99A-27 |
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M. J. MILLER, R. Barrangou, I. Austdal, B. Cargile, and T. R. Klaenhammer. Southeast Dairy Foods Research Ctr., North Carolina State Univ., Dept. of Food Science, Box 7624, Raleigh, NC 27695-7624 Lactobacillus acidophilus is a probiotic organism that displays the ability to utilize a variety of carbohydrates including fructooligosaccharides (FOS). Prebiotics, including FOS, stimulate the growth of beneficial commensals in the gastrointestinal tract. FOS are a diverse family of fructose polymers used commercially in food products and nutritional supplements. The dietary intake averages 2.5 grams of oligofructose daily, originating primarily from wheat and onion. Previous studies identified the FOS utilization operon in L. acidophilus and determined that transcription of the operon was induced by FOS and repressed by glucose, suggesting carbon catabolite repression. Our objective was to construct an expression plasmid inducible in the gastrointestinal tract upon feeding the prebiotic FOS, based on the FOS operon promoter. We constructed an expression vector (pFOS::GUS) with the FOS promoter fused to gusA3, a reporter gene derived from the beta-glucuronidase (gusA) identified from L. gasseri. For comparison, the constitutive P6 promoter was also fused to gusA3 (pP6::GUS). Subsequently, L. acidophilus was transformed with pFOS::GUS and pP6::GUS. Transformants were evaluated for GUS activity in the presence of two different carbon sources, glucose and FOS. For L. acidophilus transformants containing pFOS::GUS, growth in glucose as the sole carbon source resulted in an increase of 1.4X the activity of native L. acidophilus (GUS negative). L. acidophilus transformants containing pFOS::GUS grown in FOS as the sole carbon source have 2.5X the GUS activity compared to glucose as the sole carbon source. For L. acidophilus transformants with pP6::GUS, the GUS activity was at least 10X nativeL. acidophilus in both conditions indicating strong constitutive expression of gusA by the P6 promoter. The FOS promoter is an inducible expression system for the probiotic L. acidophilus with relatively low expression under non-inducing conditions. Such a vector could offer several potential applications including oral delivery of enzymes, proteins or vaccines.
Session 99A, Biotechnology: General
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