High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.
Alkaline pectate lyase
Alkaline protease
Bacillus subtilis
Expression vectors
Pullulanase
pUC980
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
07 Feb 2020
07 Feb 2020
Historique:
received:
04
12
2019
accepted:
27
01
2020
entrez:
8
2
2020
pubmed:
8
2
2020
medline:
23
9
2020
Statut:
epublish
Résumé
pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.
Sections du résumé
BACKGROUND
BACKGROUND
pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed.
RESULTS
RESULTS
The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports.
CONCLUSION
CONCLUSIONS
The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.
Identifiants
pubmed: 32028973
doi: 10.1186/s12934-020-1296-5
pii: 10.1186/s12934-020-1296-5
pmc: PMC7006159
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
25Subventions
Organisme : the National Natural Science Fund of China
ID : 31701534
Organisme : the Tianjin outstanding talent training program, the Tianjin Science & Technology Planning Project
ID : 14ZCZDSY00157
Organisme : the Tianjin outstanding talent training program, the Tianjin Science & Technology Planning Project
ID : 15PTCYSY00020
Organisme : Yantai Marine economy innovation development demonstration project
ID : YHCX-SW-L-201703
Références
Curr Microbiol. 2007 Aug;55(2):89-93
pubmed: 17624574
Genome Res. 2015 Aug;25(8):1147-57
pubmed: 26063738
Plasmid. 2008 Sep;60(2):118-24
pubmed: 18582938
Ann N Y Acad Sci. 1991 Dec 27;646:69-77
pubmed: 1809207
Proteins. 2014 Sep;82(9):1685-93
pubmed: 24375572
Microb Cell Fact. 2017 Feb 20;16(1):32
pubmed: 28219382
J Appl Microbiol. 2004;96(3):569-78
pubmed: 14962137
EMBO J. 1987 Dec 1;6(12):3863-9
pubmed: 3123220
Appl Environ Microbiol. 1991 Apr;57(4):901-9
pubmed: 2059048
Appl Microbiol Biotechnol. 2017 Apr;101(7):2919-2929
pubmed: 28028551
DNA. 1986 Jun;5(3):219-25
pubmed: 3013549
Appl Microbiol Biotechnol. 2013 Jul;97(14):6113-27
pubmed: 23749118
Biochem Soc Trans. 1995 Aug;23(3):442S
pubmed: 8566331
Plasmid. 1988 May;19(3):231-41
pubmed: 2852818
FEMS Microbiol Lett. 2002 Apr 9;209(2):237-41
pubmed: 12007811
Mol Gen Genet. 1987 Jun;208(1-2):349-52
pubmed: 3112524
J Biotechnol. 2015 Sep 20;210:8-14
pubmed: 26116135
Can J Microbiol. 2004 Jan;50(1):1-17
pubmed: 15052317
Plasmid. 2005 Nov;54(3):241-8
pubmed: 16005967
Int J Biol Macromol. 2019 Sep 15;137:973-981
pubmed: 31295482
Front Microbiol. 2014 Dec 09;5:687
pubmed: 25538698
Wiley Interdiscip Rev RNA. 2018 Nov;9(6):e1500
pubmed: 30074293
J Biotechnol. 1999 Jun 11;72(1-2):185-95
pubmed: 12680365
Protein Expr Purif. 2006 Apr;46(2):189-95
pubmed: 16125412
Methods Enzymol. 2011;498:399-406
pubmed: 21601687
Biotechnol Lett. 2010 Jan;32(1):119-24
pubmed: 19728109
Microb Cell Fact. 2015 May 21;14:72
pubmed: 25990516
Microb Biotechnol. 2012 Mar;5(2):214-25
pubmed: 21895995
Plasmid. 1985 Nov;14(3):235-44
pubmed: 3006103
Science. 2017 Feb 24;355(6327):
pubmed: 28209641
J Bacteriol. 1983 Jun;154(3):1184-94
pubmed: 6406425
Lett Appl Microbiol. 2017 Sep;65(3):192-198
pubmed: 28631335
J Biosci Bioeng. 2019 Jan;127(1):8-15
pubmed: 30228040
Mol Gen Genet. 1988 Nov;214(3):482-9
pubmed: 3146018
Proc Natl Acad Sci U S A. 1978 Mar;75(3):1428-32
pubmed: 418412
Science. 1996 Nov 1;274(5288):777-80
pubmed: 8864116
Nucleic Acids Res. 2019 Apr 23;47(7):e40
pubmed: 30767015
Biomed Res Int. 2016;2016:3073949
pubmed: 27073802
N Biotechnol. 2016 May 25;33(3):372-9
pubmed: 26820123
Nucleic Acids Res. 1988 May 25;16(10):4389-406
pubmed: 2837734
Mol Microbiol. 1999 Aug;33(3):466-75
pubmed: 10417638
Microb Cell Fact. 2014 May 03;13:63
pubmed: 24885003
Sci Rep. 2017 Jan 10;7:40587
pubmed: 28071737
J Biotechnol. 2016 Apr 20;224:14-7
pubmed: 26953743
J Microbiol Methods. 2006 Jun;65(3):476-87
pubmed: 16216354
Annu Rev Biochem. 2013;82:25-54
pubmed: 23746253
Nucleic Acids Res. 1994 Sep;22(17):3485-7
pubmed: 7524021
Res Microbiol. 2004 Oct;155(8):605-10
pubmed: 15380546
J Microbiol Biotechnol. 2014 Apr;24(4):431-9
pubmed: 24375416
Mol Gen Genet. 1988 May;212(2):232-40
pubmed: 2841567
Annu Rev Biochem. 2017 Jun 20;86:515-539
pubmed: 28375743
Adv Biochem Eng Biotechnol. 2004;86:47-82
pubmed: 15088763
World J Microbiol Biotechnol. 2017 Oct 3;33(10):190
pubmed: 28975516
Plasmid. 2013 Jul;70(1):42-51
pubmed: 23415796