Patent ID: 8652772

Claim:
A yeast produced by a process comprising: (a) transforming yeast cells with a replicative and integrative plasmid comprising an autonomous replicating sequence, exogenous DNA, and a first selection marker; and (b) repeatedly replicating the cells from step (a) to produce a number of generations of progeny cells while selecting for cells which include the selection marker, promoting the retention of the replicative and integrative plasmid in subsequent generations of the progeny cells to produce progeny cells having multiple integrated copies of the exogenous DNA, and wherein the method further includes the step of repeatedly replicating the progeny cells to produce a number of generations of progeny cells in the absence of selection for cells which include the selection marker, so as to promote the loss of the plasmid in subsequent generations of progeny cells and recovering the cells each containing multiple copies of the exogenous DNA integrated into its chromosomal DNA, wherein the cells are yeast cells, wherein the exogenous DNA encodes proteins having xylose reductase activity, xylitol dehydrogenase activity, and xylulokinase activity, which also serve as the first selection marker, wherein the yeast cells comprise at least 10 copies of the exogenous DNA integrated into chromosomal DNA, wherein the yeast ferments xylose to ethanol, and wherein fermentation activity does not decrease after culture in non-selective medium for 20 generations.