Effects of Different Integration Sites on the Expression of Exogenous Alkaline Protease in Bacillus amyloliquefaciens

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

Abstract

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

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.

Figures/Tables 13

References 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