Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains

Multicopy integration frequencies and distribution of copy numbers in transformed K. lactis cells. (A) K. lactis cells were transformed with pKLAC1 (white bars) or pGBN19 (black bars) containing four different heterologous genes (for HSA, MBP, Gluc, and EK_L). (B) Cells were transformed with pGBN19-HSA, and transformants were selected on 200 to 1,000 μg G418 ml⁻¹. In both panels A and B, the values indicate the percentages of colonies in a sample population of n transformants that tested positive for multicopy integration by whole-cell PCR.

Vector copy number determination in multiply integrated strains. (A and B) The vector copy number was determined by digestion of genomic DNA with SpeI and AflII (restriction sites that flank the insertion site) and separation of large DNA fragments by pulsed-field gel electrophoresis and Southern blotting. The locations of hybridization probes are shown with black (pGBN19-HSA) or open (pKLAC1-HSA) bars. Single-copy integrants yield a 14.8-kb fragment. This fragment increases in size by 8.1 kb per additional inserted vector (e.g., 22.9 kb for two copies, 31 kb for three copies, etc). (B) A sample Southern blot showing copy number determination for eight strains. (C) Distribution of integrated vector copy numbers determined by both PCR and Southern blotting in sample populations of K. lactis transformants selected by growth on acetamide (white bars) or G418 (black bars). The values indicate the percentages of colonies in each sample transformant population that contained each copy number.

Frequencies of formation of strains coexpressing multiple heterologous proteins. K. lactis cells were cotransformed with two (A), three (B), or four (C) pKLAC1 vectors containing various heterologous genes using growth on 5 mM acetamide for selection. Shown are the percentages of strains in sample transformant populations (n) that produced each protein (HSA, MBP, Gluc, and EK_L).

Cosecretion of HSA and MBP proteins. Shown is a Coomassie-stained SDS-polyacrylamide gel electrophoresis gel with resolution of 13 μl of spent culture medium from nine random transformants producing both HSA and MBP proteins (lanes 4 to 12). Spent medium (13 μl) from cultures of reference strains that produce only HSA (YCT384) or MBP (YCT463) are shown in lanes 2 and 3, respectively. Lane 1 contains a broad-range protein standard (New England BioLabs).