Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces...

The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short- and long-term culture. The vectors carried an expression/secretion cassette consisting of the Saccharomyces cerevisiae SUC2 gene under the control of the S. cerevisiae alpha-factor promoter and leader. The first set of vectors contained the entire pKD1 sequence linearized at either the unique EcoRI or the unique SphI site of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity rapidly dropped while the plasmid-bearing population increased from 60% to 100%. This apparently contradictory behavior was due to structural instability. The enzyme restriction patterns of recovered plasmid DNA retained the pKD1 band while the band containing the SUC2 cassette had decreased substantially in size. To overcome this structural instability, a vector carrying the pKD1 replication origin and the cis-acting stability locus (lacking the inverted repeats) was employed in a pKD1+ (but otherwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While the new vector was significantly more stable, initial invertase activity was substantially lower than that for the vectors containing the full pKD1 sequence. Southern hybridization confirmed that this decrease was primarily due to reduced copy number. The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suitable for long-term (e.g., fed-batch or continuous) culture.