-Acetylserine
Serine acetyltransferase
(SAT)
Cysteine
Cysteine desulfhydrase
(CD)
Degradation
1) Enhancing the biosynthetic activity
Functional improvement of serine acetyltransferase (SAT)
2) Weakening the degradation pathway
Identification and gene disruption of Cys desulfhydrase (CD)
A reaction catalyzed by Cysteine Desulfhydrase (CD)
COOH
CD
H2N-C-H
COOH
C=O
CH2
SH
CH3
L-Cysteine
Pyruvate
+
NH3
+
H2S is generated during fermentation !!
Cys degradation is occurred !!
Cys degradation pathway is unknown ??
Analysis of Cys degradation pathway
H2 S
Identification of the E. coli CDs by activity staining
Native-PAGE
CD activity staining
Cys
CD
H2S + BiCl3
(1)
(2)
=
BiSO4
Black bands
(3)
(4)
(5)
At least, five CD proteins are newly detected in E. coli.
E. coli CD (1)
Determine the N-terminus sequence
(1)
1
Purified sample
E. coli Tryptophanase (TNase)
Wild-type tnaA-disruptant
Vector
Vector
+ tnaA
15
MENFKHLPEPFRIRV・・・
MENFKHLPEPFRIRV・・・
A reaction catalyzed by TNase (the tanA product)
L-Tryptophan → Indole + Pyruvate + NH3
( 1 ) TNase
(2)?
TNase (the tnaA product) is one of the E. coli CDs.
E. coli CD (2)
＜CD reaction＞
COOH
( 1 ) TNase
(2)
COOH
CD
H2N-C-H
C=O + NH3 + H2S
CH2
CH3
SH
L-Cysteine
Pyruvate
＜Cystathionine -lyase (CBL; the metC product) reaction＞
COOH
L-Cysteine
O-Succinyl-homoserine
COOH
H2N-C-H
CH2
H2C
COOH
H2N-C-H
S
CH2
Cystathionine
CBL
COOH
H2N-C-H
C=O + NH3 +
CH3
Pyruvate
CH2
CH2
SH
Homocysteine
L-Methionine
The CD and CBL reactions are the same.
CBL accepts Cys as a substrate in vitro.
CBL functions as a CD ?
E. coli CD (3) - (5)
Use of an E. coli library containing 4,388 kinds of open reading frame (ORF)
lacZp
X
: lacZ promoter
Vector
X
: ORF (total 4,388)
Cmr
CD activity staining
pCN24-X
+ cysK + cysM + malY
( 1 ) TNase
( 2 ) CBL
( 3 ) O-Acetylserine sulfhydlase-A (OASS-A; cysK)
( 4 ) MalY regulatory protein (malY)
( 5 ) O-Acetylserine sulfhydlase-B (OASS-B; cysM)
OASS (-A, -B) and MalY protein are identified as the E. coli CDs.
List of the E. coli CDs
( 1 ) Tryptophanase
(TNase; the tnaA product) Trp-degrading enzyme
(1)
( 2 ) Cystathionine -lyase
(2)
(CBL; the metC product) Cystathionine-degrading enzyme
( 3 ) O-Acetylserine sulfhydlase-A
(OASS-A; the cysK product) Cys-synthesizing enzyme
( 4 ) MalY regulatory protein
(3)
(5)
(4)
(the malY product) transcriptional regulator in mal expression
( 5 ) O-Acetylserine sulfhydlase-B
(OASS-B; the cysM product) isomer of OASS-A !?
Five CD proteins were identified in E. coli…
Total CD activity
Genotype
total CD activity (mU/mg)
Wild-type
20.6
tnaA
15.7
metC
15.0
cysK
18.2
cysM
17.9
malY
15.3
 tnaA metC
9.6
tnaA metC cysM malY
9.1
tnaA metC cysK cysM malY
8.7
・Total CD activities of all mutants were lower than wild-type.
・Even the quintet mutant still had a low level of CD activity.
Cys production in the CD gene disruptants
1600
Cysteine productivity(mg / L)
1400
1200
Wild-type
tnaA mutsnt
1000
metC mutsnt
800
cysM mutsnt
malY mutant
600
4 genes mutant
400
200
0
0
24
48
72
96
Culture time (hr)
・Cys production in these mutants was higher than that in wild-type.
・CD gene disruption is effective in the production of Cys by E. coli.
Growth of E. coli cells in the presence of Cys
Cys inhibits the growth of E. coli cells.
LB + 30 mM Cys
4.5
4.0
Growth (OD610)
3.5
2.5
Wild-type
tnaA mutant
metC mutant
2.0
cysK mutant
1.5
cysM mutant
3.0
malY mutant
1.0
0.5
0
0
3
6
9
12
15
18
21
24
Culture time (hr)
・The tnaA disruptant was significantly inhibited.
・TNase is a key enzyme in Cys degradation in E. coli ??
TNase induction by Cys
Native-PAGE
Cys
(mM)
0
10
Northern blotting
SDS-PAGE
Cys
(mM)
0
10
Cys
(mM)
0
10
(kDa)
94・
67・
1.7kb
43・
23s rRNA
30・
20・
･TNase synthesis is induced by Cys.
･TNase contributes mainly to Cys degradation.
16s rRNA
Identification and gene disruption of the E. coli CDs
1) Identification of the E. coli CDs
( 1 ) Tryptophanase
(TNase; the tnaA product) Trp-degrading enzyme
( 2 ) Cystathionine -lyase
(CBL; the metC product) Cystathionine-degrading enzyme
( 3 ) O-Acetylserine sulfhydlase-A
(OASS-A; the cysK product) Cys-synthesizing enzyme
( 4 ) MalY regulatory protein
(the malY product) transcriptional regulator in mal expression
( 5 ) O-Acetylserine sulfhydlase-B
(OASS-B; the cysM product) isomer of OASS-A !?
2) Construction of the CD gene disruptants
The gene disruption is significantly effective for Cys production.
3) TNase contributes primarily to Cys degradation.
Genome information-based
Identification and analysis of the Cys transporter
Enhancing the export system
Glucose
L-Cysteine
Bcr
Yamada et al., Appl. Environ. Microbiol., 72, 4735-4742 (2006)
TolC
Natthawut et al., Appl. Microbiol. Biotechnol., in press.
Poster
Enhancement of Cys export system
Imbalance of cellular
oxidation-reduction state
Cys overproducer
Mutant SAT gene
Cys accumulation
CD gene
Growth inhibition
Cys export
Mutant SAT gene
Cys transporter gene
Improvement of Cys
productivity ?
CD gene
・Identification and analysis of Cys transporter
・Evaluation of Cys transporter on Cys productivity
Screening of Cys transporter
The growth of E. coli cells is inhibited by excess Cys (30 mM).
E. coli cells with a lower level of CD activity would be much more
sensitive to Cys due to Cys accumulation.
The transporter that exports Cys and reverses the growth inhibition
Px
Wild-type
pUC118-X
Ampr
32 putative drug transporter genes
Growth (OD610)
Transporter X
naA disruptant
+ transporter gene
tnaA disruptant
Screening of Cys transporter
Culture time (hr)
6
emrAB
Growth (OD610)
5
Wild-type
4
acrD, acrEF, bcr, cusA,
emrKY, ybjYZ, yojIH
3
2
tnaA disruptant
1
0
0
6
12
Culture time (hr)
18
24
Genes that reversed the growth inhibition of tnaA disruptant by Cys:
acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH
Intracellular Cys contents of E. coli cells
Transporter X
pUC118-X
Ampr
Intracellular Cys content (mg/L/OD610)
Cys（30 mM）
3
2
1
0
vector vector
Cys
Wild-type
emrAB emrKY yojIH
acrEF
bcr
cusA
acrD ybjYZ
tnaA disruptant
Genes that decreased intracellular Cys level of tnaA disruptant:
acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH
List of Cys exporter candidates
Gene
Function
bcr
Bicyclomycin resistance
emrAB
Multidrug resistance
emrKY
Multidrug resistance
acrEF
Acriflavin resistance
acrD
Acriflavin resistance,
Aminoglycosides efflux
cusA
Putative copper transporter
ybjYZ
Putative transporter
yojIH
Putative transporter
No one knows whether these genes are involved in amino acid export.
＜ Cys transport assay ＞
+
[35S]-Cys
Cys uptake activity
＜Cys uptake ＞
Cys uptake （nmol/mg cell wt）
Cell suspension
vector
2.0
ybjYZ
ydeD
bcr
1.0
0
0
10
Remaining labeled Cys content
＜Cys export＞
Increased export：
bcr, acrEF, emrAB, (ydeD)
Cys export (%)
100
bcr, ybjYZ,(ydeD)
30
Time (min)
⇒ Cys export rate
Reduced uptake：
20
80
60
bcr
ydeD
40
acrEF emrAB
vector
20
＜ Cys uptake↓、Cys export↑ ＞
bcr, ydeD
0
0
10
20
30
Time (min)
Bcr overexpression promotes Cys export in E. coli cells.
Cys production by E. coli cells expressing bcr
Mutant SAT gene
Enhancing Cys synthesis
Pbcr
bcr
pUC118-bcr
Enhancing Cys export
Cys
Concentration of Cys （mg/L/OD562）
pACYC-M256I
1000
+ bcr
800
600
400
+ vector (pUC118)
200
0
24
Cys
48
Culture time (h)
Bcr overexpression contributes to Cys production.
72
32 putative drug transporter genes
Growth inhibition, Intracellular Cys level
Identify the Bcr protein as a Cys transporter
Export activity, Specificity, Cys production
Bcr derives energy for Cys export from the proton gradient, and Cys
may be the only amino acid exported by Bcr.
Bcr overexpression contributes to Cys production
Future plans
Functional analysis (transcriptional regulation, physiological role)
Improved function (export activity, substrate specificity)
Molecular breeding of Cys overproducer
L-Serine
+ Acetyl-CoA
SO4 (external)
Ser acetyltransferase
Activated
by OAS
O-Acetylserine
Feedback
inhibition by Cys
Enhance Cys
biosynthesis
2-
S2L-Cysteine
Appl. Environ. Microbiol., 64, 1607, 1998;
FEBS Lett., 452, 323, 1999;
FEMS Microbiol. Lett., 179, 453, 1999;
J. Biochem., 136, 629, 2004;
FEMS Microbiol. Lett., 255, 156, 2006;
Protein Eng. Des. Sel., 19, 163, 2006 etc.
Cys transporter
Export
Cys desulfhydrase
Enhance Cys transport
Degradation (NH3, H2S, Pyr.)
Weaken Cys degradation
MINI-REVIEW
Appl. Environ. Microbiol., 72, 4735, 2006;
Appl. Microbiol. Biotechnol., in press.
FEMS Microbiol. Lett., 217, 103, 2002;
Appl. Microbiol. Biotechnol., 62, 239, 2003;
Appl. Environ. Microbiol., 71, 4149, 2005 etc.
Appl. Microbiol. Biotechnol., 73, 48, 2006
Real Scientists !!
Fukui Pref. Univ. (1995-2006)
Shin-ichiro Kobayashi
Chitose Kobayashi
Naoki Awano
Akemi Kohdoh
Tomohiro Oikawa
Keiko Haisa
Mizue Yamazaki
Yutaka Haitani
Hiroyuki Yamazawa
Kyoko Inubushi
Eri Maeda
Dr. Masaaki Noji
Dr. Kazuki Saito
(Chiba Univ.)
Dr. Kunihiko Nishino
Dr. Akihito Yamaguchi
(Osaka Univ.)
Dr. Hirotada Mori
(NAIST)
Dr. Masaru Wada
(Fukui Pref. Univ.)
Dr. Shigeru Nakamori
(Fukui Pref. Univ.)
Ajinomoto Co., Inc.
NAIST (2006-)
Natthawut Wiriyathanawudhiwong
Zhao-Di Li
Dr. Iwao Ohtsu
大腸菌 SAT とシステイン生産の研究の流れ
研究者
研究概要
SATの
フィードバック阻害
システイン生産量
(mg/L)
Kredich
(1983)
Cys による制御の証明
(野生株)
感受性
Denk et al.
(1987)
SAT・一次構造の決定
Met256Ile 変異株の分離
感受性低下
30
Nakamori et al.
(1998)
Met256X の構築
Cys 分解能低下株
感受性低下
600 〜 800
Takagi et al.
(1999)
PCR ランダム変異の導入
Cys 分解能低下株
感受性さらに低下
シロイヌナズナ SAT
遺伝子の導入
非感受性
本研究
0
〜 1,000
?
シロイヌナズナ SAT を用いたシステイン生合成系の強化
Construction of mutant SATs
E. coli chromosome
PCR
pBluescript
2.9 kb
EcoRV
cysE (1.2 kb)
Ampr
pCE
4.1 kb
＜Site-directed mutagenesis＞
Wild-type cysE
Primer for introducing mutation
(Met256X)
PCR
Ampr
pCEX
4.1 kb
Mutant cysE
Selection of the Cys-overproducing strains
Transformants expressing
the mutant SAT gene
Replica
E. coli JM39 (the Cys auxotroph)
M9 agar plates + Amp
Halo formation of the Cys auxotroph
Cys-overproducing strains
25 mutants → the DNA sequence
Amino acid and DNA substitutions in the E. coli SAT
Mutant
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Amino acid substitution (Base substitution)
E7V (A→ T)
E7D (A→ T)
N12I (A→ T)
N12I (A→ T)
A17D (C→ A)
T19A (A→ G)
E24K (G→ A)
S29C (A→ T)
N40S (A→ G)
M48V (A→ G)
N51K (C→ A)
E68V (A→ T)
W119X (G→ A)
A127T (G→ A)
V138M (G→ A)
E166G (A→ G)
T167K (C→ A)
D173N (G→ A)
D173G (A→ G)
M201R (T→ G)
M201T (T→ C)
Q228P (A→ C)
P252R (C→ G)
S253L (C→ T)
M256V (A→ G)
L27P (T →C)
F131L (T →A)
S43R (T→A)
P232L (C →T)
R197H (G→A)
Q258P (A →C)
C23W (T→G)
L36F (C →T)
L120W (T→G)
R91H (G→A)
V130G (T→G)
M201V (A→G)
G270R (G→A)
H233Y (C→T)
D271G (A →G)
E. coli CD (2)
Wild-type
vector
metC disruptant
vector
+ metC
( 1 ) TNase (the tnaA product)
( 2 ) CBL (the metC product)
CBL (the metC product) is one of the E. coli CDs.
Construction of the CD gene-disruptant
Ampr
pEL3 Δ-X
A
B
A B
Ampr
ori (ts)
ori (ts)
A
B
X
42℃, LB medium + Amp
Homologous recombination
A
B
Ampr
ori (ts)
A B
X
X
37℃
LB medium
E. coli chromosome
A
E. coli chromosome
B
Plasmid deletion → Amp-sensitive
・Construct the multiple CD gene disruptant
・Check the disruption by PCR and CD activity staining
Pye et al., J. Biol. Chem., 279, 40729-40736 (2004)
cysM 遺伝子産物・OASS-B について
Cys 生合成経路において、O-acetylserine と S2- から Cys を合成する
酵素 O-acetylserine sulfhydlase-A (OASS-A) のアイソマーと推定されて
いるが、その機能解析は全く行われていなかった
L-Serine + Acetyl-CoA
SAT
O-Acetylserine
OASS-A
SO4 2(external)
遺伝子名
遺伝子の長さ (bp)
タンパク質名
OASS-B !?
972
912
(OASS-A)
(OASS-B)
323
303
2-
ホモロジー (アミノ酸)
L-Cysteine
機能
H2O
cysM
O-acetylserine sulfhydlase-A O-acetylserine sulfhydlase-B
アミノ酸の長さ (aa)
S
cysK
CD
発現制御
38%一致, 53%相似
Cys合成
Cys合成 !?
CD !?
CD
CysBとN-Acetylserine
による正の制御
CysBとN-Acetylserine
による制御!?
ダイマーを形成
硫黄取り込みパ−ミアーゼと
クラスターを形成
Methionine
degradation
その他
SATとコンプレックス形成
Cys 分解能低下株の tnaA 領域 DNAシーケンス解析
野生株
Cys
(mM)
0
10
Cys 分解能
低下株
0
10
TNase
CBL
+1
P
tnaC
tnaA
P : プロモーター
+1 : 転写開始点
tnaC : リーダーペプチド
tnaA : TNase ORF
変異点なし !!
転写調節因子に変
＜bcr 産物の排出メカニズム＞
bcr 産物：MF 型トランスポーターで、bicyclomycin 耐性に関与
排出機構はプロトン濃度勾配による能動輸送
Cys 取込み活性（nmol/mg
dcw）
アンカプラーで活性が阻害
（carbonylcyanide m-chlorophenylhydrazone; CCCP）
＜取込み活性＞
2.5
CCCP の添加により、
取込み活性が減少
100
1.5
ベクター（＋CCCP）
1.0
bcr（-CCCP）
0.5
bcr（＋CCCP）
10
20
時間 （min）
30
Cys 排出率（%）
ベクター（-CCCP）
2.0
0
＜取込み活性＞
＜排出率＞
80
bcr（-CCCP）
60
ベクター（-CCCP）
ベクター（＋CCCP）
40
20
bcr（＋CCCP）
0
10
20
時間 （min）
30
⇒未知の取込み系を阻害？
＜排出率＞
CCCP の添加により、bcr
高発現株で、排出率が減少
⇒bcr 産物の排出能を阻害
bcr 産物のCys 排出機構は、プロトン濃度勾配による能動輸送
＜bcr産物の基質特異性の解析＞
＜Cys 排出率＞
Cys 排出率
（%）
100
80
bcr 高発現株
60
40
ベクターのみ
20
0
Cys 同様に、他のアミノ酸について
排出率を測定
10
20
30
時間 （min）
＜使用アミノ酸
＞
親水性アミノ酸：
Pro, Ser
疎水性アミノ酸： Leu, Val
酸性アミノ酸 ： Glu
塩基性アミノ酸： Arg
含硫アミノ酸 ： Met
bcr 高発現株、ベクター導入株
でアミノ酸排出率に差はなかっ
た
アミノ酸の性質、構造に関係なくCysを特異的に排出
Fig. 1
A
L
+ 15 mM Cys
C
tolC
BW25113
BW25113 (pLS219)
(pLSTolC)
tolC
(pLS219)
(pLSTolC)
B
250 K
150 K
100 K
75 K
L
BW25113 (pCA24N)
+ 15 mM Cys
50 K
TolC
LamB
37 K
OmpF+C
OmpA
(pTolC)
tolC
(pCA24N)
(pTolC)
YncD
Fig. 2
A
B
L
TolC
AcrA
H+
AcrB
BW25113
ΔtolC
ΔacrA
ΔacrB
ΔacrE
ΔacrF
ΔemrA
ΔemrB
ΔmacA
ΔmacB
+ 10 mM Cys
Fig. 3
A
BW25113
ΔtolC
ΔompA
ΔompC
ΔompF
ΔompT
ΔompX
L
+ 10 mM Cys
B
ΔtolC (pCA24N)
(pTolC)
(pOmpA)
(pOmpC)
(pOmpF)
(pOmpT)
(pOmpX)
L
+ 10 mM Cys
Fig. 4
B
3.5
3.5
3
3
2.5
2.5
Growth (OD660)
Growth (OD660)
A
2
1.5
2
1.5
1
1
0.5
0.5
0
0
0
5
10
15
Culture time (h)
20
25
0
5
10
15
Culture time (h)
20
25
Fig. 5
B
6
40
Growth (OD660)
30
4
25
3
20
15
2
10
1
5
0
0
0
20
Culture time (h)
40
Concn of glucose (g/liter)
35
5
Concn of L-cysteine plus L-cystine (mg/liter)
A
150
120
90
60
30
0
0
20
Culture time (h)
40
Fig. 6
B
Concn of L-cysteine plus L-cystine (mg/liter)
A
5
Growth (OD660)
4
3
2
1
1250
1000
750
500
250
0
0
0
20
Culture time (h)
40
0
20
Culture time (h)
40
Fig. 7
+ DTT
LB
BW25113 / pCA24N
ΔdsbA / pCA24N
ΔtolC / pCA24N
ΔtolC / pTolC
ΔtolC / pDsbA
5 mM
10 mM