Construction of a genomic library of the lactose utilising yeast Kluyveromyces marxianus var. fragilis for transformation into Saccharomyces cerevisiae

Abstract

The object of this project was to create a genomic library of an industrial Kluyveromyces marxianus var. fragilis strain as a preliminary step to cloning the genes responsible for lactose utilisation into Saccharomyces cerevisiae. Lactose is the major carbohydrate component of whey, a major byproduct of the dairy industry. Currently most whey is dumped which is both a waste of a potential resource and is a source of environmental contamination. Lactose could in theory be used a substrate for the production of ethanol however traditional brewing strains of yeast (mostly S.cerevisiae strains) cannot utilise lactose. One possible approach to get around this problem would be to clone the genes responsible for lactose utilisation from a lactose utilising yeast (in this case the genes LAC4 and LAC12 from K.marxianus var.fragilis) into S.cerevisiae. As a preliminary step in the cloning LAC4 and LAC12 into S.cerevisiae a genomic library of an industrial strain of the lactose-utilising yeast, K.marxianus var. fragilis was constructed. Partially Sau3AI digested, size selected fragments of K.marxianus var.fragilis DNA were cloned into the Saccharomyces-Eschericia coli shuttle vector, YEp 13 and the resulting recombinant plasmids were transformed into E.coli DH5α. The resulting library was found to contain approximately 15 000 recombinant plasmid bearing clones with an average insert size of 7.7kb. This library thus contains approximately 116 OOOkb of DNA which is equivalent to approximately 8 times the genome size of K.marxianus var. fragilis. The two genes of interest, LAC4 (which encodes β-galactosidase) and LAC12 (which encodes lactose permease), are closely linked, being divergently transcribed from an approximately 2.6kb intergenic region, and are coregulated. If these genes are treated as a single entity then there is a greater than 99% chance that this library will contain the genes of interest. However since the genes of interest occur on a region of DNA approximately 10kb in size, which is greater than the average insert size, the chances of successfully cloning the required genes in a single DNA fragment will be much reduced. However should an attempt be made to 'shot-gun' clone these genes into S.cerevisiae a procedure for high efficiency transformation of S.cerevisiae with the plasmid YEp13 has been demonstrated. Using the spheroplast transformation method a transformation efficiency of 10⁶ transformants/µg YEp13 DNA was obtained.