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 SciencesNara Institute of Science and Technology

Amino acid

Microbial production of amino acids

Microorganism Market (tons/y)

L-GlutamateL-LysineL-PhenylalanineL-ThreonineL-GlutamineL-Arginine

C. glutamicumC. glutamicumC. glutamicum / E. coliE. coliC. glutamicumC. glutamicum

1,000,000250,000

8,0004,0001,3001,200

(L-Cysteine)(DL-Methionine)

(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

Environmental issues

Direct fermentation of glucose

A variety of applications

Increase of demand

Pseudomonas thiazoliniphilum ：　　 DL-ATC (2-aminothiazoline-4-carboxylic acid) 　 → →　 L-CysteineCysteine desulfhydrase ：　　 L-ClCH2CHNH2COOH + Na2S + H2O → L-Cysteine + NaCl + NaOH

Metabolism and its regulation of Cys in E. coli

L-Serine + Acetyl-CoA

O-Acetyl-L-serine

SO42- ( external )

S2-

L-Cysteine

L-Methionine

CD

SAT

OASS

SO42-

SO32-

APS

PAPS

Glutathione

degradation( Pyruvate, NH3, H2S )

・ Feedback inhibition of SAT by Cys

・ Cys degradation catalyzed by CD

・ No oversynthesis

・ No accumulation

Feedbackinhibition

In E. coli cells…

SH

CH2

H2N-C-H

COOH

L-Cysteine (Cys)

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)

Direct fermentation of Cys from glucose

Glucose Serine O-Acetylserine Cysteine

Acetyl-CoA H2SMethionine

Degradation

Serine acetyltransferase(SAT)

Cysteine desulfhydrase(CD)

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

Serine acetyltransferase (SAT) of E. coli

＜ Feedback inhibition of SAT activity by Cys ＞

Ser

Acetyl-CoA

Cysteine(endproduct)

SAT(inactive)

SAT

Activesite

Allostericsite

＋Ser

Acetyl-CoA

Enzyme Substrate ES-complex

0

25

50

75

100

Rel

ativ

e ac

tivi

ty (

%)

L-Cysteine conc. (M)

25 50 75 100

AlteredN C

Met256X

N CTruncated

Wild-typeN C

Met256

＜ site-directed mutagenesis by PCR ＞

Amino acid substitution of Met256 of the E. coli SAT

Analysis of feedback inhibition and Cys productivity <1st PCR> A

256All +

B

256All-

<2nd PCR>

Ligation

B

A

＜ mixed primers ＞5’-AATGGAT GGG GACCAGC-3’

CCC

TTT AAA

＜ Strain ＞ E. coli JM39-8 (SAT-deficient and Cys non-utilizing)

＜ Medium ＞ Cys production medium (1L) (pH 7.0)

GlucoseNa2S2O3NH4Cl

FeSO4・7H2O

MnCl2・4H2O

CaCO3

15 g

10 g

0.01 g0.01 g

0.1 g each

20 g

30 g

Production of cysteine plus cystine

＜ Cultivation ＞ 30℃, Sakaguchi-flask, shaking

＜ Determination of cysteine + cystine ＞

Bioasay (Pediococcus acidilactici IFO3076)

KH2PO4MgSO4・7H2O

2 g1 g

Gly, L-Ile, L-Leu, L-Met

Cys production by expression of the mutant SATs

Cys overproduction was achieved by expressing the mutant SAT.

CySH + Cys (mg/L)

pCE

M256A

M256R

M256D

M256E

M256S

M256W

M256V

M256Stop

0.5

24.1

32.1

24.2

17.3

27.0

18.6

25.6

31.3

Activity remaining in the presence of

100 M cysteine (%)

Amino acidresidue at

position 256

ND

790 ± 380

600 ± 80

580 ± 50

710 ± 270

610 ± 40

610 ± 70

560 ± 70

730 ± 110

Met (Wild-type)

Ala

Arg

Asp

Glu

Ser

Trp

Val

-*

*, termination codon at position 256

Plasmid

., Nakamori et al Appl. Environ. Microbiol., 64, 1607-1611 (1998)

EcoRI XbaI

pUC19

pHC Transformation of E. coli JM39-8

＜ 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

E. coli wild-type SAT gene (cysE)

Error-prone PCR

Error-prone PCR random mutagenesis into E. coli SAT

EcoRI XbaI

EcoRI XbaI

pCE

Several amino acid residues other than Met256 are responsible for t

he feedback inhibition by Cys and the overproduction of Cys.

PlasmidCySH + Cys

(mg/L)

pCE

pCE M256A

pHC 6

pHC 7

pHC 8

pHC 10

pHC 11

pHC 12

pHC 13

ND

790

210 ± 170

330 ± 70

260 ± 50

990 ± 200

740 ± 120

50 ± 20

960 ± 460

0.9

24.1

51.9

78.2

37.1

20.9

16.3

33.2

28.9

Activity remaining in the presence of

100 M cysteine (%)

Amino acid substitution

M256I

N51K, R91H, H233Y

E166G, M201V

T167K

M201R

M201T

P252R

S253L

Takagi et al., FEBS Lett., 452, 323-327 (1999)

Characteristics of the E. coli mutant SATs

SAT-m SAT-p wild-type SAT

SAT-p

SAT-m

SAT-c

Localization Feedback inhibition

Chloroplast

Mitochondria

Cytoplasm

Insensitive

Insensitive

Sensitive

SATs of Arabidopsis thaliana

A. thaliana

SAT

The E. coli cysE promoter

pEAS-m, pEAS-p

E. coli

The A. thaliana SAT-m or SAT-p gene

＜ Expression plasmids for the SAT cDNA ＞ ＜ Western analysis for the SAT expression ＞

The A. thaliana SATs are expressed in E. coli cells.

Ampr

cysEp

(Noji et al., J. Biol. Chem., 273, 32739-32745, 1998)

0

pEAS-m pCEpCEM256IpEAS-p

plasmid

SAT A. thaliana SAT-m

A. thaliana SAT-p

E. coli Met256Ile

E. coli wild-type

27.9 21.3 88.0 2,273SAT activity

(mU/min/mg)

Relative activity (%) for L-cysteine added (M)

10

100

100100

100

100100

100

10088

24

1001.5

ND

Comparison of catalytic properties of recombinant SATs

The A. thaliana SATs were insensitive to feedback inhibition.

ND : Not detected.

SAT

SAT-m SAT-p E. coli Met256Ile

( A ) Growth (OD562)

( B ) L-Cysteine produced (mg/L)

( B ) / ( A )

0.91 ± 0.02 0.77 ± 0.10 0.64 ± 0.12

1,580 ± 100 1,660 ± 200 870 ± 160

1,750 ± 100 2,140 ± 200 1,360 ± 70

Cys production by recombinant strains

Expression of two cDNAs encoding SAT-m and SAT-p in E. coli cells significantly increased the Cys productivity.

Takagi et al., FEMS Microbiol. Lett., 179, 453-459 (1999)

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)

Enhancement of Cys biosynthetic activity

Kai et al., Prot. Eng. Des. Sel., 19, 163-167 (2006)

Arg89-Asp96

Ser

1) Enhancing the biosynthetic activity Functional improvement of serine acetyltransferase (SAT)

2) Weakening the degradation pathway Identification and gene disruption of Cys desulfhydrase (CD)

Direct fermentation of Cys from glucose

Glucose Serine O-Acetylserine Cysteine

Acetyl-CoA H2SMethionine

Degradation

Serine acetyltransferase(SAT)

Cysteine desulfhydrase(CD)

A reaction catalyzed by Cysteine Desulfhydrase (CD)

Cys degradation pathway is unknown ??

H2S is generated during fermentation !!

Cys degradation is occurred !!

Analysis of Cys degradation pathway

L-Cysteine

CD

CH3

C=O

COOH

+ H2S+NH3

PyruvateSH

CH2

H2N-C-H

COOH

CysCD

H2S + BiCl3

BiSO4

=

Black bands

CD activity staining

Identification of the E. coli CDs by activity staining

At least, five CD proteins are newly detected in E. coli.

( 1 )

( 2 )

( 4 )( 5 )

( 3 )

Native-PAGE

E. coli CD (1)

( 1 ) TNase

( 2 ) ?

Purified sample 　　　 MENFKHLPEPFRIRV ・・・E. coli Tryptophanase (TNase) 　 MENFKHLPEPFRIRV ・・・

1 15

Determine the N-terminus sequence

Wild-type tnaA-disruptant

+ tnaAVectorVector L-Tryptophan → Indole + Pyruvate + NH3

A reaction catalyzed by TNase (the tanA product)

TNase (the tnaA product) is one of the E. coli CDs.

( 1 )

E. coli CD (2)

COOH

H2N-C-H

CH2

H2C S CH2

H2N-C-H

COOHCBL

Cystathionine HomocysteinePyruvate

NH3

CH3

C=O

COOH

+ +

COOH

CH2

CH2

H2N-C-H

SH

L-Cysteine

O-Succinyl-homoserine

L-Methionine

＜ Cystathionine -lyase (CBL; the metC product) reaction ＞

L-Cysteine

CDH2S

SH

CH2

H2N-C-H

COOH

Pyruvate

NH3

CH3

C=O

COOH

+ +

＜ CD reaction ＞

The CD and CBL reactions are the same. CBL accepts Cys as a substrate in vitro.

CBL functions as a CD ?

( 2 )

( 1 ) TNase

E. coli CD (3) - (5)

Use of an E. coli library containing 4,388 kinds of open reading frame (ORF)

X

pCN24-X

: ORF (total 4,388)

: lacZ promoter

X

CmrCD activity staining

( 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)

Vector + cysK + cysM + malY

OASS (-A, -B) and MalY protein are identified as the E. coli CDs.

lacZp

( 1 )

( 2 )

( 4 )( 5 )

( 3 )

List 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 !?

Five CD proteins were identified in E. coli…

total CD activity (mU/mg)Genotype

20.6

15.7

15.0

Wild-type

tnaA

metC

cysK

cysM

malY

tnaA metC

tnaA metC cysM malY

tnaA metC cysK cysM malY

18.2

17.9

15.3

9.1

8.7

Total CD activity

9.6

・ Total CD activities of all mutants were lower than wild-type.

・ Even the quintet mutant still had a low level of CD activity.

0

200

400

600

800

1000

1200

1400

1600

0 24 48 72 96Culture time (hr)

Cys

tein

e p

rod

uct

ivit

y(m

g / L

)

Cys production in the CD gene disruptants

Wild-type

tnaA mutsnt

malY mutant

metC mutsnt

4 genes mutant

cysM mutsnt

・ 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 CysCys inhibits the growth of E. coli cells.

LB + 30 mM Cys

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 6 24123 9 15 2118Culture time (hr)

Gro

wth

(O

D61

0) Wild-typetnaA mutant

malY mutant

metC mutant

cysK mutant

cysM mutant

・ The tnaA disruptant was significantly inhibited. ・ TNase is a key enzyme in Cys degradation in E. coli ??

23s rRNA

16s rRNA

Native-PAGE

0 10

SDS-PAGE Northern blotting

0 0 1010 Cys (mM)

Cys (mM)

Cys (mM)

1.7kb

TNase induction by Cys

･ TNase synthesis is induced by Cys.･ TNase contributes mainly to Cys degradation.

・・

・

・・

67

43

30

20

94(kDa)

1) Identification of the E. coli CDs

2) Construction of the CD gene disruptants 　 The gene disruption is significantly effective for Cys production.

3) TNase contributes primarily to Cys degradation.

Identification and gene disruption 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 !?

Genome information-based

Identification and analysis of the Cys transporter

Enhancing the export system

L-CysteineGlucose

Yamada et al., Appl. Environ. Microbiol., 72, 4735-4742 (2006)

Natthawut et al., Appl. Microbiol. Biotechnol., in press.

Poster

Bcr

TolC

・ Identification and analysis of Cys transporter

・ Evaluation of Cys transporter on Cys productivity

Enhancement of Cys export system

Growth inhibition

Cys overproducer

Cys export

Improvement of Cys productivity ?

Mutant SAT gene

CD gene

Mutant SAT gene

CD gene

Cys transporter gene

Imbalance of cellular

oxidation-reduction state

Cys accumulation

Screening of Cys transporter

E. coli cells with a lower level of CD activity would be much more sensitive to Cys due to Cys accumulation.

The growth of E. coli cells is inhibited by excess Cys (30 mM).

The transporter that exports Cys and reverses the growth inhibition

+ transporter gene

pUC118-X

Transporter X

32 putative drug transporter genes

Screening of Cys transporter

tnaA disruptant

Wild-type

Gro

wth

(O

D61

0)

Culture time (hr)

naA disruptant

Px

Ampr

Genes that reversed the growth inhibition of tnaA disruptant by Cys:

acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH

0

1

6

5

4

3

2

0 6 24 18

Gro

wth

(O

D61

0)

Culture time (hr)

emrAB

Wild-type

tnaA disruptant

acrD, acrEF, bcr, cusA, emrKY, ybjYZ, yojIH

12

Intracellular Cys contents of E. coli cells

Cys （ 30 mM ）

Cys

Genes that decreased intracellular Cys level of tnaA disruptant:

acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH

Transporter X

tnaA disruptant Wild-type

Intr

acel

lula

r C

ys c

onte

nt

(mg/

L/O

D61

0)

2

emrAB emrKY yojIH acrEF bcr cusA acrD ybjYZvector

1

0

vector

3

pUC118-X

Ampr

List of Cys exporter candidates

bcr

emrAB

emrKY

acrEF

acrD

cusA

ybjYZ

yojIH

FunctionGene

Bicyclomycin resistance 　

Multidrug resistance

Multidrug resistance

Acriflavin resistance

Acriflavin resistance, 　　　　 Aminoglycosides efflux

Putative copper transporter

Putative transporter

Putative transporter

No one knows whether these genes are involved in amino acid export.

＜ Cys transport assay ＞

+ [35S]-Cys

Remaining labeled Cys content

⇒ Cys export rate

ydeD

30

vector

ybjYZ

bcr

0

1.0

2.0

10 20

Time (min)

0

Cys

up

tak

e （

nm

ol/m

g ce

ll w

t）

acrEF emrAB vector

100

0

80

60

40

20

10 3020

Time (min)

bcr

ydeD

0

Cys

exp

ort

(%)

Reduced uptake ：bcr, ybjYZ,(ydeD)

Increased export ：

bcr, acrEF, emrAB, (ydeD)

Bcr overexpression promotes Cys export in E. coli cells.

＜ Cys uptake ＞

＜ Cys export ＞

＜ Cys uptake↓ 、 Cys export↑ ＞

bcr, ydeD

Cell suspension

Cys uptake activity

Bcr overexpression contributes to Cys production.

0

400

200

1000

800

600

24 7248

Culture time (h)

+ bcr

+ vector (pUC118)

Con

cen

trat

ion

of

Cys

（m

g/L

/OD

562）

pUC118-bcr

bcr

Enhancing Cys synthesis

Enhancing Cys export

Cys

Cys

pACYC-M256I

Mutant SAT gene

Pbcr

Cmr

Am

p r

Cys production by E. coli cells expressing bcr

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

32 putative drug transporter genes

Functional analysis (transcriptional regulation, physiological role)

Improved function (export activity, substrate specificity)

Molecular breeding of Cys overproducer

Growth inhibition, Intracellular Cys level

Export activity, Specificity, Cys production

Identify the Bcr protein as a Cys transporter

L-Serine Acetyl-CoA +

O-Acetylserine

Ser acetyltransferase SO42- (external)

S2-

L-Cysteine

Cys desulfhydrase

Degradation (NH3, H2S, Pyr.)

Cys transporterExport

Activated by OAS

Enhance Cys transportEnhance Cys transport

Weaken Cys degradationWeaken Cys degradation

Enhance Cys biosynthesis

Enhance Cys biosynthesis

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.

FEMS Microbiol. Lett., 217, 103, 2002;Appl. Microbiol. Biotechnol., 62, 239, 2003; Appl. Environ. Microbiol., 71, 4149, 2005 etc.

Appl. Environ. Microbiol., 72, 4735, 2006;Appl. Microbiol. Biotechnol., in press.

Feedback inhibition by Cys

Appl. Microbiol. Biotechnol., 73, 48, 2006MINI-REVIEW

Dr. Masaru Wada(Fukui Pref. Univ.)

Dr. Shigeru Nakamori(Fukui Pref. Univ.)

Real Scientists !!Real Scientists !!

Dr. Hirotada Mori(NAIST)

Fukui Pref. Univ. (1995-2006)Shin-ichiro KobayashiChitose KobayashiNaoki AwanoAkemi KohdohTomohiro OikawaKeiko HaisaMizue YamazakiYutaka HaitaniHiroyuki YamazawaKyoko InubushiEri Maeda

NAIST (2006-)Natthawut WiriyathanawudhiwongZhao-Di LiDr. Iwao Ohtsu

Dr. Kunihiko NishinoDr. Akihito Yamaguchi(Osaka Univ.)

Dr. Masaaki NojiDr. Kazuki Saito(Chiba Univ.)

Ajinomoto Co., Inc.

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) 感受性 0

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)

本研究 非感受性 ?

Cys による制御の証明 ( 野生株 )

感受性低下 600 〜 800Met256X の構築Cys 分解能低下株

シロイヌナズナ SAT を用いたシステイン生合成系の強化

シロイヌナズナ SAT遺伝子の導入

Takagi et al.

(1999)感受性さらに低下 〜 1,000

PCR ランダム変異の導入Cys 分解能低下株

E. coli chromosome

PCR

cysE (1.2 kb)

Wild-type cysEAmpr

Primer for introducing mutation

(Met256X) PCR

＜ Site-directed mutagenesis ＞

EcoRVpBluescript2.9 kb

Mutant cysE

Construction of mutant SATs

Ampr

pCE4.1 kb

pCEX4.1 kb

Transformants expressing the mutant SAT gene

E. coli JM39 (the Cys auxotroph)Replica

M9 agar plates + Amp

Halo formation of the Cys auxotroph

Selection of the Cys-overproducing strains

Cys-overproducing strains25 mutants → the DNA sequence

Amino acid and DNA substitutions in the E. coli SAT

1 E7V (A→ T) L27P (T →C) S43R (T →A) D271G (A →G) 2 E7D (A→ T) F131L (T →A) P232L (C →T) 3 N12I (A→ T) 4 N12I (A→ T) R197H (G →A) 5 A17D (C→ A) Q258P (A →C) 6 T19A (A→ G) C23W (T →G) 7 E24K (G→ A) L36F (C →T) 8 S29C (A→ T) 9 N40S (A→ G) L120W (T →G)10 M48V (A→ G)11 N51K (C→ A) R91H (G →A) H233Y (C →T)12 E68V (A→ T)13 W119X (G→ A)14 A127T (G→ A) V130G (T →G)15 V138M (G→ A)16 E166G (A→ G) M201V (A →G)17 T167K (C→ A)18 D173N (G→ A)19 D173G (A→ G) G270R (G →A)20 M201R (T→ G)21 M201T (T→ C)22 Q228P (A→ C)23 P252R (C→ G)24 S253L (C→ T)25 M256V (A→ G)

Mutant Amino acid substitution (Base substitution)

Wild-type metC disruptant

+ metCvectorvector

( 1 ) TNase (the tnaA product)

( 2 ) CBL (the metC product)

CBL (the metC product) is one of the E. coli CDs.

E. coli CD (2)

X

Ampr

Ampr

Ampr

ori (ts) ori (ts)

ori (ts)X

X

LB medium

37℃

Homologous recombination

42℃, LB medium + Amp

pEL3 Δ-X

Construction of the CD gene-disruptant

E. coli chromosome Plasmid deletion → Amp-sensitive

A B A B

B B

B

B

A A

A

A

E. coli chromosome

・ 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

L-Serine Acetyl-CoA +

O-Acetylserine 　 SO4 2-

(external)

S 2-

L-Cysteine

Methionine

H2O CD

degradation

SAT

OASS-A OASS-B !?

アミノ酸の長さ (aa)

遺伝子名

遺伝子の長さ (bp)

タンパク質名

cysK cysM

972 912

O-acetylserine sulfhydlase-A

　　 (OASS-A)O-acetylserine sulfhydlase-B　　 (OASS-B)

323 303

ホモロジー ( アミノ酸 )

機能 Cys 合成CD !?

Cys 合成 !?

CD

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

38% 一致 , 53% 相似

発現制御 CysB と N-Acetylserineによる正の制御

CysB と N-Acetylserineによる制御 !?

その他 SAT とコンプレックス形成ダイマーを形成 硫黄取り込みパ−ミアーゼと

クラスターを形成

Cys 分解能低下株の tnaA 領域 DNA シーケンス解析

tnaC tnaAP+1

変異点なし !! 転写調節因子に変異 !?

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

TNase

野生株Cys 分解能

低下株

CBL

0 10 0 10 Cys(mM)

＜ 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

アンカプラーで活性が阻害

排出機構はプロトン濃度勾配による能動輸送

CCCP の添加により、取込み活性が減少

0

1.0

0.5

2.5

2.0

1.5

10 3020

時間 （ min）

Cys 取込

み活性

（nmol

/mg dc

w）

＜取込み活性＞

⇒未知の取込み系を阻害？

0

40

20

100

80

60

10 3020

Cys 排

出率（

%）

＜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

＜取込み活性＞

＜排出率＞

（ carbonylcyanide m-chlorophenylhydrazone; CCCP ）

ベクター（＋ CCCP）

bcr （ -CCCP ）

ベクター（ -CCCP）

bcr （＋ CCCP ）

ベクター（ -CCCP）

bcr （＋ CCCP ）

bcr （ -CCCP ）

ベクター（＋ CCCP）

時間 （ min）

＜ bcr 産物の基質特異性の解析＞

ベクターのみ

bcr 高発現株

0

40

20

100

80

60

10 3020

Cys 排出率

（%）

＜ Cys 排出率＞

時間 （ min）

Cys 同様に、他のアミノ酸について排出率を測定

＜使用アミノ酸＞

アミノ酸の性質、構造に関係なく Cys を特異的に排出

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

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

含硫アミノ酸　： Met

Fig. 1

OmpF+COmpA

TolCLamB

YncD

37 K

50 K

75 K100 K

150 K250 K

tolC BW25113

pLS2

19

pLST

olC

pLS2

19

pLST

olC

A

B

CL + 15 mM Cys

(pLS219)

(pLSTolC)

BW25113

(pLS219)

(pLSTolC)

tolC

L + 15 mM Cys

(pCA24N)

(pTolC)

BW25113

(pCA24N)

(pTolC)

tolC

Fig. 2

BW25113ΔtolCΔacrAΔacrBΔacrEΔacrFΔemrAΔemrBΔmacAΔmacB

L + 10 mM CysB

TolC

AcrB

H+AcrA

A

Fig. 3

L + 10 mM CysBW25113ΔtolCΔompAΔompCΔompFΔompTΔompX

L + 10 mM CysΔtolC (pCA24N)

(pTolC)(pOmpA)(pOmpC)(pOmpF)(pOmpT)(pOmpX)

A B

Fig. 4

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25

Culture time (h)

Gro

wth

(O

D66

0)

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25

Culture time (h)G

row

th (

OD

660)

A B

Fig. 5

0

30

60

90

120

150

0 20 40Culture time (h)

Con

cn o

f L

-cys

tein

e pl

us L

-cys

tine

(m

g/lit

er)

A B

0

1

2

3

4

5

6

0 20 40

0

5

10

15

20

25

30

35

40G

row

th (

OD

660)

Con

cn o

f gl

ucos

e (

g/lit

er)

Culture time (h)

Fig. 6

0

250

500

750

1000

1250

0 20 40

Con

cn o

f L

-cys

tein

e pl

us L

-cys

tine

(m

g/lit

er)

Culture time (h)

A B

0

1

2

3

4

5

0 20 40

Gro

wth

(O

D66

0)

Culture time (h)

Fig. 7

BW25113 / pCA24N

ΔdsbA / pCA24N

ΔtolC / pCA24N

ΔtolC / pTolC

ΔtolC / pDsbA

LB 5 mM 10 mM

+ DTT