Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins
Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins
The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.
Bacterial Proteins/genetics, Bacteroides/genetics, Base Sequence, Chromosomes, Bacterial/genetics, Cloning, Molecular, DNA, Bacterial/genetics, Endo-1,4-beta Xylanases/genetics, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genes, Reporter, Genetic Engineering/methods, Humans, Molecular Sequence Data, Operon, Peptides/metabolism, Promoter Regions, Genetic, Recombinant Proteins/genetics, Reverse Transcriptase Polymerase Chain Reaction, Transcription Initiation Site, Trefoil Factor-3, Xylans/metabolism
3165-3174
Hamady, Zaed Z R
545a1c81-276e-4341-a420-aa10aa5d8ca8
Farrar, Mark D
5b6b2aad-3bb6-400f-9ae3-cb8f9be8cd57
Whitehead, Terence R
6d7a75b8-215f-4602-afba-07c97be26ba4
Holland, Keith T
59cafad5-1008-4848-aef6-bc9b0a249839
Lodge, J Peter A
c97dd104-0cb2-4f05-a008-9b9af01e4820
Carding, Simon R
1104c711-b95b-4ed5-9cc6-6d09f1f268ea
October 2008
Hamady, Zaed Z R
545a1c81-276e-4341-a420-aa10aa5d8ca8
Farrar, Mark D
5b6b2aad-3bb6-400f-9ae3-cb8f9be8cd57
Whitehead, Terence R
6d7a75b8-215f-4602-afba-07c97be26ba4
Holland, Keith T
59cafad5-1008-4848-aef6-bc9b0a249839
Lodge, J Peter A
c97dd104-0cb2-4f05-a008-9b9af01e4820
Carding, Simon R
1104c711-b95b-4ed5-9cc6-6d09f1f268ea
Hamady, Zaed Z R, Farrar, Mark D, Whitehead, Terence R, Holland, Keith T, Lodge, J Peter A and Carding, Simon R
(2008)
Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.
Microbiology (Reading, England), 154 (Pt 10), .
(doi:10.1099/mic.0.2008/019109-0).
Abstract
The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.
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Published date: October 2008
Keywords:
Bacterial Proteins/genetics, Bacteroides/genetics, Base Sequence, Chromosomes, Bacterial/genetics, Cloning, Molecular, DNA, Bacterial/genetics, Endo-1,4-beta Xylanases/genetics, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genes, Reporter, Genetic Engineering/methods, Humans, Molecular Sequence Data, Operon, Peptides/metabolism, Promoter Regions, Genetic, Recombinant Proteins/genetics, Reverse Transcriptase Polymerase Chain Reaction, Transcription Initiation Site, Trefoil Factor-3, Xylans/metabolism
Identifiers
Local EPrints ID: 455118
URI: http://eprints.soton.ac.uk/id/eprint/455118
ISSN: 1350-0872
PURE UUID: 825605ce-ce23-4a1b-b121-8bf75869c393
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Date deposited: 10 Mar 2022 17:31
Last modified: 17 Mar 2024 04:12
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Author:
Zaed Z R Hamady
Author:
Mark D Farrar
Author:
Terence R Whitehead
Author:
Keith T Holland
Author:
J Peter A Lodge
Author:
Simon R Carding
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