利用内源元件在谷氨酸棒杆菌中分泌表达木聚糖酶
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摘要
以来自于谷氨酸棒杆菌内源AH6启动子和5′UTR及其前38 bp结合合适的Shine-Dalgarno (SD)序列,构建双顺反子表达载体对木聚糖酶进行表达。为了能够实现分泌表达,选取了来自谷氨酸棒杆菌的两种分泌途径的信号肽,分别为Tat型的CgR0949及Sec型的CspB信号肽。在实现分泌表达之后,对其进行5 L发酵罐的扩大培养以提高分泌量。并对纯化的木聚糖酶进行了部分酶学性质的研究,包括最适催化pH及酸碱耐受性;最适催化温度及热稳定性。结果表明:在上述表达体系中,以CgR0949为信号肽木聚糖酶不能分泌到胞外;木聚糖酶能在CspB信号肽的引导下分泌到胞外,分泌表达量为486.2 U/mL。木聚糖酶的分泌量在5 L发酵罐水平上达到1 648.7 U/mL,是摇瓶培养的3.4倍。该木聚糖酶的最适反应pH为4.5,最适温度为45℃;在pH 4–11范围内4℃处理24 h酶活保持在80%以上;在50℃前处理15 min酶活保持在95%以上,超过60℃则酶活迅速下降至20%及其以下。上述结果表明,谷氨酸棒杆菌内源元件能有效用于木聚糖酶的分泌表达,扩大培养能进一步提升木聚糖酶的分泌量。该双顺反子表达体系能为外源蛋白在谷氨酸棒杆菌中的分泌表达提供一种可用的工具。此外,通过酶学性质的研究可进一步提高木聚糖酶的催化效率。
We constructed bicistronic expression system containing AH6 promoter, 5′ UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno(SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat(CgR0949) and Sec(CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃,respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4–11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C.glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.
We constructed bicistronic expression system containing AH6 promoter, 5′ UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno(SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat(CgR0949) and Sec(CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃,respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4–11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C.glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.
引文
[1]Juturu V,Wu JC.Microbial xylanases:engineering,production and industrial applications.Biotechnol Adv,2012,30(6):1219-1227.
[2]Pa?s G,Berrin JG,Beaugrand J.GH11 xylanases:structure/function/properties relationships and applications.Biotechnol Adv,2012,30(3):564-592.
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[4]Guan CR,Cui WJ,Cheng JT,et al.Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.Microb Cell Fact,2015,14(1):150.
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[9]Liu XX,Zhang W,Zhao ZH,et al.Protein secretion in Corynebacterium glutamicum.Crit Rev Biotechnol,2017,37(4):541-551.
[10]Kalinowski J,Bathe B,Bartels D,et al.The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins.JBiotechnol,2003,104(1/3):5-25.
[11]Zhao ZH,Liu XX,Zhang W,et al.Construction of genetic parts from the Corynebacterium glutamicum genome with high expression activities.Biotechnol Lett,2016,38(12):2119-2126.
[12]Liu XX,Zhao ZH,Sun Y,et al.Selection of endogenous expression elements from Corynebactrium glutamicum.Microbiol China,2016,43(8):1671-1678(in Chinese).刘秀霞,赵子豪,孙杨,等.谷氨酸棒杆菌内源表达元件的筛选.微生物学通报,2016,43(8):1671-1678.
[13]Yim SS,An SJ,Choi JW,et al.High-level secretory production of recombinant single-chain variable fragment(scFv)in Corynebacterium glutamicum.Appl Microbiol Biotechnol,2014,98(1):273-284.
[14]Zou YL,Chen T,Feng LL,et al.Enhancement of5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in Corynebacterium glutamicum.Biotechnol Lett,2017,39(9):1369-1374.
[15]Zhang SH,Liu DY,Mao ZT,et al.Model-based reconstruction of synthetic promoter library in Corynebacterium glutamicum.Biotechnol Lett,2018,40(5):819-827.
[16]Liu YH,Lin S,Liu K,et al.High-level expression of the Streptomyces mobaraense CICC 11018transglutaminase in Corynebacterium glutamicum ATCC 13032.Appl Biochem Microbiol,2014,50(5):456-462.
[17]Rytter JV,Helmark S,Chen J,et al.Synthetic promoter libraries for Corynebacterium glutamicum.Appl Microbiol Biotechnol,2014,98(6):2617-2623.
[18]Yim SS,An SJ,Kang M,et al.Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum.Biotechnol Bioeng,2013,110(11):2959-2969.
[19]Zhang W,Zhao ZH,Yang YK,et al.Construction of an expression vector that uses the aph promoter for protein expression in Corynebacterium glutamicum.Plasmid,2017,94:1-6.
[20]Shi F,Luan MY,Li YF.Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producingγ-aminobutyrate in Corynebacterium glutamicum.AMB Express,2018,8(1):61.
[21]Kikuchi Y,Date M,Itaya H,et al.Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869.Appl Environ Microbiol,2006,72(11):7183-7192.
[22]Suzuki N,Watanabe K,Okibe N,et al.Identification of new secreted proteins and secretion of heterologous amylase by C.glutamicum.Appl Microbiol Biotechnol,2009,82(3):491-500.
[23]Watanabe K,Tsuchida Y,Okibe N,et al.Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences.Microbiology,2009,155:741-750.
[24]van der Rest ME,Lange C,Molenaar D.A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.Appl Microbiol Biotechnol,1999,52(4):541-545.
[25]Liu XX,Zhao ZH,Zhang W,et al.Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum.Eng Life Sci,2017,17(10):1118-1125.
[26]Mart??n JF,Barreiro C,González-Lavado E,et al.Ribosomal RNA and ribosomal proteins in corynebacteria.J Biotechnol,2003,104(1/3):41-53.
[27]Mutalik VK,Guimaraes JC,Cambray G,et al.Precise and reliable gene expression via standard transcription and translation initiation elements.Nat Methods,2013,10(4):354-360.
[28]Matsuda Y,Itaya H,Kitahara Y,et al.Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.Microb Cell Fact,2014,13(1):56.
[29]An SJ,Yim SS,Jeong KJ.Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide.Protein Expr Purif,2013,89(2):251-257.
[30]Liu TY,Zhang JG.High-level expression and characterization of Aspergillus niger ATCC 1015xylanase B in Komagataella phaffii.Appl Biolog Chem,2018,61(4):373-381.
[2]Pa?s G,Berrin JG,Beaugrand J.GH11 xylanases:structure/function/properties relationships and applications.Biotechnol Adv,2012,30(3):564-592.
[3]Chen AN,Sun Y,Zhang W,et al.Downsizing a pullulanase to a small molecule with improved soluble expression and secretion efficiency in Escherichia coli.Microb Cell Fact,2016,15(1):9.
[4]Guan CR,Cui WJ,Cheng JT,et al.Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.Microb Cell Fact,2015,14(1):150.
[5]Meissner D,Vollstedt A,van Dijl JM,et al.Comparative analysis of twin-arginine(Tat)-dependent protein secretion of a heterologous model protein(GFP)in three different Gram-positive bacteria.Appl Microbiol Biotechnol,2007,76(3):633-642.
[6]Hermann T.Industrial production of amino acids by coryneform bacteria.J Biotechnol,2003,104(1/3):155-172.
[7]Zhang XM,Dou WF,Xu HY,et al.Metabolic flux analysis L-serine synthesis by Corynebacterium glutamicum SYPS-062.Chin J Biotech,2010,26(10):1363-1371(in Chinese).张晓梅,窦文芳,许泓瑜,等.谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析.生物工程学报,2010,26(10):1363-1371.
[8]Huan XJ,Li K,Shi F,et al.Overexpression of Corynebacterium glutamicum NAD kinase improves the L-isoleucine biosynthesis.Chin J Biotech,2012,28(9):1038-1047(in Chinese).还晓静,李坤,史锋,等.谷氨酸棒杆菌NAD激酶的过表达对L-异亮氨酸合成的促进作用.生物工程学报,2012,28(9):1038-1047.
[9]Liu XX,Zhang W,Zhao ZH,et al.Protein secretion in Corynebacterium glutamicum.Crit Rev Biotechnol,2017,37(4):541-551.
[10]Kalinowski J,Bathe B,Bartels D,et al.The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins.JBiotechnol,2003,104(1/3):5-25.
[11]Zhao ZH,Liu XX,Zhang W,et al.Construction of genetic parts from the Corynebacterium glutamicum genome with high expression activities.Biotechnol Lett,2016,38(12):2119-2126.
[12]Liu XX,Zhao ZH,Sun Y,et al.Selection of endogenous expression elements from Corynebactrium glutamicum.Microbiol China,2016,43(8):1671-1678(in Chinese).刘秀霞,赵子豪,孙杨,等.谷氨酸棒杆菌内源表达元件的筛选.微生物学通报,2016,43(8):1671-1678.
[13]Yim SS,An SJ,Choi JW,et al.High-level secretory production of recombinant single-chain variable fragment(scFv)in Corynebacterium glutamicum.Appl Microbiol Biotechnol,2014,98(1):273-284.
[14]Zou YL,Chen T,Feng LL,et al.Enhancement of5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in Corynebacterium glutamicum.Biotechnol Lett,2017,39(9):1369-1374.
[15]Zhang SH,Liu DY,Mao ZT,et al.Model-based reconstruction of synthetic promoter library in Corynebacterium glutamicum.Biotechnol Lett,2018,40(5):819-827.
[16]Liu YH,Lin S,Liu K,et al.High-level expression of the Streptomyces mobaraense CICC 11018transglutaminase in Corynebacterium glutamicum ATCC 13032.Appl Biochem Microbiol,2014,50(5):456-462.
[17]Rytter JV,Helmark S,Chen J,et al.Synthetic promoter libraries for Corynebacterium glutamicum.Appl Microbiol Biotechnol,2014,98(6):2617-2623.
[18]Yim SS,An SJ,Kang M,et al.Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum.Biotechnol Bioeng,2013,110(11):2959-2969.
[19]Zhang W,Zhao ZH,Yang YK,et al.Construction of an expression vector that uses the aph promoter for protein expression in Corynebacterium glutamicum.Plasmid,2017,94:1-6.
[20]Shi F,Luan MY,Li YF.Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producingγ-aminobutyrate in Corynebacterium glutamicum.AMB Express,2018,8(1):61.
[21]Kikuchi Y,Date M,Itaya H,et al.Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869.Appl Environ Microbiol,2006,72(11):7183-7192.
[22]Suzuki N,Watanabe K,Okibe N,et al.Identification of new secreted proteins and secretion of heterologous amylase by C.glutamicum.Appl Microbiol Biotechnol,2009,82(3):491-500.
[23]Watanabe K,Tsuchida Y,Okibe N,et al.Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences.Microbiology,2009,155:741-750.
[24]van der Rest ME,Lange C,Molenaar D.A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.Appl Microbiol Biotechnol,1999,52(4):541-545.
[25]Liu XX,Zhao ZH,Zhang W,et al.Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum.Eng Life Sci,2017,17(10):1118-1125.
[26]Mart??n JF,Barreiro C,González-Lavado E,et al.Ribosomal RNA and ribosomal proteins in corynebacteria.J Biotechnol,2003,104(1/3):41-53.
[27]Mutalik VK,Guimaraes JC,Cambray G,et al.Precise and reliable gene expression via standard transcription and translation initiation elements.Nat Methods,2013,10(4):354-360.
[28]Matsuda Y,Itaya H,Kitahara Y,et al.Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.Microb Cell Fact,2014,13(1):56.
[29]An SJ,Yim SS,Jeong KJ.Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide.Protein Expr Purif,2013,89(2):251-257.
[30]Liu TY,Zhang JG.High-level expression and characterization of Aspergillus niger ATCC 1015xylanase B in Komagataella phaffii.Appl Biolog Chem,2018,61(4):373-381.