解淀粉芽胞杆菌不同整合位点对外源碱性蛋白酶表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0755

摘要/Abstract

摘要：

为探究基因组上不同整合位点对外源碱性蛋白酶表达的影响,基于解淀粉芽胞杆菌TCCC 111018基因组信息,通过预测基因组上复制起始位点OriC确定了yaah基因整合位点;与此同时,通过分析6个胞外蛋白酶基因（epr、vpr、mpr、apr、bpr和nprE）单独缺失后对外源碱性蛋白酶酶活力的影响,并对发酵上清液中主要分泌蛋白的分析和质谱鉴定,确定了nprE和amyE基因位置为另外两个整合位点。在确定的3个整合位点处分别整合克劳氏芽胞杆菌来源的碱性蛋白酶基因aprE,并对整合菌株的碱性蛋白酶酶活力进行对比分析,结果表明整合菌株解淀粉芽胞杆菌18-ΔY∷aprE 和18-ΔN∷aprE碱性蛋白酶酶活力分别达到784.36 U/mL和1 289.09 U/mL,分别比原始菌株提高了15%和88.9%,实时荧光定量PCR和SDS-PAGE凝胶电泳表明18-ΔN∷aprE的碱性蛋白酶的转录水平和表达量均最高,这表明在nprE基因位点整合碱性蛋白酶基因可以有效提高碱性蛋白酶表达量,为进一步构建工业酶生产菌株奠定了基础。

关键词: 解淀粉芽胞杆菌, 整合位点, 碱性蛋白酶, 异源表达

Abstract:

In order to explore the effects of different gene integration sites on the expression of exogenous alkaline protease in the genome of Bacillus amyloliquefaciens,based on the genomic information of B. amyloliquefaciens TCCC 111018,the integration site of yaah gene was determined by predicting the replication initiation site OriC on the genome. In addition,the effects of 6 extracellular protease genes（epr,vpr,mpr,apr,bpr and nprE）after deleted individually on the activity of exogenous alkaline protease were analyzed,and the main secreted proteins in the fermentation supernatant were analyzed and identified by mass spectrometry. It is determined that the positions of the nprE and amyE genes are the other two integration sites. The alkaline protease gene aprE,from Bacillus clausii was integrated at three integration sites,then the alkaline protease activity of the integrated strain was compared and analyzed. The results showed that the alkaline protease activity of the integrated strain B.amyloliquefaciens 18-ΔY∷aprE and 18-ΔN∷aprE reached 784.36 U/mL and 1 289.09 U/mL,respectively,which were 15% and 88.9% higher than that of the original strain. Real-time fluorescent quantitative PCR and SDS-PAGE gel electrophoresis indicated that the transcription level and expression level of the alkaline protease of 18-ΔN∷aprE were the highest.The results showed that the integration of alkaline protease gene at nprE gene site effectively improved the expression of alkaline protease quantity,which laid a foundation for the further construction of industrial enzyme production strain.

Key words: Bacillus amyloliquefaciens, integration sites, alkaline protease, heterologous expression

牛馨, 张莹, 王茂军, 刘文龙, 路福平, 李玉. 解淀粉芽胞杆菌不同整合位点对外源碱性蛋白酶表达的影响[J]. 生物技术通报, 2022, 38(4): 253-260.

NIU Xin, ZHANG Ying, WANG Mao-jun, LIU Wen-long, LU Fu-ping, LI Yu. Effects of Different Integration Sites on the Expression of Exogenous Alkaline Protease in Bacillus amyloliquefaciens[J]. Biotechnology Bulletin, 2022, 38(4): 253-260.

图/表 13

参考文献 29

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菌株/质粒Strain/Plasmid	特性/目的Characteristics/Purpose	来源Source
菌株Strain
E.coli EC135 pM.Bam	对质粒DNA进行甲基化修饰 Plasmid DNA methylation modifcation	中科院微生物研究所 Institute of Microbiology,Chinese Academy of Sciences
E.coli JM109	用于敲除质粒的构建Construction of knockout vectors	本实验室Author’s lab
B.amyloliquefaciens111018	出发菌株Parent host	本实验室Author’s lab
B.amyloliquefaciens18-ΔM	mpr基因缺失mpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔB	bpr基因缺失bpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔE	epr基因缺失epr gene deletion	本研究This work
B.amyloliquefacien18-ΔV	vpr基因缺失vpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔN	nprE基因缺失nprE gene deletion	本研究This work
B.amyloliquefaciens18-ΔA	apr基因缺失apr gene deletion	本研究This work
B.amyloliquefaciens18-ΔN∷aprE	在nprE位置整合aprE Integration of aprE in nprE site	本研究This work
B.amyloliquefaciens18-Δα∷aprE	在amyE位置整合aprE Integration of aprE in amyE site	本研究This work
B.amyloliquefaciens18-ΔY∷aprE	在yaah位置整合aprEIntegration of aprE in yaah site	本研究This work
质粒Plasmid
pWH-T2	穿梭表达载体Shuttle expression vector	湖北大学Hubei University
pLY-2	aprE表达载体aprE expression cassette	本实验室Author’s lab

菌株/质粒Strain/Plasmid	特性/目的Characteristics/Purpose	来源Source
菌株Strain
E.coli EC135 pM.Bam	对质粒DNA进行甲基化修饰 Plasmid DNA methylation modifcation	中科院微生物研究所 Institute of Microbiology,Chinese Academy of Sciences
E.coli JM109	用于敲除质粒的构建Construction of knockout vectors	本实验室Author’s lab
B.amyloliquefaciens111018	出发菌株Parent host	本实验室Author’s lab
B.amyloliquefaciens18-ΔM	mpr基因缺失mpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔB	bpr基因缺失bpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔE	epr基因缺失epr gene deletion	本研究This work
B.amyloliquefacien18-ΔV	vpr基因缺失vpr gene deletion	本研究This work
B.amyloliquefaciens18-ΔN	nprE基因缺失nprE gene deletion	本研究This work
B.amyloliquefaciens18-ΔA	apr基因缺失apr gene deletion	本研究This work
B.amyloliquefaciens18-ΔN∷aprE	在nprE位置整合aprE Integration of aprE in nprE site	本研究This work
B.amyloliquefaciens18-Δα∷aprE	在amyE位置整合aprE Integration of aprE in amyE site	本研究This work
B.amyloliquefaciens18-ΔY∷aprE	在yaah位置整合aprEIntegration of aprE in yaah site	本研究This work
质粒Plasmid
pWH-T2	穿梭表达载体Shuttle expression vector	湖北大学Hubei University
pLY-2	aprE表达载体aprE expression cassette	本实验室Author’s lab

引物Primer	序列Sequence（5'-3'）
Up-F	TTAACGAATTCCTGCAGCCCGGGCAGAAAGACCA- TCCACAC
Up-R	TCATCCGCCAAAGCAGATAACGGATGATTC
Down-F	CAGCCCTCAGCTGACTGAAAAACCATTTATCATTG
Down-R	ATCCTTTGATCTTTTCTACGAGCTCCCGCTTATTAA- G ACGGC
djh-up-F	TAGCACACGTTTCATTGCAAATC
djh-up-R	CCGACTGCGCAAAAGACATAATC
djh-down-F	CCGACTGCGCAAAAGACATAATC
djh-down-R	TCTGAGCGCAGAAACGG
sjh-F	TAGCACACGTTTCATTGCAAATC
sjh-R	TCTGAGCGCAGAAACGG

引物Primer	序列Sequence（5'-3'）
Up-F	TTAACGAATTCCTGCAGCCCGGGCAGAAAGACCA- TCCACAC
Up-R	TCATCCGCCAAAGCAGATAACGGATGATTC
Down-F	CAGCCCTCAGCTGACTGAAAAACCATTTATCATTG
Down-R	ATCCTTTGATCTTTTCTACGAGCTCCCGCTTATTAA- G ACGGC
djh-up-F	TAGCACACGTTTCATTGCAAATC
djh-up-R	CCGACTGCGCAAAAGACATAATC
djh-down-F	CCGACTGCGCAAAAGACATAATC
djh-down-R	TCTGAGCGCAGAAACGG
sjh-F	TAGCACACGTTTCATTGCAAATC
sjh-R	TCTGAGCGCAGAAACGG

引物Primer	序列Sequence（5'-3'）
16S-F	GGGCTACACACGTGCTACAATGG
16S-R	GTATTCACCGCGGCATGCTG
aprE-F	GAGCACATACCCAGGTTCAACG
aprE -R	GTTGCCGCTTCTGCATTGAC