Constitutive expression of a heterologous Eubacterium ruminantium xylanase gene (xynA) in Butyrivibrio fibrisolvens.

Abstract

An Eubacterium ruminantium xylanase gene (xynA) was inserted into pYK4, a shuttle vector replicable in both Escherichia coli and Butyrivibrio fibrisolvens, and the resultant chimeric plasmid (pYK4XT) was electroporated into B. fibrisolvens OB156C in an attempt to obtain a more xylanolytic B. fibrisolvens. Electrotransformants were screened by the development of erythromycin resistance, followed by an activity staining and Southern hybridization. The presence of mRNA from xynA in the transformant, B. fibrisolvens NO4, was confirmed by Northern hybridization. Xylanase activity of the transformant NO4 was apparently enhanced regardless of carbon sources in the medium. When grown on glucose or cellobiose. NO4 had approximately 5-6 times higher intracellular activity than the parent OB156C on a culture volume basis as well as protein basis. The transformant showed extracellular xylanase activity much higher (between 7- and 10(4)-fold) than the parent. Transformant NO4 recorded the highest activity when grown on xylan. Most (> 90%) of the activity was extracellular. The extracellular activity was 2-fold greater in NO4. These findings indicate that the introduced xynA was expressed constitutively and the xylanase protein was exported into the culture supernatant. Growth of NO4 on glucose was similar to that of OB156C, which suggests little extra load for plasmid maintenance and foreign xylanase production in the transformant. The plasmid pYK4XT was maintained stably in the transformant for more than 100 generations.