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.
Hamady
Zaed Z R
ZZR
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Farrar
Mark D
MD
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Whitehead
Terence R
TR
Fermentation Biotechnology Research, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
Holland
Keith T
KT
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Lodge
J Peter A
JPA
School of Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, UK.
Carding
Simon R
SR
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001439:Bacteroides / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009876:Operon; D010455:Peptides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024363:Transcription Initiation Site; D000071165:Trefoil Factor-3; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010455:Peptides; D011994:Recombinant Proteins; C082495:TFF3 protein, human; D000071165:Trefoil Factor-3; D014990:Xylans; D043364:Endo-1,4-beta Xylanases