Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (4): 253-260.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0755
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NIU Xin1(), ZHANG Ying1, WANG Mao-jun1, LIU Wen-long2, LU Fu-ping1, LI Yu1()
Received:
2021-06-10
Online:
2022-04-26
Published:
2022-05-06
Contact:
LI Yu
E-mail:niuxin0968@outlook.com;liyu@tust.edu.cn
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 |
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 |
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 |
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 |
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 |
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 |
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
[1] |
Ohmiya K, Tanimura S, Yashi TK, et al. Application of immobilized alkaline protease to cheese-making[J]. J Food Sci, 1979, 44(6):1584-1588.
doi: 10.1111/j.1365-2621.1979.tb09095.x URL |
[2] |
Raval VH, Pillai S, Rawal CM, et al. Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria[J]. Process Biochem, 2014, 49(6):955-962.
doi: 10.1016/j.procbio.2014.03.014 URL |
[3] |
Mahmoudian M. Biocatalytic production of chiral pharmaceutical intermediates[J]. Biocatal Biotransformation, 2000, 18(2):105-118.
doi: 10.3109/10242420009015240 URL |
[4] | Zhou C, Qin HL, Chen XJ, et al. A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry[J]. Sci Rep, 2018, 8(1):1-18. |
[5] |
Omrane Benmrad M, Moujehed E, Ben Elhoul M, et al. A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101[J]. Int J Biol Macromol, 2016, 91:961-972.
doi: 10.1016/j.ijbiomac.2016.06.025 pmid: 27296442 |
[6] |
Baweja M, Tiwari R, Singh PK, et al. An alkaline protease from Bacillus pumilus MP 27:functional analysis of its binding model toward its applications as detergent additive[J]. Front Microbiol, 2016, 7:1195.
doi: 10.3389/fmicb.2016.01195 pmid: 27536284 |
[7] |
Sauer C, Syvertsson S, Bohorquez LC, et al. Effect of genome position on heterologous gene expression in Bacillus subtilis:an unbiased analysis[J]. ACS Synth Biol, 2016, 5(9):942-947.
doi: 10.1021/acssynbio.6b00065 URL |
[8] |
Bryant JA, Sellars LE, Busby SJ, et al. Chromosome position effects on gene expression in Escherichia coli K-12[J]. Nucleic Acids Res, 2014, 42(18):11383-11392.
doi: 10.1093/nar/gku828 pmid: 25209233 |
[9] |
Kurat CF, Yeeles JTP, Patel H, et al. Chromatin controls DNA replication origin selection, lagging-strand synjournal, and replication fork rates[J]. Mol Cell, 2017, 65(1):117-130.
doi: 10.1016/j.molcel.2016.11.016 URL |
[10] |
Couturier E, Rocha EPC. Replication-associated gene dosage effects shape the genomes of fast-growing bacteria but only for transcription and translation genes[J]. Mol Microbiol, 2006, 59(5):1506-1518.
pmid: 16468991 |
[11] |
Zhou C, Zhou H, Li D, et al. Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709[J]. Microb Cell Fact, 2020, 19(1):45.
doi: 10.1186/s12934-020-01307-2 URL |
[12] |
Tsirigotaki A, De Geyter J, Šoštaric N, et al. Protein export through the bacterial Sec pathway[J]. Nat Rev Microbiol, 2017, 15(1):21-36.
doi: 10.1038/nrmicro.2016.161 pmid: 27890920 |
[13] |
Mulder KCL, Bandola J, Schumann W. Construction of an artificial secYEG operon allowing high level secretion of α-amylase[J]. Protein Expr Purif, 2013, 89(1):92-96.
doi: 10.1016/j.pep.2013.02.008 URL |
[14] |
Ren GH, Cao LC, Kong W, et al. Efficient secretion of the β-galactosidase Bgal1-3 via both tat-dependent and tat-independent pathways in Bacillus subtilis[J]. J Agric Food Chem, 2016, 64(28):5708-5716.
doi: 10.1021/acs.jafc.6b01735 URL |
[15] |
Watzlawick H, Altenbuchner J. Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system[J]. AMB Express, 2019, 9(1):158.
doi: 10.1186/s13568-019-0884-4 pmid: 31571017 |
[16] | Zhang G, Wang W, Deng A, et al. A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria[J]. PLoS Genet, 2012, 8(9):e1002987. |
[17] |
McKenney PT, Driks A, Eichenberger P. The Bacillus subtilis endospore:assembly and functions of the multilayered coat[J]. Nat Rev Microbiol, 2013, 11(1):33-44.
doi: 10.1038/nrmicro2921 pmid: 23202530 |
[18] |
Zhang J, Xu X, Li X, et al. Reducing the cell Lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens[J]. Int J Biol Macromol, 2021, 167:777-786.
doi: 10.1016/j.ijbiomac.2020.11.193 URL |
[19] |
Ling Lin Fu, Zi Rong Xu, Wei Fen Li, et al. Protein secretion pathways in Bacillus subtilis:implication for optimization of heterologous protein secretion[J]. Biotechnol Adv, 2007, 25(1):1-12.
pmid: 16997527 |
[20] |
Zhang X, Xu ZY, Liu S, et al. Improving the production of salt-tolerant glutaminase by integrating multiple copies of mglu into the protease and 16S rDNA genes of Bacillus subtilis 168[J]. Molecules, 2019, 24(3):592.
doi: 10.3390/molecules24030592 URL |
[21] | Mo QS, Tian Y, Zhang HT, et al. Using 16S rDNA as target site for homologous recombination to improve the alkaline protease production of Bacillus alcalophilus[J]. Adv Mater Res, 2014, 886:349-354. |
[22] |
Wang H, Zhang X, Qiu J, et al. Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system[J]. J Ind Microbiol Biotechnol, 2019, 46(1):113-123.
doi: 10.1007/s10295-018-2089-2 URL |
[23] |
Kawamura F, Doi RH. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases[J]. J Bacteriol, 1984, 160(1): 442-444.
doi: 10.1128/jb.160.1.442-444.1984 pmid: 6434524 |
[24] |
Nakamura A, Toyama N, Kitamura A, et al. Use of a triple protease-deficient mutant of Bacillus subtilis as a host for secretion of a B. subtilis cellulase and TEM beta-lactamase[J]. Agric Biol Chem, 1991, 55(9):2367-2374.
pmid: 1368741 |
[25] |
Abe S, Yasumura A, Tanaka T. Regulation of Bacillus subtilis aprE expression by glnA through inhibition of scoC and σ D -dependent degR expression[J]. J Bacteriol, 2009, 191(9):3050-3058.
doi: 10.1128/JB.00049-09 URL |
[26] |
Kada S, Ishikawa A, Ohshima Y, et al. Alkaline serine protease AprE plays an essential role in poly-γ-glutamate production during natto fermentation[J]. Biosci Biotechnol Biochem, 2013, 77(4):802-809.
doi: 10.1271/bbb.120965 URL |
[27] |
Gupta R, Gigras P, Mohapatra H, et al. Microbial α-amylases:a biotechnological perspective[J]. Process Biochem, 2003, 38(11):1599-1616.
doi: 10.1016/S0032-9592(03)00053-0 URL |
[28] |
Wang P, Wang PL, Tian J, et al. A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens[J]. Sci Rep, 2016, 6(1):1-10.
doi: 10.1038/s41598-016-0001-8 URL |
[29] |
Reuβ DR, Altenbuchner J, Mäder U, et al. Large-scale reduction of the Bacillus subtilis genome:consequences for the transcriptional network, resource allocation, and metabolism[J]. Genome Res, 2017, 27(2):289-299.
doi: 10.1101/gr.215293.116 URL |
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