PARTIAL-PKD1 PLASMIDS PROVIDE ENHANCED STRUCTURAL STABILITY FOR HETEROLOGOUS PROTEIN-PRODUCTION IN KLUYVEROMYCES-LACTIS

The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short-and long-term culture. The vectors carri ed an expression/secretion cassette consisting of the Saccharomyces ce revisiae SUC2 gene under the control of the S. cerevisiae alpha-factor promoter and leader. The first set of vectors contained the entire pK D1 sequence linearized at either the unique EcoRI or the unique SphI s ite of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity rapidly droppe d while the plasmid-bearing population increased from 60% to 100%. Thi s 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 s ubstantially in size. To overcome this structural instability, a vecto r carrying the pKD1 replication origin and the cis-acting stability lo cus (lacking the inverted repeats) was employed in a pKD1(+) (but othe rwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While the new vector was sign ificantly more stable, initial invertase activity was substantially lo wer than that for the vectors containing the full pKD1 sequence. South ern hybridization confirmed that this decrease was primarily due to re duced copy number. The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suita ble for long-term (e.g. fed-batch or continuous) culture.