1. 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

2. Amino acid Microbial production of amino acids MicroorganismMarket (tons/y) L -Glutamate L -Lysine L -Phenylalanine L -Threonine L -Glutamine L -Arginine C. glutamicum C. glutamicum / E. coli E. coli C. glutamicum 1,000,000 250,000 8,000 4,000 1,300 1,200 ( L -Cysteine) ( DL -Methionine) (1,500) (350,000) No direct-fermentation process for sulfur-containing amino acids (Cys, Met) has yet been achieved.

3. 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 -Cysteine Cysteine desulfhydrase ： L -ClCH 2 CHNH 2 COOH + Na 2 S + H 2 O → L -Cysteine + NaCl + NaOH

4. Metabolism and its regulation of Cys in E. coli L -Serine + Acetyl-CoA O -Acetyl- L -serine SO 4 2- ( external ) S 2- L-Cysteine L-Methionine CD SAT OASS SO 4 2- SO 3 2- APS PAPS Glutathione degradation ( Pyruvate, NH 3, H 2 S ) ・ Feedback inhibition of SAT by Cys ・ Cys degradation catalyzed by CD ・ No oversynthesis ・ No accumulation Feedback inhibition In E. coli cells… SH CH 2 H 2 N-C-H COOH L -Cysteine (Cys)

5. 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 GlucoseSerineO-Acetylserine Cysteine Acetyl-CoA H2SH2S Methionine Degradation Serine acetyltransferase (SAT) Cysteine desulfhydrase (CD)

6. 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 Active site Allosteric site ＋ Ser Acetyl-CoA EnzymeSubstrateES-complex 0 25 50 75 100 Relative activity (%) L-Cysteine conc. (  M) 255075100

7. Altered N C Met256X NC Truncated Wild-type NC Met256 ＜ site-directed mutagenesis by PCR ＞ Amino acid substitution of Met256 of the E. coli SAT Analysis of feedback inhibition and Cys productivity A 256All + B 256All- Ligation B A ＜ mixed primers ＞ 5’-AATGGATGGG GACCAGC-3’ CCC TTT AAA

8. ＜ Strain ＞ E. coli JM39-8 (SAT-deficient and Cys non-utilizing) ＜ Medium ＞ Cys production medium (1L) (pH 7.0) Glucose Na 2 S 2 O 3 NH 4 Cl FeSO 4 ・ 7H 2 O MnCl 2 ・ 4H 2 O CaCO 3 15 g 10 g 0.01 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) KH 2 PO 4 MgSO 4 ・ 7H 2 O 2 g 1 g Gly, L -Ile, L -Leu, L -Met

9. 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 acid residue 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., Nakamoriet alAppl. Environ. Microbiol., 64, 1607-1611 (1998)

10. EcoRIXbaI pUC19 pHC Transformation of E. coli JM39-8 ＜ Reaction mixture ＞ 10 mM Tris-HCl ( pH 8.3 ) 50 mM KCl 1.5 mM MgCl 2 0.01 M  -mercaptoethanol 10% DMSO 0.5 mM MnCl 2 0.5  M forward primer 0.5  M reverse primer 0.2mM dATP 1mM dGTP, dCTP, dTTP each 1UTaq DNA polymerase E. coli wild-type SAT gene (cysE) Error-prone PCR Error-prone PCR random mutagenesis into E. coli SAT EcoRIXbaI EcoRIXbaI pCE

11. Several amino acid residues other than Met256 are responsible for the feedback inhibition by Cys and the overproduction of Cys. Plasmid CySH + 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 Takagiet al.,FEBS Lett., 452, 323-327 (1999) Characteristics of the E. coli mutant SATs

12. SAT-m SAT-pwild-type SAT SAT-p SAT-m SAT-c LocalizationFeedback inhibition Chloroplast Mitochondria Cytoplasm Insensitive Sensitive SATs of Arabidopsis thaliana ・大腸菌でシロイヌナズナ SAT は発現・機能する ・シロイヌナズナの SAT はフィードバック阻害非感受性 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. Amp r cysE p (Nojiet al.,J. Biol. Chem., 273, 32739-32745, 1998)

13. 0 pEAS-mpCEpCEM256IpEAS-p plasmid SAT A. thaliana SAT-m A. thaliana SAT-p E. coli Met256Ile E. coli wild-type 27.921.388.02,273 SAT activity (mU/min/mg) Relative activity (%) for L -cysteine added (  M) 10 100 88 24 100 1.5 ND Comparison of catalytic properties of recombinant SATs The A. thaliana SATs were insensitive to feedback inhibition. ND : Not detected.

14. SAT SAT-m SAT-p E. coli Met256Ile ( A ) Growth (OD 562 ) ( B ) L -Cysteine produced (mg/L) ( B ) / ( A ) 0.91 ± 0.020.77 ± 0.100.64 ± 0.12 1,580 ± 1001,660 ± 200870 ± 160 1,750 ± 1002,140 ± 2001,360 ± 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)

15. 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

16. Kai et al., Prot. Eng. Des. Sel., 19, 163-167 (2006) Arg89-Asp96 Ser

18. 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 GlucoseSerineO-Acetylserine Cysteine Acetyl-CoA H2SH2S Methionine Degradation Serine acetyltransferase (SAT) Cysteine desulfhydrase (CD)

19. A reaction catalyzed by Cysteine Desulfhydrase (CD) Cys degradation pathway is unknown ?? H 2 S is generated during fermentation !! Cys degradation is occurred !! Analysis of Cys degradation pathway L -Cysteine CD CH 3 C=O COOH + H2SH2S + NH 3 Pyruvate SH CH 2 H 2 N-C-H COOH

20. Cys CD H 2 S + BiCl 3 BiSO 4 = 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

21. E. coli CD (1) ( 1 ) TNase ( 2 ) ? Purified sample MENFKHLPEPFRIRV ・・・ E. coli Tryptophanase (TNase) MENFKHLPEPFRIRV ・・・ 1 15 Determine the N-terminus sequence Wild-typetnaA-disruptant + tnaA Vector L -Tryptophan → Indole + Pyruvate + NH 3 A reaction catalyzed by TNase (the tanA product) TNase (the tnaA product) is one of the E. coli CDs. ( 1 )

22. E. coli CD (2) COOH H 2 N-C-H CH 2 H2CH2CS H 2 N-C-H COOH CBL Cystathionine Homocysteine Pyruvate NH 3 CH 3 C=O COOH + + CH 2 H 2 N-C-H SH L -Cysteine O -Succinyl-homoserine L -Methionine ＜ Cystathionine  -lyase (CBL; the metC product) reaction ＞ L -Cysteine CD H2SH2S SH CH 2 H 2 N-C-H COOH Pyruvate NH 3 CH 3 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

23. 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 Cm r CD 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. lacZ p

24. ( 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…

25. 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.

26. 0 200 400 600 800 1000 1200 1400 1600 0 24487296 Culture time (hr) Cysteine productivity(mg / 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.

27. Growth of E. coli cells in the presence of Cys Cys 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 24 12 3 9152118 Culture time (hr) Growth (OD 610 ) Wild-type tnaA 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 ??

28. 23s rRNA 16s rRNA Native-PAGE 0 10 SDS-PAGE Northern blotting 0010 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)

29. 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 !?

30. Genome information-based Identification and analysis of the Cys transporter Enhancing the export system L -Cysteine Glucose Yamada et al., Appl. Environ. Microbiol., 72, 4735-4742 (2006) Natthawut et al., Appl. Microbiol. Biotechnol., in press. Poster Bcr TolC

31. ・ 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

32. 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 Growth (OD 610 ) Culture time (hr) naA disruptant PxPx Amp r

33. 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 Growth (OD 610 ) Culture time (hr) emrAB Wild-type tnaA disruptant acrD, acrEF, bcr, cusA, emrKY, ybjYZ, yojIH 12

34. 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 disruptantWild-type Intracellular Cys content (mg/L/OD 610 ) 2 emrABemrKYyojIHacrEFbcr cusAacrD ybjYZ vector 1 0 3 pUC118-X Amp r

35. List of Cys exporter candidates bcr emrAB emrKY acrEF acrD cusA ybjYZ yojIH FunctionGene Bicyclomycin resistance Multidrug resistance Acriflavin resistance Acriflavin resistance, Aminoglycosides efflux Putative copper transporter Putative transporter No one knows whether these genes are involved in amino acid export.

36. ＜ Cys transport assay ＞ + [ 35 S]-Cys Remaining labeled Cys content ⇒ Cys export rate ydeD 30 vector ybjYZ bcr 0 1.0 2.0 1020 Time (min) 0 Cys uptake （ nmol/mg cell wt ） acrEF emrAB vector 100 0 80 60 40 20 103020 Time (min) bcr ydeD 0 Cys export (%) 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

37. Bcr overexpression contributes to Cys production. 0 400 200 1000 800 600 247248 Culture time (h) + bcr + vector (pUC118) Concentration of Cys （ mg/L/OD 562 ） pUC118-bcr bcr Enhancing Cys synthesis Enhancing Cys export Cys pACYC-M256I Mutant SAT gene P bcr Cm r Amp r Cys production by E. coli cells expressing bcr

38. 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

39. L -SerineAcetyl-CoA + O-Acetylserine Ser acetyltransferase SO 4 2- (external) S2-S2- L -Cysteine Cys desulfhydrase Degradation (NH 3, H 2 S, Pyr.) Cys transporter Export Activated by OAS Enhance Cys transport Weaken Cys degradation 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, 2006 MINI-REVIEW

40. Dr. Masaru Wada (Fukui Pref. Univ.) Dr. Shigeru Nakamori (Fukui Pref. Univ.) Real Scientists !! Dr. Hirotada Mori (NAIST) 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 NAIST (2006-) Natthawut Wiriyathanawudhiwong Zhao-Di Li Dr. Iwao Ohtsu Dr. Kunihiko Nishino Dr. Akihito Yamaguchi (Osaka Univ.) Dr. Masaaki Noji Dr. Kazuki Saito (Chiba Univ.) Ajinomoto Co., Inc.

42. 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 〜 800 Met256X 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

43. E. coli chromosome PCR cysE (1.2 kb) Wild-type cysE Amp r Primer for introducing mutation (Met256X) PCR ＜ Site-directed mutagenesis ＞ EcoRV pBluescript 2.9 kb Mutant cysE Construction of mutant SATs Amp r pCE 4.1 kb pCEX 4.1 kb

44. 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 strains 25 mutants → the DNA sequence

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

46. Wild-typemetC disruptant + metC vector ( 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)

47. X Amp r 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 AB 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

48. Pye et al., J. Biol. Chem., 279, 40729-40736 (2004)

49. cysM 遺伝子産物・ OASS-B について L -SerineAcetyl-CoA + O -Acetylserine SO 4 2- (external) S 2- L -Cysteine Methionine H2OH2O CD degradation SAT OASS-AOASS-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 cysKcysM 972 912 O -acetylserine sulfhydlase-A (OASS-A) O -acetylserine sulfhydlase-B (OASS-B) 323303 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 S 2- 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

50. Cys 分解能低下株の tnaA 領域 DNA シーケンス解析 tnaC tnaA P +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 Cys (mM)

51. ＜ 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 30 20 時間 （ min ） Cys 取込み活性（ nmol/mg dcw ） ＜取込み活性＞ ⇒未知の取込み系を阻害？ 0 40 20 100 80 60 103020 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 ）

52. ＜ 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 103020 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

53. Fig. 1 OmpF+C OmpA TolC LamB YncD 37 K 50 K 75 K 100 K 150 K 250 K  tolC BW25113 pLS219 pLSTolC pLS219 pLSTolC A B C L+ 15 mM Cys (pLS219) (pLSTolC) BW25113 (pLS219) (pLSTolC)  tolC L+ 15 mM Cys (pCA24N) (pTolC) BW25113 (pCA24N) (pTolC)  tolC

54. Fig. 2 BW25113 ΔtolC ΔacrA ΔacrB ΔacrE ΔacrF ΔemrA ΔemrB ΔmacA ΔmacB L + 10 mM Cys B TolC AcrB H+H+ AcrA A

55. Fig. 3 L+ 10 mM Cys BW25113 ΔtolC ΔompA ΔompC ΔompF ΔompT ΔompX L+ 10 mM Cys ΔtolC (pCA24N) (pTolC) (pOmpA) (pOmpC) (pOmpF) (pOmpT) (pOmpX) AB

56. Fig. 4 Culture time (h) Growth (OD 660 ) Culture time (h) Growth (OD 660 ) AB

57. Fig. 5 Culture time (h) Concn of L -cysteine plus L -cystine (mg/liter) AB Growth (OD 660 ) Concn of glucose (g/liter) Culture time (h)

58. Fig. 6 Concn of L -cysteine plus L -cystine (mg/liter) Culture time (h) AB Growth (OD 660 ) Culture time (h)

59. Fig. 7 BW25113 / pCA24N ΔdsbA / pCA24N ΔtolC / pCA24N ΔtolC / pTolC ΔtolC / pDsbA LB5 mM10 mM + DTT