启动子串联及改造提高FAD为辅基的葡萄糖脱氢酶在Bacillus subtilis中的表达
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摘要
以黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)为辅基的葡萄糖脱氢酶(glucose dehydrogenase with FAD,FAD-GDH,EC 1.1.99.10),与辅基结合紧密,催化效率高,是临床检测血糖指标的新型诊断用酶。将Burkholderia cepacia的FAD-GDH基因(gdh)构建含单启动P_(HpaⅡ)的穿梭质粒p MA5-1,在蛋白酶缺陷型菌株Bacillus subtilis WB600中表达。为了获得该酶的高效表达,采用启动子串联及改造策略考察产酶情况。将4种启动子(P_(amyQ’),P_(43),P_(gsiB),P_(opuaa))分别与质粒上自带的启动子P_(HpaⅡ)串联,结果表明P_(HpaⅡ)-P_(amyQ’)串联组合获得的FAD-GDH胞内酶活最高,为2 497 U/L,是串联前单启动子的2.7倍。为了减少发酵过程中,葡萄糖和甘油对产酶的抑制作用,在串联组合的基础上删去P_(amyQ’)启动子中与碳代谢调控蛋白结合的cre位点,使胞内产酶水平提高至3 626 U/L,说明cre位点的去除能够减少碳代谢产物对启动子转录的抑制。本研究为新型诊断用酶FAD-GDH的菌种改造和工业化生产应用提供参考与借鉴。
Glucose dehydrogenase(FAD-GDH,EC1.1.99.10),conjugated tightly with flavin adenine dinucleotide,is a novel diagnostic enzyme for the clinical detection of blood glucose.A protease-defective strain Bacillus subtilis WB600 was used as a host to construct a shuttle plasmid pMA5-1 containing a single promoter P_(HpaII)for expression of FAD-GDH gene(gdh)from Burkholderia cepacia.The use of promoters in series and transformation strategies to investigate enzyme production.Four promoters(P_(amyQ'),P_(43),P_(gsiB),P_(opuaa))were ligated with the promoter P_(HpaII)on the plasmid,respectively.The results showed that the intracellular enzymatic activity of FAD-GDH was highest with P_(HpaII)-P_(amyQ')tandem,which was 2 497 U/L.In order to reduce the inhibitory effect of glucose and glycerol on enzyme production during fermentation,on the basis of tandem combination,the cre sites binding to carbon metabolism regulatory proteins in P_(amyQ')promoter were deleted,and the intracellular enzyme production level was increased to 3 626 U/L,indicating that the removal of cre sites can reduce the inhibition of carbon metabolism products on promoter transcription.This study provides a reference for genetic modification and industrial production of a new diagnostic enzyme(FAD-GDH)
Glucose dehydrogenase(FAD-GDH,EC1.1.99.10),conjugated tightly with flavin adenine dinucleotide,is a novel diagnostic enzyme for the clinical detection of blood glucose.A protease-defective strain Bacillus subtilis WB600 was used as a host to construct a shuttle plasmid pMA5-1 containing a single promoter P_(HpaII)for expression of FAD-GDH gene(gdh)from Burkholderia cepacia.The use of promoters in series and transformation strategies to investigate enzyme production.Four promoters(P_(amyQ'),P_(43),P_(gsiB),P_(opuaa))were ligated with the promoter P_(HpaII)on the plasmid,respectively.The results showed that the intracellular enzymatic activity of FAD-GDH was highest with P_(HpaII)-P_(amyQ')tandem,which was 2 497 U/L.In order to reduce the inhibitory effect of glucose and glycerol on enzyme production during fermentation,on the basis of tandem combination,the cre sites binding to carbon metabolism regulatory proteins in P_(amyQ')promoter were deleted,and the intracellular enzyme production level was increased to 3 626 U/L,indicating that the removal of cre sites can reduce the inhibition of carbon metabolism products on promoter transcription.This study provides a reference for genetic modification and industrial production of a new diagnostic enzyme(FAD-GDH)
引文
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[27]徐书景,张跃灵,张妍,等.改进重叠延伸PCR技术构建定点双突变[J].中国生物工程杂志,2010,30(10):49-54.
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[2]ITO K,OKUDA S,JUNKO,et al. Designer fungus FAD glucose dehydrogenase capable of direct electron transfer[J]. Biosensors and Bioclectronics,2019,123:114-123.
[3]GALANT A L,KAUFMAN R C,WILSON J D. Glucose:detection and analysis[J]. Food Chemistry,2015,188:149-160.
[4]KOMORI H,INAKA K,FURUBAYASHI N,et al. Crystallographic analysis of FAD-dependent glucose dehydrogenase[J]. Acta Crystallographica Section F-Structural Biology Communications,2015,71(8):1 017-1 019.
[5]YANG Yufeng,HUANG Lei,WANG Jufang,et al. Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus[J]. Enzyme and Microbial Technology,2015,68:43-49.
[6]YOSHIDA H,SAKAI G,MORI K,et al. Structural analysis of fungus-derived FAD glucose dehydrogenase[J].Scientific Reports,2015,5(1):13 498.
[7]周利伟.青霉来源葡萄糖脱氢酶的克隆、表达及其酶学性质研究[D]:北京:中国农业科学院,2012.
[8]田岛辽子,一柳敦,市川惠一,等.黄素结合型葡萄糖脱氢酶:中国,201080004947. X.[P]. 2010-04-19.
[9]YANG Yufeng,HUANG Lei,WANG Jufang,et al. Efficient expression, purification, and characterization of a novel FAD-dependent glucose dehydrogenase from Aspergillus terreus in Pichia pastoris[J]. Journal of Microbiology and Biotechnology,2014,24(11):1 516-1 524.
[10]YADA T,MIYAMOTO K. FAD-conjugated glucose dehydrogenase:United states,9976125[P]. 2016-10-20.
[11]CHRISTOPH S,PETRA S,MIRIAM K,et al. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J]. Microbial Cell Factories,2011,10:106.
[12]MURATA K,AKATSUKA W,SADAKANE T,et al. Glucose oxidation catalyzed by FAD-dependent glucose dehydrogenase within Os complex-tethered redox polymer hydrogel[J]. Electrochimica Acta,2014,136:537-541.
[13]余小霞,田健,刘晓青,等.枯草芽孢杆菌表达系统及其启动子研究进展[J].生物技术通报,2015,31(2):35-44.
[14]ZHANG K,SU L,DUAN X,et al. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system[J]. Microbial Cell Factories,2017,16(1):32-47.
[15]KANG H K,JANG J H,SHIM J H,et al. Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters[J]. World Journal of Microbiology and Biotechnology, 2010, 26(10):1 915-1 918.
[16]GUAN C,CUI W,CHENG J,et al. Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis[J]. New Biotechnology,2016,33(3):372-379.
[17]BRUDER M,MOO Y M,CHUNG D A,et al. Elimination of carbon catabolite repression in clostridium acetobutylicum-a journey toward simultaneous use of xylose and glucose[J]. Applied Microbiology and Biotechnology,2015,99(18):7 579-7 588.
[18]NICHOLSON W L,PARK Y K,HENKIN T M. Catabolite repression resistant mutations of the Bacillus subtilis alpha amylase promoter affect transcription levels and are in an operator-like sequence[J]. Journal of Molecular Biology,1987,198(4):609-618.
[19]NAGARAJAN D R,KRISHANA C. Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes[J]. Protein Expression Purification,2010,70(1):122-128.
[20]ZYPRIAN E,MATZURA H. Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid p UB110 and development of a gram-positive expression vector system[J]. DNA, 1986, 5(3):219-225.
[21]GUPTA M,RAO K K. Phosphorylation of Deg U is essential for activation of amy E expression in Bacillus subtilis[J]. Journal of Biosciences,2014,39(5):747-752.
[22]WANG P Z,DOI R H. Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases[J].Biological Chemistry,1984,259(13):8 619-8 625.
[23]KIM J H,HUANG B Y,ROH J. Comparison of Papr E,Pamy E and P43 promoter strength forβ-galactosidase and staphylokinase expression in Bacillus subtilis[J]. Biotechnolgy and Bioprocess Engineering,2008,13(3):313-318.
[24]MAUL B,VLKER U,RIETHDORF S,et al.σB-dependent regulation of gsi B in response to multiple stimuli in Bacillus subtilis[J]. Molecular&General Genetics,1995,248(1):114-120.
[25]PACCEZ J D,LUIZ W B,SBROGIO A M E,et al. Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery[J]. Vaccine,2006,24(15):2 935-2 943.
[26]PROMCHAI R,PROMDONKOY B,TANAPONGPIPAT S,et al. A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis[J]. Journal of Biotechnology,2016,222:86-93.
[27]徐书景,张跃灵,张妍,等.改进重叠延伸PCR技术构建定点双突变[J].中国生物工程杂志,2010,30(10):49-54.
[28]ZHANG K,SU L,DUAN X,et al. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system[J]. Microbial Cell Factories,2017,16(1):32-47.