Acta Microbiologica Sinica 51 11 1476-1484 4 November 2011 ISSN 0001-6209 CN 11-1995 / Q http / / journals. im. ac. cn / actamicrocn Research Paper γ- L- 8-193 1 2 1 1 1 1* 1 100101 2 100049 L- L- L- 8-193 Corynebacterium crenatum 8-193 γ- EC 2. 7. 2. 11 γ-glutamyl kinase prob prob PCR prob prob C. crenatum 8-193 prob 8-193-ΔproB 8-193-ΔproB 8-193-ΔproB PCR γ- prob 8-193-ΔproB 9. 6% L- 13. 6% α- prob 8-193 γ- L- Q814 A 0001-6209 2011 11-1476-09 L- L- L- N- 1-2 L- γ- 3 γ- L- 1 4-7 N- L- car L- argi 8 * Tel / Fax + 86-10-64807415 E-mail dingjy@ sun. im. ac. cn 1986 - E-mail mypicture@ 163. com 2011-05-04 2011-05-27
γ- L- 8-193. / 2011 51 11 1477 N- 9 10 L- 8-193 γ- L- L- 1 Fig. 1 Diagram of the central metabolism and the biosynthesis of several amino acids of C. glutamicum. Abbreviations PEPCk PEP carboxykinase PEPCx PEP carboxylase PYC pyruvate carboxylase argj ornithine acetyltransferase ProB γ-glutamyl kinase NAG N- Acetyl-glutamate γ-gp γ-glutamylphosphate. Dashed arrows indicate pathways consisting of several reactions solid arrows indicate single reactions. 1 1. 1 1. 1. 3 1 LB 12 1. 1. 1 E. coli C. crenatum 2LB 4% 1 1. 1. 2 C. crenatum 13 3LB 10% TaKaRa L- BBI 4 PEP Sigma 20 g NH 4 2 SO 4 7 g ATP Bio Basic Inc NADH K 2 HPO 4 3H 2 O 0. 3 g KH 2 PO 4 0. 1 g MgSO 4 7H 2 O Ameresco 0. 4 g NaCl 0. 1 g FeSO 4 7H 2 O 25 mg MnSO 4 H 2 O MJ PTC-150 25 mg CaCl 2 H 2 O 50 mg 0. 4 mg BIO-RAD MicroPulser TM Beckman DU800 Alphalmager EC Agilent 1200 0. 2 mg 50 mg 400 mg 8-193-
1478 Xiaoman Li et al. / Acta Microbiologica Sinica 2011 51 11 Table 1 1 The strains and plasmids used in this work Strains or plasmids Characteristics Source Escherichia coli DH5α C. crenatum φ80 LacZΔM15 deor reca1 enda1 hsdr17 Stored in this lab 8-193 his - SG R This lab 8-193-ΔproB his - SG R pro - 8-193 with prob knock-out This study 8-193-ΔproB-C his - SG R 8-193-ΔproB complemented with pbs57-prob This study Plasmids pmd19-t T-vector 2. 7 kb Amp R lacz TaKaRa Co. pk18mobsacb Mobilizable E. coli vector Km R Suc S Sch fer A 11 pt-fprob pmd19-t containing prob upstream fragment from 8-193 This study pt-bprob pmd19-t containing prob downstream fragment from 8-193 This study pt-prob pmd19-t containing 1. 2kb PCR fragment of 8-193 prob gene This study pk18-fprob pk18mobsacb containing prob upstream fragment from 8-193 This study pk18-δprob pk18mobsacb containing prob in-frame deletion fragment ΔproB from 8-193 This study pbs57 E. coli-c. glutamicum shuttle expression vector Km R This lab pbs57-prob pbs57 containing prob from 8-193 to generate prob expression or complementation for 8-193-ΔproB This study his - no cell growth without histidine addition on minimal medium SG sulfaguanidine pro - no cell growth without proline addition on minimal medium. ΔproB ph 7. 0 5 2 60g NH 4 2 SO 4 35g K 2 HPO 4 3H 2 O 1 g Table 2 Primers for gene amplification and gene disruption KH 2 PO 4 0. 5 g MgSO 4 7H 2 O 0. 4 g FeSO 4 7H 2 O Primers Sequences 5' 3' Size / bp Restriction site 20 mg MnSO 4 H 2 O 20 mg 0. 1 mg 3 8-193-ΔproB ph 7. 0 1. 2% E. coli 0. 2 mg CaCO 30 g 50 mg 400 mg P2 P1 AAGTGACAGAGCTTCCCGAC TCTAGA TTATGCGCGCCTG GCGTAGTTGG TTTAAA TAAGGAAGCTA 29 37 XbaⅠ DraⅠ 37 C. crenatum 30 100 μg / ml P3 50 μg / ml GGT AAGCTT AGCCAGCGCC CTCGCATTGT 29 HindⅢ CGG TCTAGA ACTGCGCCAG 1. 2 DNA P4 AGGACACAAC E. coli 12 E. coli 29 XbaⅠ CaCl 2 C. crenatum GAC TCTAGA TGATGATGGC P5 29 XbaⅠ 1. 3 prob PCR 1. 3. 1 C. glutamicum R γ- prob GenBank accession number AP009044 8-193 prob prob 2 Primer premier 5. 0 P3 P4 prob FproB P5 P6 prob BproB 1. 3. 2 PCR 20 μl Tris-HCl ph 7. 0 2 98 10 s 68 1 min 30 0. 5 μl EasyTaq DNA 72 10 min P6 GCGGTGGAAG CAC GAATTC TGGCTGTCAG GAAGTGGCTC 29 EcoRⅠ Boxed bases indicate restriction sites bold bases indicate Shine-Dalgarno sequence underlined bases indicate homologous sequences with prob. 1. 4 P1 P2 prob 1. 4. 1 γ- 8-193 8-193-ΔproB 8-193-ΔproB-C 4 30 12 h 50 mmol / L 13500 r / min 15 min. 14 37 90 min
γ- L- 8-193. / 2011 51 11 1479 1 ml 55 g / L FeCl 3 20 g / L 1. 5. 3 SBA-40C 21 ml / L HCl γ- A 535 γ- 1. 5. 4 12000 r / min 250 L mol - 1 cm - 1 γ- Agilent 1200 1 nmol γ- 1. 4. 2 8-193 8- Agilent A 40mmol / L NaH 2 PO 4 ph7. 8 B 193-ΔproB 4 30 ACN MeOH 45 45 10 v / v / v 18 2 DAD 338 nm 262 nm 1. 5. 5 19 80 13500 r / min 15 min 10 min 12000 r / min 10 min 0. 22 μm 15 A 340 NADH Agilent 1200 6220 L mol - 1 cm - 1 2. 75 mmol / L H 2 SO 4 0. 6 ml / min 1 nmol NADH NAD + 50 10 μl DAD 210 nm 1. 4. 3 8-193 8-193-ΔproB 4 30 12 h 50 mmol / L Tris-HCl ph 6. 3 50 mmol / L NaCl 2 HEPES 100 mmol / L 20% - 20 PCR 1. 2 kb 3% CTAB pmd19-t E. coli DH5α 0. 3% 1 min LB prob 1246 bp 1221 bp 16 A 340 C. crenatum 8-193 prob 1 nmol NADH 1. 4. 4 100% GenBank 17 JF897613 1. 5 1. 5. 1 8-193 8-193-ΔproB prob DraⅠ XbaⅠ 4 30 300 r / min 12 h 12 h 100 mmol / L Tris- HCl ph 7. 5 10% 5 coli DH5α LB 30 300 r / min 12 h prob pbs57-1. 5. 2 1 mol / L HCl prob 600 nm 2. 2 pk18-δprob 10 min 0. 22 μm Zorbax Eclipse-AAA 2. 0 ml / min 40 18 Aminex HPX-87H BioRad 2 2. 1 prob 8-193 DNA P1 P2 pt-prob ORF GTG 406 C. glutamicum R prob 99. 34% 8 DraⅠ Xba Ⅰ pt-prob pbs57 prob E. 8-193 DNA P3
1480 Xiaoman Li et al. / Acta Microbiologica Sinica 2011 51 11 P4 P5 P6 PCR 1. 1 kb 2. 4 prob pmd19-t E. coli DH5α LB PCR pt-fprob pt-bprob HindⅢ XbaⅠ pt-fprob FproB Hind Ⅲ Xba Ⅰ 8-193-ΔproB pk18mobsacb FproB PCR 8-193 600bp E. coli DH5α LB 2 prob pk18-fprob Xba E. coli DH5α LB prob DNA pk18-δprob prob 2 Fig. 2 prob PCR 2. 3 prob of prob of 8-193 2. PCR fragment of prob of 8-193-ΔproB. pk18-δprob 8-193 LB 8-193 8-193-ΔproB 8-193-ΔproB-C pk18-δprob 8-193 30 48 h pk18-δprob DNA DNA 8-193-ΔproB 3 8-193 8-193-ΔproB-C 3 3 prob pk18-δprob sacb DNA Ⅰ EcoRⅠ pt-bprob BproB Xba Ⅰ EcoR Ⅰ pk18- FproB BproB PCR prob prob 3 prob 3 8-193 8-193-ΔproB 8-193-ΔproB-C 8-193-ΔproB 2. 1 pbs57-prob Fig. 3 The growth of 8-193 8-193-ΔproB and 8-193-ΔproB-C on prob 8-193-ΔproB minimal medium. Ⅰ Minimal medium containing histidine Ⅱ LB Minimal medium containing histidine and proline. a C. crenatum 8-193- 8-193 b C. crenatum 8-193-ΔproB c C. crenatum 8-193- ΔproB-C ΔproB prob 8-193 8-193-ΔproB DNA P3 P6 PCR PCR Characterization of defined prob knock-out mutant by PCR analysis. M. DNA marker Trans5K DNA Marker 1. PCR fragment ΔproB-C.
γ- L- 8-193. / 2011 51 11 1481 γ- 8-193-ΔproB 8-193 18. 35 ± 0. 22 U / mg 8-193-ΔproB-C 23. 19 ± 0. 93 U / mg 26. 38% prob 8-193-ΔproB 8-193-ΔproB prob 2. 5 prob 8-193 prob 8-193 L- 8-193-ΔproB 8-193 8-193 9. 6% 4-A L- 8-193-ΔproB 8-193 8-193-ΔproB L- 8-193 13. 78g / L 4 8-193 13. 6% 4-B Fig. 4 prob 8-193 production profiles. 3 8-193 8-193-ΔproB A B Time course of 8-193 and 8-193-ΔproB fermentation A cell growth profiles B glucose consumption and L-arginine Strains 3 Table 3 Comparison of fermentation parameters of the two strains Parameters P Arg / g / L X max / g / L Q S / g / L h R X / g / L h Y P / S g / g Y X / S g / g Φ g / L h 8-193 12. 13 10. 12 0. 83 0. 14 0. 20 0. 17 0. 17 8-193-ΔproB 13. 78 9. 32 1. 00 0. 13 0. 23 0. 16 0. 19 P Arg Arginine Production X max The maximum Cell Dry weight Q S Glucose consumption rate R X Average Growth Rate Y P / S Arginine yield on Glucose Y X / S Biomass yield on Glucose Φ Arginine productivity. 4 8-193-ΔproB 8-193-ΔproB 8-193 70. 3% 31. 2% α- 145% 194% 70% 9. 1% 31. 4% 49. 7% 5
1482 Xiaoman Li et al. / Acta Microbiologica Sinica 2011 51 11 Table 4 4 The analysis of by-product amino acids in fermentation broth c Amino acid / mg / L Strains Aspartate Asparagine Isoleucine Lysine Threonine 8-193 137. 86 ± 9. 93 30. 13 ± 0. 89 1858. 23 ± 66. 08 86. 70 ± 6. 71 308. 90 ± 54. 58 8-193-ΔproB 154. 07 ± 5. 56 33. 20 ± 1. 57 2249. 27 ± 59. 39 130. 35 ± 4. 67 370. 99 ± 22. 77 Methionine Glutamate Glutamine Citrulline Serine 8-193 78. 00 ± 8. 37 8. 22 ± 1. 21 4. 15 ± 0. 68 749. 77 ± 3. 57 6. 13 ± 1. 10 8-193-ΔproB 98. 90 ± 6 22 20. 10 ± 1. 83 12. 22 ± 1. 10 1278. 29 ± 18. 05 6. 87 ± 0. 31 Tyrosine Phenylalanine Alanine Valine Leucine 8-193 52. 35 ± 6. 55 55. 80 ± 9. 20 1546. 10 ± 75. 68 5. 01 ± 0. 62 28. 17 ± 2. 40 8-193-ΔproB 51. 14 ± 2. 67 55. 14 ± 3. 39 1350. 72 ± 43. 65 4. 65 ± 0. 30 28. 92 ± 1. 90 Table 5 5 The analysis of organic acids in fermentation broth c Organic acid / g / L Strains Citirc acid Acetic acid Lactic acid Succinic acid Malic acid 8-193 0. 47 ± 0. 05 3. 72 ± 0. 02 2. 22 ± 0. 35 4. 63 ± 0. 38 1. 54 ± 0. 04 8-193-ΔproB 0. 48 ± 0. 07 3. 80 ± 0. 13 3. 78 ± 0. 34 2. 33 ± 0. 44 2. 02 ± 0. 42 Pyruvate α-ketoglutaric acid PEP 8-193 0. 42 ± 0. 01 0. 33 ± 0. 12 0. 00637 ± 0. 00046 8-193-ΔproB 0. 32 ± 0. 03 0. 30 ± 0. 00 0. 00437 ± 0. 00034 prob α- 3 α- L- 8-193 γ- prob L- L- prob 2. 6 γ- L- prob 8-193 L- 13. 6% prob 8-193- α- ΔproB prob α- 75% 44% 6 8-193-ΔproB L- 6 8-193 8-193-ΔproB 50% α- Table 6 Specific activity of PEPCx and PYC of 8-193 and 8-193-ΔproB Strains PEPCx / U / mg protein Specific enzyme activity PYC / U / mg cell dry weight 8-193 94. 4 ± 6. 9 57. 2 ± 3. 6 8-193-ΔproB 165. 2 ± 12. 0 82. 3 ± 13. 9
γ- L- 8-193. / 2011 51 11 1483 5 Xu Y Labedan B Glansdorff N. Surprising Arginine 15-16 prob Biosynthesis a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms. Micorobiology and molecular biology review 2007 71 36-47. 6. prob Reengineering of a Corynebacterium glutamicum L- 8-193-ΔproB Arginine and L-Citrulline Producer. Applied and prob Environmental Microbiology 2009 75 1635-1641. prob 10. N-. Acta Microbiologica Sinica 2006 46 1 90-94. 11 Sh fer A Tauch A J ger W Kalinowski J Thierbach 8-193-ΔproB G Pühler. A. Small mobilizable multi-purpose cloning ArgR vectors derived from the Escherichia coli plasmids pk18 argr and pk19 selection of defined deletions in the N- argb chromosome of Corynebacterium glutamicum. Gene 22-8- 1994 145 1 69-73. 12 Sambrook J Russell DW.. 193-ΔproB argr argb.. 2002. 13 Jang KH Britz ML. Improved electrotransformation frequencies of Corynebacterium glutamicum using cellsurface mutants. Biotechnology Letters 2000 22 539-1 Flynn NE Meininger CJ Haynes TE Wu G. The 545. metabolic basis of arginine nutrition and 14 Hayzer DJ Leisinger T. The gene-enzyme relationships pharmacotherapy. Biomedicine & Pharmacotherapy of proline biosynthesis in Escherichia coli. Journal of 2002 56 9 427-438. 2 de Jonge WJ Kwikkers KL te Velde AA van Deventer SJ Nolte MA Mebius RE Ruijter JM Lamers MC general microbiology 1980 118 2 287-293. 15 O' Regan M Thierbach G Bachmann B Villeval D Lepage P Viret JF Lemoine Y. Cloning and nucleotide Lamers WH. Arginine deficiency affects early B cell sequence of the phosphoenolpyruvate carboxylase-coding maturation and lymphoid organ development in transgenic gene of Corynebacterium glutamicum ATCC13032. Gene mice. Journal of Clinical Investigation 2002 110 1989 77 2 237-251. 10 1539-1548. 3 Bian K Murad F. Nitric oxide NO - biogeneration regulation and relevence to human diseases. Frontiers in 16. CD945. Acta Microbiologica Sinica bioscience 2003 8 264-278. 4 Lu CD. Pathways and regulation of bacterial arginine metabolism and perspectives for obtaining arginine overproducing strains. Applied Microbiology and Biotechnology 2006 70 261-272.. Food Science 2005 26 35-39. 8-193 prob 8-7 193-ΔproB. L- argh 20-21 prob γ-. China Biotechnology proa 1kb proa 2010 30 9 49-55. 8 Tuchman M Rajagopal BS McCann MT Malamy MH. -5- proc Enhanced production of arginine and urea by genetically prob engineered Escherichia coli K-12 strains. Applied and 600bp Environmental Microbiology 1997 63 33-38. 600bp prob ORF 9 Ikeda M Mitsuhashi S Tanaka K Hayashi M. 2003 43 2 214-219. 17 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976 72 248-254.
1484 Xiaoman Li et al. / Acta Microbiologica Sinica 2011 51 11 18 Henderson JW Ricker RD Bidlingmeyer BA Woodward C. Rapid Accurate Sensitive and Reproducible HPLC Analysis of Amino Acids. AAA Techical note P5 2000 Publication No 5980-1193. 19 Chinnici F Spinabelli U Riponi C Amati A. Optimization of the determination of organic acids and sugars in fruit juices by ion-exclusion liquid chromatography. Food Composition and Analysis 2005 18 121-130. 20 Ankri S Serebrijski I Reyes O Leblon G. Mutations in the Corynebacterium glutamicum proline biosynthetic pathway A natural bypass of the proa step. Journal of Bacteriology 1996 178 15 4412-4419. 21 Serebrijski I Wojcik F Reyes O Leblon G. Multicopy Suppression by Asd Gene and Osmotic Stress-Dependent Complementation by Heterologous proa in proa Mutants. Journal of Bacteriology 1995 177 24 7255-7260. 22 Lee SY Park JM Lee JH Chang ST Park JS Kim YH Min J. Interaction of Transcriptional Repressor ArgR with Transcriptional Regulator FarR at the argb Promoter Region in Corynebacterium glutamicum. Applied and Environmental Microbiology 2011 77 3 711-718. 23 Lee SY Shin HS Park JS Kim YH Min J. Proline reduces the binding of transcriptional regulator ArgR to upstream of argb in Corynebacterium glutamicum. Applied Microbiology and Biotechnology 2010 86 1 235-242. Effect of gamma-glutamyl kinase gene knock-out on metabolism in L-arginine-producing strain Corynebacterium crenatum 8-193 Xiaoman Li 1 2 Zhi Zhao 1 Yingzi Zhang 1 Yu Wang 1 Jiuyuan Ding 1* 1 Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China 2 Graduate School of the Chinese Academy of Sciences Beijing 100049 China Abstract Objective In order to optimize precursor supply for L-arginine biosynthesis we constructed a Corynebacterium crenatum 8-193 mutant with gamma-glutamyl kinase gene prob in-frame deletion. The effects of prob knock-out on physiological characteristics of the mutant were investigated. Methods The upstream and downstream fragments of prob were cloned from C. crenatum 8-193 chromosome and ligated to integration vector. The mutant C. crenatum 8-193-ΔproB was obtained by homologous recombination. The mutant phenotype can be reversed by complementation with prob gene from the expression vector. The physiological characteristics of the mutant were investigated by measurement of the activities of phosphoenolpyruvate carboxylase PEPCx and pyruvate carboxylase PYC. Results The prob gene in-frame deletion was screened and confirmed by PCR gamma-glutamyl kinase determination and complementation. The mutant lost the ability of growth on minimal medium without proline addition. The prob knock-out mutant resulted a decrease of cell mass by 9. 6% and an increase of L-arginine accumulation by 13. 6% compared with that of the parent strain. The analysis of by-products of fermentation broth showed that the concentrations of glutamate-related and aspartate-related amino acids increased and the concentrations of α-ketoglutaric acid PEP and succinic acid decreased. The specific activities of PEPCx and PYC increased in 8-193-ΔproB. Conclusion The prob gene knock-out of the strain 8-193 blocked branch catabolism of L-glutamate and improved efficiency of the glucose utilization and L-arginine accumulation. Keywords Corynebacterium crenatum gamma-glutamyl kinase gene knock-out L-arginine Corresponding author. Tel / Fax + 86-10-64807415 E-mail dingjy@ sun. im. ac. cn Received 4 May 2011 / Revised 27 May 2011