生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 253-260.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0755
牛馨1(), 张莹1, 王茂军1, 刘文龙2, 路福平1, 李玉1()
收稿日期:
2021-06-10
出版日期:
2022-04-26
发布日期:
2022-05-06
通讯作者:
李玉,女,博士,教授,研究方向:工业酶制剂开发;E-mail: liyu@tust.edu.cn作者简介:
牛馨,女,硕士研究生,研究方向:发酵工程;E-mail: niuxin0968@outlook.com
基金资助:
NIU Xin1(), ZHANG Ying1, WANG Mao-jun1, LIU Wen-long2, LU Fu-ping1, LI Yu1()
Received:
2021-06-10
Published:
2022-04-26
Online:
2022-05-06
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。
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.
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 |
表1 研究中使用的菌株和质粒
Table 1 Bacterial strains and plasmids used in this study
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 |
表2 vpr基因缺失的引物设计
Table 2 Primers for vpr gene deletion
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 |
表3 用于实时荧光定量PCR的引物
Table 3 Primers for real-time fluorescent quantitative PCR
引物Primer | 序列Sequence(5'-3') |
---|---|
16S-F | GGGCTACACACGTGCTACAATGG |
16S-R | GTATTCACCGCGGCATGCTG |
aprE-F | GAGCACATACCCAGGTTCAACG |
aprE -R | GTTGCCGCTTCTGCATTGAC |
图5 TCCC 111018发酵液上清SDS-PAGE检测 M:1kb marker;1,2:TCCC 111018的发酵上清液
Fig.5 SDS-PAGE detection of TCCC 111018 fermentation broth supernatant M:1kb marker. 1 and 2:Fermentation supernatant of TCCC 111018
RT | Mass | NCBI ID | Compound name |
---|---|---|---|
1 | 58458.9 | P00692 | Alpha-amylase |
1.1 | 218451.1 | Q8TF72 | Protein Shroom3 |
1.2 | 77037.6 | O18740 | Keratin,type I cytoskeletal 9 |
1.3 | 56182.7 | B0TWF7 | Chromosomal replication initiator protein DnaA |
1.4 | 11230.8 | A5CR30 | 50S ribosomal protein L21 |
1.5 | 151424.3 | A4VHM3 | DNA-directed RNA polymerase subunit beta |
1.6 | 40412.9 | QQ4FN99 | Peptide chain release factor 1 |
1.7 | 18115.2 | A0NDK8 | Pro-corazonin |
1.8 | 100713.8 | Q65CL1 | Catenin alpha-3 |
1.9 | 35715.8 | G8GV69 | Dioxygenase easH |
2 | 22703.3 | Q6AGN3 | Nucleoside triphosphate pyrophosphatase |
2.1 | 33273.6 | A0R5C5 | UDP-glucose 4-epimerase |
2.2 | 35380.5 | Q7TVA4 | D-fructose 1,6-bisphosphatase class 2 |
2.3 | 107560.6 | A1A5S1 | Pre-mRNA-processing factor 6 |
2.4 | 75310.5 | A6TEG6 | Penicillin-binding protein activator LpoA |
2.5 | 229710 | P33144 | Separin |
2.6 | 43491.7 | A5GS98 | Ferrochelatase |
2.7 | 13835.7 | A9BNG3 | 30S ribosomal protein S6 |
2.8 | 52452.5 | A1UEN7 | Cobyric acid synthase |
2.9 | 48850.9 | Q28RY2 | Trigger factor |
3 | 40629.4 | Q5ZJ37 | Rab9 effector protein with kelch motifs |
3.1 | 86619.2 | P49051 | S-layer protein sap |
表4 质谱结果分析
Table 4 Analysis of mass spectrometry results
RT | Mass | NCBI ID | Compound name |
---|---|---|---|
1 | 58458.9 | P00692 | Alpha-amylase |
1.1 | 218451.1 | Q8TF72 | Protein Shroom3 |
1.2 | 77037.6 | O18740 | Keratin,type I cytoskeletal 9 |
1.3 | 56182.7 | B0TWF7 | Chromosomal replication initiator protein DnaA |
1.4 | 11230.8 | A5CR30 | 50S ribosomal protein L21 |
1.5 | 151424.3 | A4VHM3 | DNA-directed RNA polymerase subunit beta |
1.6 | 40412.9 | QQ4FN99 | Peptide chain release factor 1 |
1.7 | 18115.2 | A0NDK8 | Pro-corazonin |
1.8 | 100713.8 | Q65CL1 | Catenin alpha-3 |
1.9 | 35715.8 | G8GV69 | Dioxygenase easH |
2 | 22703.3 | Q6AGN3 | Nucleoside triphosphate pyrophosphatase |
2.1 | 33273.6 | A0R5C5 | UDP-glucose 4-epimerase |
2.2 | 35380.5 | Q7TVA4 | D-fructose 1,6-bisphosphatase class 2 |
2.3 | 107560.6 | A1A5S1 | Pre-mRNA-processing factor 6 |
2.4 | 75310.5 | A6TEG6 | Penicillin-binding protein activator LpoA |
2.5 | 229710 | P33144 | Separin |
2.6 | 43491.7 | A5GS98 | Ferrochelatase |
2.7 | 13835.7 | A9BNG3 | 30S ribosomal protein S6 |
2.8 | 52452.5 | A1UEN7 | Cobyric acid synthase |
2.9 | 48850.9 | Q28RY2 | Trigger factor |
3 | 40629.4 | Q5ZJ37 | Rab9 effector protein with kelch motifs |
3.1 | 86619.2 | P49051 | S-layer protein sap |
图9 整合菌株AmyE的SDS-PAGE检测 M:1 kb marker;Δα:菌株18-Δα∷aprE的发酵液上清;ΔY:菌株18-ΔY∷aprE的发酵液上清;ΔN:菌株18-ΔN∷aprE的发酵液上清;18:菌株TCCC 111018的发酵液上清
Fig.9 SDS-PAGE detection of AmyE in the integrated strains M:1 kb marker. Δα:Fermentation supernatant of strain 18-Δα∷aprE. ΔY:Fermentation supernatant of strain 18-ΔY∷aprE. ΔN:Fermentation supernatant of strain 18-ΔN∷aprE. 18:Fermentation supernatant of strain TCCC 111018
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