Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Abstract

The strong LAC4 promoter (P LAC4 ) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P LAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P LAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P LAC4 . Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ϳ87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type P LAC4 . We created a novel integrative expression vector (pKLAC1) containing the P LAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.

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

“…The K. lactis integrative expression vector pKLAC1, which has been previously described (2), contains the Aspergillus nidulans acetamidase gene (amdS), which permits cell growth on medium containing acetamide. To create pGBN19, the neomycin gene was excised from pGBN1 (2) with a SmaI-BamHI digest and cloned into the same sites of pKLAC1.…”

“…EK L activity was quantitated by measuring cleavage of fluorogenic peptide substrate as previously described (2). Gluc activity was measured by mixing 25 l of spent culture medium and 50 l of 1ϫ Gaussia luciferase assay buffer (New England BioLabs) in a Microfluor black flat-bottom microtiter plate (Thermo Labsystems, Franklin, MA).…”

“…An alternative selection strategy involves the use of acetamide in nitrogen-free medium to impose a nitrogen source selection upon transformed cells (11,12). In this method, a gene encoding an Aspergillus acetamidase (amdS), present on an expression vector (2), confers the ability of a transformed cell to process acetamide to ammonia for use as a source of nitrogen. Furthermore, cells that have lost or have been cured of the amdS gene can be counterselected on medium containing fluoroacetamide.…”

“…This strategy has been used to construct selection marker-free strains for use in food industry processes (11,12). Acetamide selection has been used to transform various filamentous fungi, including Aspergillus, Neurospora, Penicillium, Fusarium, and Trichoderma species, as well as the yeasts K. lactis and Saccharomyces cerevisiae (2,5,(9)(10)(11)(12)(18)(19)(20).…”

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) .…”