A. Gene deletion strategy

The advanced cloning-free PCR-based allele replacement strategy (Erdeniz et al., 1997) has been followed in the present study. The principle of this strategy is graphically depicted in Figure 1a. In the gene targeting experiment performed in the present study, the uracil auxotrophy (Ura-) of CEN.PK 113-5D strain has been exploited. Thus, cells where the gene targeting substrate integrated correctly (see lower left part of Figure 1b) were easily detected, as these cells had obtained a new selectable feature, i.e., URA3 gene (URA3; uracil gene from another yeast species, K. lactis), and were selected by plating on uracil deficient synthetic complete media (SC-Ura) in a positive selection procedure. A complex gene targeting substrate has been used for the deletion of the FMP43 gene, as after positive selection, the URA3 marker has been stably integrated at the target site, and the strain couldn't thus be re-targeted by using the URA3 gene as a marker. Therefore, the URA3 marker was flanked by a direct repeat in the complex gene targeting substrate (see lower part of Figure 1a). In vivo recombination between these two direct repeat individual units could eliminate the URA3 gene from the genome (see right part of Figure 1b). After this event, called pop-out recombination, the strain becomes Ura-again, and can thus be further manipulated by a new round of URA3-based targeting. Gene targeting substrates have been constructed based on PCR methods. In total, five individual pieces of DNA need to be fused to create the substrate. The fusions required to make the complete gene targeting substrates require PCR and adaptamer technology as well as in vivo homologous recombination (see Figure 1). The core part of the substrate, the URA3 gene flanked by a direct repeat, is constant in all experiments. For that reason, a plasmid, pWJ1042 has been used that contains the central three-partite part of the gene targeting substrate. To construct the complete gene targeting substrate, the sequence upstream of the target site needs to be fused to one end of the core targeting substrate, and the sequence downstream of the target site needs to be fused to the other end of the core substrate.