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2008 NAIST-UM (BTI) Synmposium

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.

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2008 NAIST-UM (BTI) Synmposium

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  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. Microbial production of amino acids Amino acid Microorganism Market (tons/y) -Glutamate C. glutamicum 1,000,000 L -Lysine C. glutamicum 250,000 L -Phenylalanine C. glutamicum / E. coli 8,000 L -Threonine E. coli 4,000 L -Glutamine C. glutamicum 1,300 L -Arginine C. glutamicum 1,200 L ( -Cysteine) (1,500) L ( -Methionine) (350,000) DL 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 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

  4. SO42- ( external ) L-Serine + Acetyl-CoA SO42- APS SAT COOH PAPS H2N-C-H O-Acetyl-L-serine SO32- CH2 S2- OASS SH L-Cysteine CD Glutathione degradation ( Pyruvate, NH3, H2S ) L-Methionine Metabolism and its regulation of Cys in E. coli L-Cysteine (Cys) Feedback inhibition ・Feedback inhibition of SAT by Cys ・Cys degradation catalyzed by CD In E. coli cells… ・No oversynthesis ・No accumulation

  5. Direct fermentation of Cys from glucose Methionine Acetyl-CoA H2S Glucose Serine O-Acetylserine Cysteine Serine acetyltransferase (SAT) 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)

  6. Enzyme Substrate ES-complex 100 Ser Active site Allosteric site ＋ 75 Acetyl-CoA Relative activity (%) 50 SAT Ser 25 Cysteine (endproduct) Acetyl-CoA 0 25 50 75 100 L-Cysteine conc. (M) SAT (inactive) 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＞

  7. ＜mixed primers＞ 5’-AATGGAT GGG GACCAGC-3’ CCC AAA TTT ＜site-directed mutagenesis by PCR＞ Amino acid substitution of Met256 of the E. coli SAT Analysis of feedback inhibition and Cys productivity 2 56All- <1st PCR> A B 2 56All + A Wild-type Met256 <2nd PCR> N C B Met256X Altered N C Truncated Ligation N C

  8. 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 Na S O 15 g 2 2 3 NH Cl 10 g 4 KH PO 2 g 2 4 MgSO 7H O ・ 1 g 4 2 FeSO 7H O ・ 0.01 g 4 2 MnCl 4H O 0.01 g ・ 2 2 Gly, -Ile, -Leu, -Met 0.1 g each L L L CaCO 20 g 3 ＜Cultivation＞ 30℃, Sakaguchi-flask, shaking ＜Determination of cysteine + cystine＞ Bioasay (Pediococcus acidilactici IFO3076)

  9. Activity remaining Amino acid CySH + Cys in the presence of residue at (mg/L) 100 M cysteine (%) position 256 Nakamori et al Appl. Environ. Microbiol ., 64, 1607-1611 (1998) ., Cys production by expression of the mutant SATs Plasmid pCE Met (Wild-type) 0.5 ND 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 M256Stop -* 31.3 730 ± 110 *, termination codon at position 256 Cys overproduction was achieved by expressing the mutant SAT.

  10. ＜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 m 0.5 M reverse primer m 0.2mM dATP 1mM dGTP, dCTP, dTTP each 1U Taq DNA polymerase Error-prone PCR random mutagenesis into E. coli SAT E. coliwild-type SAT gene (cysE) pCE Error-prone PCR EcoRI XbaI EcoRI XbaI pUC19 EcoRI XbaI Transformation of E. coli JM39-8 pHC

  11. Activity remaining CySH + Cys Amino acid in the presence of (mg/L) substitution 100 M cysteine (%) Takagi et al ., FEBS Lett ., 452, 323-327 (1999) Characteristics of the E. coli mutant SATs Plasmid ND 0.9 pCE 790 24.1 pCE M256A M256I 210 170 51.9 pHC 6 ± N 51 K , R 91 H , H 233 Y 330 70 78.2 pHC 7 ± E 166G, M201V 260 50 37.1 pHC 8 ± T167K 990 200 20.9 pHC 10 ± M201R 740 120 16.3 pHC 11 ± M201T 50 20 33.2 pHC 12 ± P252R 960 460 28.9 pHC 13 ± S253L Several amino acid residues other than Met256 are responsible for the feedback inhibition by Cys and the overproduction of Cys.

  12. (Noji et al ., J. Biol. Chem ., 273, 32739-32745, 1998) SATs of Arabidopsis thaliana SAT Localization Feedback inhibition SAT-m Mitochondria Insensitive SAT-p Chloroplast Insensitive SAT-c Cytoplasm Sensitive ＜Expression plasmids for the SAT cDNA＞ ＜Western analysis for the SAT expression＞ cysEp E. coli A. thaliana pEAS-m, pEAS-p SAT-m SAT-p wild-type SAT Ampr The A. thaliana SAT-m or SAT-p gene The E. coli cysE promoter The A. thaliana SATs are expressed in E. coli cells. ・ 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

  13. Comparison of catalytic properties of recombinant SATs plasmid pEAS-m pEAS-p pCEM256I pCE E. coli wild-type A. thaliana SAT-m A. thaliana SAT-p E. coli Met256Ile SAT SAT activity (mU/min/mg) 27.9 21.3 88.0 2,273 Relative activity (%) for L-cysteine added (M) 0 100 100 100 100 10 100 100 88 1.5 100 100 100 24 ND ND : Not detected. The A. thaliana SATs were insensitive to feedback inhibition.

  14. 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.

  15. 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)

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

  17. Direct fermentation of Cys from glucose Methionine Acetyl-CoA H2S Glucose Serine O-Acetylserine Cysteine Serine acetyltransferase (SAT) 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)

  18. COOH COOH CD H2N-C-H C=O NH3 + + H2S CH2 CH3 SH Pyruvate L-Cysteine A reaction catalyzed by Cysteine Desulfhydrase (CD) H2S is generated during fermentation !! Cys degradation is occurred !! Cys degradation pathway is unknown ?? Analysis of Cys degradation pathway

  19. ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) Identification of the E. coli CDs by activity staining Native-PAGE CD activity staining CD H2S + BiCl3 Cys BiSO4 = Black bands At least, five CD proteins are newly detected in E. coli.

  20. E. coli CD (1) Determine the N-terminus sequence ( 1 ) 1 15 Purified sampleMENFKHLPEPFRIRV・・・ E. coli Tryptophanase (TNase)MENFKHLPEPFRIRV・・・ A reaction catalyzed by TNase (the tanA product) Wild-type tnaA-disruptant L-Tryptophan → Indole + Pyruvate + NH3 + tnaA Vector Vector ( 1 )TNase ( 2 ) ? TNase (the tnaA product) isone of the E. coli CDs.

  21. E. coli CD (2) ＜CD reaction＞ COOH COOH CD ( 1 ) TNase H2N-C-H + + H2S NH3 C=O ( 2 ) CH2 CH3 SH Pyruvate L-Cysteine ＜Cystathionine b-lyase (CBL; the metC product)reaction＞ COOH COOH H2N-C-H COOH H2N-C-H COOH CBL L-Cysteine CH2 H2N-C-H CH2 + + NH3 C=O O-Succinyl-homoserine CH2 H2C S CH2 CH3 SH Cystathionine Pyruvate Homocysteine L-Methionine The CD and CBL reactions are the same. CBL accepts Cys as a substrate in vitro. CBL functions as a CD ?

  22. E. coli CD (3) -(5) Use of an E. coli library containing 4,388 kinds of open reading frame (ORF) lacZp X : ORF (total 4,388) X CD activity staining : lacZ promoter Cmr pCN24-X + cysK + cysM + malY Vector ( 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.

  23. 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 !? ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) Five CD proteins were identified in E. coli…

  24. Total CD activity total CD activity (mU/mg) Genotype 20.6 Wild-type tnaA metC cysK cysM malY  tnaA metC tnaA metC cysM malY tnaA metC cysK cysM malY 15.7 15.0 18.2 17.9 15.3 9.6 9.1 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.

  25. Cys production in the CD gene disruptants 1600 1400 1200 Wild-type tnaA mutsnt 1000 metC mutsnt Cysteine productivity(mg / L) 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.

  26. 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 3.5 Wild-type 3.0 tnaA mutant metC mutant 2.5 Growth (OD610) cysK mutant 2.0 cysM mutant 1.5 malY mutant 1.0 0.5 0 3 0 24 9 12 15 18 21 6 Culture time (hr) ・The tnaA disruptant was significantly inhibited. ・TNase is a key enzyme in Cys degradation in E. coli ??

  27. TNase induction by Cys Native-PAGE Northern blotting SDS-PAGE Cys (mM) Cys (mM) Cys (mM) 0 10 0 0 10 10 (kDa) ・ 94 1.7kb ・ 67 ・ 43 23s rRNA ・ 30 16s rRNA ・ 20 ･TNase synthesis is induced by Cys. ･TNase contributes mainly to Cys degradation.

  28. Identification and gene disruption of the E. coliCDs 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.

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

  30. CD gene 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 ? ・Identification and analysis of Cys transporter ・Evaluation of Cys transporter on Cys productivity

  31. 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 Transporter X pUC118-X naA disruptant + transporter gene Ampr Growth (OD610) 32 putative drug transporter genes tnaA disruptant Screening of Cys transporter Culture time (hr)

  32. 6 emrAB 5 Wild-type 4 acrD, acrEF, bcr, cusA, emrKY, ybjYZ, yojIH Growth (OD610) 3 2 tnaA disruptant 1 0 0 6 18 24 12 Culture time (hr) Genes that reversed the growth inhibition of tnaA disruptant by Cys: acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH

  33. Intracellular Cys contents of E. coli cells Cys（30mM） 3 2 Transporter X Intracellular Cys content (mg/L/OD610) pUC118-X 1 Ampr 0 vector vector cusA acrD emrAB emrKY yojIH acrEF bcr ybjYZ Cys Wild-type tnaA disruptant Genes that decreased intracellular Cys level of tnaA disruptant: acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, yojIH

  34. List of Cys exporter candidates Gene Function bcr emrAB emrKY acrEF acrD cusA ybjYZ yojIH 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.

  35. ＜ Cys transport assay ＞ ＜Cys uptake ＞ Cell suspension vector 2.0 + [35S]-Cys ybjYZ Cys uptake （nmol/mg cell wt） ydeD Cys uptake activity 1.0 bcr 0 0 10 20 30 Remaining labeled Cys content Time (min) ⇒ Cys export rate ＜Cys export＞ 100 80 Reduced uptake： bcr, ybjYZ,(ydeD) bcr 60 Cys export (%) Increased export： bcr, acrEF, emrAB, (ydeD) ydeD 40 acrEF emrAB 20 vector ＜ Cys uptake↓、Cys export↑ ＞ 0 10 20 30 0 bcr, ydeD Time (min) Bcr overexpression promotes Cys export in E. coli cells.

  36. Cys production by E. coli cells expressing bcr Mutant SAT gene pACYC-M256I Cmr 1000 + bcr Enhancing Cys synthesis 800 Pbcr 600 Concentration of Cys（mg/L/OD562） bcr pUC118-bcr 400 Ampr + vector (pUC118) Enhancing Cys export 200 Cys 0 24 48 72 Culture time (h) Cys Bcr overexpression contributes to Cys production.

  37. 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

  38. L-Serine Acetyl-CoA + Enhance Cys biosynthesis SO42- (external) Ser acetyltransferase Activated by OAS Feedback inhibition by Cys O-Acetylserine 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. S2- L-Cysteine Export Cys transporter Cys desulfhydrase Enhance Cys transport Degradation (NH3, H2S, Pyr.) Appl. Environ. Microbiol., 72, 4735, 2006; Appl. Microbiol. Biotechnol., in press. Weaken Cys degradation FEMS Microbiol. Lett., 217, 103, 2002; Appl. Microbiol. Biotechnol., 62, 239, 2003; Appl. Environ. Microbiol., 71, 4149, 2005 etc. MINI-REVIEW Appl. Microbiol. Biotechnol., 73, 48, 2006

  39. Real Scientists !! Dr. Masaaki Noji Dr. Kazuki Saito (Chiba Univ.) 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. Hirotada Mori (NAIST) Dr. Masaru Wada (Fukui Pref. Univ.) Dr. Shigeru Nakamori (Fukui Pref. Univ.) Ajinomoto Co., Inc.

  40. 大腸菌 SAT とシステイン生産の研究の流れ SATの フィードバック阻害 システイン生産量 (mg/L) 研究者 研究概要 Cys による制御の証明 (野生株) Kredich (1983) 0 感受性 Denk et al. (1987) SAT・一次構造の決定 Met256Ile 変異株の分離 30 感受性低下 Met256X の構築 Cys 分解能低下株 Nakamori et al. (1998) 600 〜 800 感受性低下 PCR ランダム変異の導入 Cys 分解能低下株 Takagi et al. (1999) 〜 1,000 感受性さらに低下 シロイヌナズナ SAT 遺伝子の導入 ? 本研究 非感受性 シロイヌナズナ SAT を用いたシステイン生合成系の強化

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

  42. 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

  43. Amino acid and DNA substitutions in the E. coli SAT Mutant Amino acid substitution (Base substitution) → → 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) →

  44. E. coli CD (2) Wild-type metC disruptant + metC vector vector ( 1 ) TNase (the tnaA product) ( 2 )CBL (the metC product) CBL (the metC product) is one of the E. coli CDs.

  45. Construction of the CD gene-disruptant Ampr A B A B ori (ts) Ampr pEL3 Δ-X ori (ts) X B A 42℃, LB medium + Amp Homologous recombination A B A B ori (ts) Ampr X X 37℃ LB medium E. coli chromosome A B E. coli chromosome Plasmid deletion → Amp-sensitive ・Construct the multiple CD gene disruptant ・Check the disruption by PCR and CD activity staining

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

  47. 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 cysK cysM 遺伝子名 SO42- (external) O-Acetylserine 912 972 遺伝子の長さ (bp) O-acetylserine sulfhydlase-A (OASS-A) O-acetylserine sulfhydlase-B (OASS-B) タンパク質名 OASS-A OASS-B !? アミノ酸の長さ (aa) 323 303 S 2- 38%一致, 53%相似 ホモロジー (アミノ酸) L-Cysteine Cys合成 !? CD Cys合成 CD !? 機能 CD H2O CysBとN-Acetylserine による制御!? CysBとN-Acetylserine による正の制御 発現制御 Methionine degradation 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 その他 SATとコンプレックス形成

  48. 転写調節因子に変異 !? 変異点なし!! Cys 分解能低下株の tnaA 領域 DNAシーケンス解析 Cys 分解能 低下株 野生株 Cys (mM) 0 0 10 10 TNase CBL +1 tnaA P tnaC 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

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