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Enhancing Expression of ywtD Gene in Bacillus licheniformis WX-02 by Two Types of Promoters(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2012 03
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Enhancing Expression of ywtD Gene in Bacillus licheniformis WX-02 by Two Types of Promoters
FU Juntao QI Gaofu LIU Jun JI Zhixia MA Xin CHEN Shouwen
(1State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China)
(2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China)
poly-γ-glutamate B. licheniformis depolymerase gene SacB promoter α-amylase gene promoter enhancing expression
Q936 : Q786

Poly-γ-glutamate (γ-PGA) is a biopolymer material that has a good application prospect. The effect of two promoters on the ywtD gene expression in Bacillus licheniformis WX-02 was investigated in this study to obtain highly expressed γ-PGA depolymerase YwtD. Two recombinants of B. licheniformis WX-02, SYT and PYT, in which ywtD gene was expressed from the control of SacB promoter and α-amylase gene promoter, respectively, were constructed. The enzyme activity assay showed that the expression of YwtD proteins were enhanced in the recombinants SYT and PYT. Both B. licheniformis recombinants SYT and PYT could reduce the molecular weight of γ-PGA from 1 000 000~1 200 000 to 800 000~900 000. In addition, PYT resulted in a 33% increase of γ-PGA production from 13.50 to 17.97 g L-1, whereas γ-PGA production of SYT reduced to 10.85 g L-1. Therefore, the promoter of α-amylase gene was more suitable for constructing the engineering strain with a higher yield and reduced molecular weight of γ-PGA.


Ashiuchi M, Shimanouchi K, Nakamura H, Kamei T, Soda K, Park C, Sung MH, Misono H. Enzymatic synthesis of high-molecular-mass poly-γ-glutamate and regulation of its stereochemistry. Appl Environ Microbiol, 2004, 70: 4249~4255
Kunioka M. Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms. Appl Microbiol Biotechnol, 1997, 47: 469~475
Richard A, Margaritis A. Rheology, oxygen transfer and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis. Biotech Bioeng, 2003, 82: 299~305
Irurzun I, Bou JJ, Peréz-Camero G, Abad C, Campos A, Muuerra S. Mark-houwink, parameters of biosynthetic poly(γ-glutamic acid) in aqueous solution. Macromol Chem Phys, 2001, 202: 3253~3256
Hirose I, Sano K, Shinoda I, Kumano M, Nakamura K, Yamane K. Proteome analysis of Bacillus subtilis extracellular proteins: A two-dimensional protein electrophoretic study. Microbiology, 2000, 146: 65~75
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 1997, 390 (6657): 249~256
Ashiuchi M, Nakamura H. Poly-γ-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity. J Mol Catal B Enzym, 2003, 23: 249~255
Kimura K, Itoh Y. Characterization of poly-γ-glutamate hydrolase encoded by a bacteriophage Genome: Possible role in phage infection of Bacillus subtilis encapsulated with poly-γ-glutamate. Appl Environ Microbiol, 2003, 69: 2491~2497
Suzuki T, Tahara Y. Characterization of the Bacillus subtilis ywtD gene, whose product is involved in γ-polyglutamic acid degradation. J Bacteriol, 2003, 185 (7): 2379~2382
Yao J, Jing J, Xu H, Liang JF, Wu Q, Feng XH, Ouyang PK. Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2. J Mol Catal B Enzym, 2009, 56: 158~164
Zhou HB (周海波). Cloning and expression of the poly-γ-glutamic acid depolymerase gene and γ-glutamyltranspeptidase gene from Bacillus licheniformis: [Master Degree Dissertation]. Wuhan: Huazhong Agricultural University (武汉: 华中农业大学), 2008
Steinmetz M, Le Coq D, Aymerich S, Gonzy-Tréboul G, Gay P. The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet, 1985, 200 (2): 220~228
Chen ZJ, Heng C, Li ZY, Liang XL, Xinchen SG. Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide. Appl Microbiol Biotechnol, 2007, 73: 1377~1381
Heng C, Chen ZJ, Du LX, Lu FP. Expression and secretion of an acid-stable a-amylase gene in Bacillus subtilis by SacB promoter and signal peptide. Biotechnol Lett, 2005, 27: 1731~1736
Niu DD (牛丹丹), Xu M (徐敏), Ma JS (马骏双), Wang ZX (王正祥). Cloning of the gene encoding a thermostable α-amylase from Bacillus licheniformis CICIM B0204 and functional identification of its promoter. Acta Microbiol Sin (微生物学报), 2006, 46 (4): 576~580
Niu DD, Zuo ZR, Shi GY, Wang ZX. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system. Microb Cell Factories, 2009, 8: 58~65
Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Meth, 1999, 34: 183~191
Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction. Biotechniques, 1990, 8 (5): 528~535
Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual. 3rd ed. 2001. 26~130
Lü ZB (吕正兵), Zhang F (张方), Xia Y (夏颖), Kan XZ (阚显照), Zhu GP (朱国萍). An improved alkaline lysis adaptive to extracting plasmid DNA of Bacillus. J Anhui Norm Univ (安徽师范大学学报), 2002, 25: 54~55
Moore S, Stein WH. A modified ninhydrin reagent for the photometric determination of amino acid and related compounds. J Biol Chem, 1954, 211: 907~913
Do JH, Chang HN, Lee SY. Efficient recovery of gamma-poly(glutamic acid) from highly viscous culture broth. Biotech Bioeng, 2001, 76: 219~223
Candela T, Fouet A. Poly-gamma-glutamate in bacteria. Mol Microbiol, 2006, 60 (5): 1091~1098
Dong C (董晨), Cao J (曹娟), Zhang J (张迹), Shen B (沈标). High-level expression of thermostable α-amylase in Bacillus subtilis. Chin J Appl Environ Biol (应用与环境生物学报), 2008, 14 ( 4 ): 534~538


Last Update: 2012-06-19