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

Abstract

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.

Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis

“…Heterologous recombinant proteins expressed by K. lactis can be sequestered intracellularly or secreted into the medium. As with P. pastoris , K. lactis secretes very low levels of native proteins, therefore secreted target proteins comprise the vast majority of the total protein present in the culture medium, which simplifi es purifi cation of the target protein (16)(17)(18) .…”

“…Using the pKLAC vector, a cDNA cassette containing the target gene under the control of the LAC4 promoter is inserted into the genome of K. lactis by using homologous recombination (16)(17)(18) . The transcriptional activity of the LAC4 promoter is induced by galactose, which K. lactis also utilizes as a carbon source for cell growth.…”

“…The transcriptional activity of the LAC4 promoter is induced by galactose, which K. lactis also utilizes as a carbon source for cell growth. Therefore, expression of the target protein is constitutively induced by cultivation in a medium containing galactose (16)(17)(18) .…”

“…Since only transformants that express amdS can utilize acetamide as a sole nitrogen source, amdS is used for selection of the desired transformants, thereby obviating the need for expensive antibiotics, such as Zeocin™ and rendering this auxotrophic selection method more cost-effective (16)(17)(18) . In addition, auxotrophic acetamide selection enriches populations of transformants in which multiple tandem copies of target cDNA are integrated into the yeast genome (16)(17)(18) . Thus, the K. lactis protein expression system is simple, cost-effective, easily scaled-up, and highly reproducible.…”

Over-expression of chloroperoxidase in Caldariomyces fumago

“…(Stratagene). As selection marker we wanted to use the acetamidase cDNA from Aspergillus nidulans as it often causes multicopy integrations (Read et al 2007). However C. fumago apparently contains an acetamidase as it can grow on minimal medium agar plates containing acetamide as sole nitrogen source (data not shown).…”