Process for preparing genetically transformed yeasts capable of producing a molecule of interest at a high titre

A method for preparing a genetically transformed yeast producing a high titre of a molecule of interest, comprising:

• (a) providing two integration plasmids with up to four expression cassettes, each integration plasmid comprising at least two expression cassettes and optionally a selectable marker, wherein the different expression cassettes encode proteins which are members of a metabolic pathway involved in the synthesis of the molecule of interest;
• (b) stably integrating multiple copies of the two integration plasmids into a population of yeast cells by co-transforming the plasmids into the yeast;
• (c) performing a primary screen to select at least 30 yeast clones, wherein selection of the clones is based on the presence of the expression cassettes, or selection for the expression of the selectable marker when such a marker is present;
• and
• (d) performing a functional secondary screen on the at least 30 yeast clones selected in the primary screen, to identify a yeast isolate producing a high titre of a molecule of interest.