TANDEM INTEGRATION OF MULTIPLE ILV5 COPIES AND ELEVATED TRANSCRIPTIONIN POLYPLOID YEAST

An industrial yeast strain was modified by introducing DNA into brewin g yeast such that the derived cells contain only yeast DNA. Thus selec table markers and bacterial sequences are not present in the final str ain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunc tional plasmid containing a dominant resistance marker. Resistant colo nies were screened for site-directed integration of the ILV5 DNA. Cand idates were examined by several methods including Southern transfer an d polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressu re. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. nea-tandem) parental strain led to incre ased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identi cal to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromoso me. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.