Studies on the plasmid stability, plasmid copy number and endo(1, 3)(1, 4) b-glucanase production by free and alginate immobilised recombinant saccharomyces cerevisiae cells

Abstract

A recombinant yeast strain, Saccharomyces cerevisiae DBY746, containing the plasmid pJG317, was grown in a variety of fermentation modes including batch, serial batch and chemostat culture incorporating a wide range of media types Plasmid pJG317 consists of a 2^-denved yeast episomal plasmid containing the gene which encodes for the bacterial enzyme endo (1,3)(1,4) P-glucanase. The concentration of enzyme produced appears to be proportional to the number of plasmid copies per cell. Specific enzyme activities were found to be in the range of 14 x 106 to 48 x lO6 U/cell for free cell culture, with a corresponding plasmid copy number of 8±0 5 to 40±6 3 copies per cell respectively. A procedure for measuring the copy number of pJG317 in S cerevisiae was developed, tested and optimised. The procedure is based on Southern hybridisation and measured the relative intensities of hybridisation of a probe to the single copy yeast chromosomal actin gene and to the multicopy plasmid pJG317. Plasmid pJG317 is quite unstable under non-selective conditions and its copy number and stability are influenced by both growth rate and nutrient supply By immobilising cells in calcium alginate gel beads, the plasmid could be stabilised and high volumetric productivities of up to 38 U/ml-h attained. Although radial gradients in biomass concentration and in percentage of plasmidcontaimng cells in the alginate gel beads were confirmed, no significant difference was found between the plasmid copy number of cells in the centre of the gel beads (36 5±6 8) and cells close to the surface of the gel beads(32 4± 33)