Figure 1.

Molecular strategies used in K. phaffii to improve gene transcription. The titer of heterologous transcripts can be improved by using strong promoters (engineered or not) or by generating clones with extra doses of the desired gene integrated in the genome or present in episomal plasmids. The extra-copies may be introduced by different approaches: sequential transformation with vectors bearing different markers, in vitro multimerization, colony screening under drug selective pressure, gene amplification by PTVA (or liquid PTVA), use of defective auxotrophic markers in minimal medium and diploidization of selected clones. Abbreviations: zeo/G418 = dominant markers which confer resistance to zeocin or G418, respectively.