Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins

• Published in: Microbiology (Society for General Microbiology) Vol. 154; no. 10; pp. 3165 - 3174
• Main Authors: Hamady, Zaed Z. R, Farrar, Mark D, Whitehead, Terence R, Holland, Keith T, Lodge, J. Peter A, Carding, Simon R
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.10.2008; Society for General Microbiology
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteroides - genetics; Bacteroides - metabolism; Bacteroides ovatus; Base Sequence; Biological and medical sciences; Biotechnology; Chromosomes, Bacterial - genetics; Cloning, Molecular; DNA, Bacterial - genetics; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Reporter; Genetic Engineering - methods; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Molecular Sequence Data; Operon; Other active biomolecules; Ovatus; Peptides - metabolism; Production of active biomolecules; Promoter Regions, Genetic; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transcription Initiation Site; Trefoil Factor-3; Xylans - metabolism; Human; Transcription; Enzyme; Identification; Genetically modified microorganism; Bacteroidaceae; Bacteroides; Glycosylases; Complementary DNA; Activator; Glycosidases; Bacteria; Hydrolases; Xylan endo-1,3-β-xylosidase; Recombinant protein; Reverse transcription polymerase chain reaction
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2008/019109-0

1 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK 2 Fermentation Biotechnology Research, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA 3 School of Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, UK Correspondence Simon R. Carding Simon.Carding{at}BBSRC.ac.uk 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 . Abbreviations: IL-2, interleukin-2; 5'RACE, rapid amplification of 5' cDNA ends; TFF-3, trefoil factor 3; TIS, transcription initiation site The GenBank accession number for the B. ovatus promoter and operon is EU334491.