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United States Department of Agriculture

Agricultural Research Service

Research Project: ANAEROBIC MICROBIOLOGICAL PROCESSES IN ANIMAL MANURE MANAGEMENT Title: Identification and Use of the Putative Bacteroides ovatus Xylanase Promoter for the Inducible Production of Recombinant Human Proteins

Authors
item Hamady, Zaed - UNIV LEEDS, UK
item Farrar, Mark - UNIV LEEDS, UK
item Whitehead, Terence
item Holland, Keith - UNIV LEEDS, UK
item Lodge, J - UNIV LEEDS, UK
item Carding, Simon - UNIV LEEDS, UK

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 10, 2008
Publication Date: October 1, 2008
Citation: Hamady, Z.Z.R., Farrar, M.D., Whitehead, T.R., Holland, K.T., Lodge, J.P.A., Carding, S.R. 2008. Identification and Use of the Putative Bacteroides ovatus Xylanase Promoter for the Inducible Production of Recombinant Human Proteins. Microbiology. 154(10):3165-3174.

Interpretive Summary: Chronic disorders of the human gastrointestinal (GI) tract, for example inflammatory bowel disease (IBD) which includes the disorders Crohn’s disease and ulcerative colitis, affect a significant proportion of the population in developed countries such as the United States. Present therapies are not curative and may cause adverse side effects during treatment. Therefore, there is a need for more targeted and controlled forms of immunotherapy. Previous work has indicated that commensal bacteria that normally inhabit the GI tract might prove useful for treating certain forms of bowel disease. Therefore, a human isolate of the commensal bacterium Bacteroides ovatus was engineered in our laboratories as a probiotic for production and secretion of various proteins important for the treatment of IBD using a xylan-inducing operon. The present work describes the identification of the xylan-operon controlling region and controlled expression of another intestinal factor important in immunotherapy for inflammatory gut diseases.

Technical 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 rapid amplification of 5' cDNA ends (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.

Last Modified: 11/23/2014
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