An important component of any yeast-based expression system is a shuttle vector that allows for the propagation of cloned genes in bacteria prior to their introduction into yeast cells for expression. In this context, yeast expression systems that utilize the strong K. lactis LAC4 promoter (PLAC4) can be adversely affected by the serendipitous function of PLAC4 in E. coli. This promoter activity can interfere with the cloning efficiency of genes whose translational products are potentially detrimental to bacteria. In the present study, we addressed this by eliminating sequences within PLAC4 that resemble the bacterial Pribnow box transcription element. We showed that PLAC4 variants that lack Pribnow box-like sequences have reduced or abolished ability to promote gene expression in E. coli but retain their full ability to function as strong promoters in yeast. Finally, we constructed a K. lactis expression vector that is based upon the PLAC4-PBI variant created in this study and used it to express the commercially important protease enterokinase.
The Pribnow box is an important component of bacterial promoters, that is, an A/T-rich region located approximately 10 nucleotides upstream from the site where transcription begins. A prior study mapped a major and a minor transcription start site associated with the K. lactis LAC4
promoter in E. coli
). Two stretches of nucleotide sequence that closely resemble the Pribnow box consensus sequence TATAAT are located at −204 to −209 (PBI) and −136 to −144 (PBII and PBIII) within PLAC4
and reside just upstream of the major and minor transcription start sites, respectively. Elimination of these Pribnow box-like sequences by targeted mutagenesis revealed that most PLAC4
-based expression in E. coli
was due to the PBI sequence associated with the major transcription start site. Interestingly, mutation of PBII and PBIII did not lead to a significant decrease in the expression of GFP in E. coli
but nevertheless led to a 62% decrease in the isolation of clones carrying loss-of-function mutations in the EKL
gene. This suggests that the cloning efficiency of detrimental genes in E. coli
can be dramatically improved even by small decreases in PLAC4
expression levels. Importantly, none of the point mutations that reduced or eliminated PLAC4
expression in E. coli
adversely affected the levels of protein expression and secretion from K. lactis
cells. This finding underscores differences in promoter elements that are required for bacterial versus yeast expression. For example, in K. lactis
initiates transcription at multiple sites (−97, −98, −105, −115, and −127) presumably due to TATA box sequences in the −169 to −173 and −226 to −234 regions (6
) that are distinct from the PBI and PBII/PBIII sequences that are located within the −204 to −209 and −136 to −144 regions, respectively.
A recent study used a different method to curtail the potentially detrimental effects of PLAC4
expression in E. coli
). In this work, a yeast intron containing translational stop codons was placed immediately downstream of the translational start codon of the desired protein. Because E. coli
cells cannot process introns, PLAC4
activity generated an mRNA containing early stop codons that prevented translation of the full-length protein in bacteria. This method was effective in lowering the PLAC4
-based expression of xylanase and lipase genes in E. coli
; however, xylanase activity was not completely abolished. The authors noted that this was likely due to alternative translational start codons that lie downstream of the inserted intron that may allow for translation of active protein fragments (9
). In contrast, the promoter variants described in the present study presumably function by blocking the ability of PLAC4
to initiate transcription in E. coli
, which provides a tighter regulation of the expression of potentially detrimental recombinant proteins.
Based on our findings, we constructed a novel K. lactis
integrative expression vector (pKLAC1) for the production of recombinant proteins. Two key elements of this vector are as follows: (i) the PLAC4-PBI
variant containing mutations in PBI to allow the assembly of DNA fragments encoding potentially toxic proteins in E. coli
and high-level protein production in yeast and (ii) an acetamidase-selectable marker gene. Expression of acetamidase in transformed yeast cells allows for their growth on medium lacking a simple nitrogen source but containing acetamide (12
). Acetamidase breaks down acetamide to ammonia, which can be utilized by cells as a source of nitrogen. An important benefit of this selection method is that it enriches transformant populations for cells that have incorporated multiple tandem integrations of a pKLAC1-based expression vector and that produce more recombinant protein than single integrations (Fig. and data not shown). We have recently shown that more than 90% of transformants that form on acetamide plates following transformation of K. lactis
strain GG799 with pKLAC1-based constructs that express HSA or the E. coli
maltose binding protein contain two to four copies of the integrated vector.
We successfully used pKLAC1 to efficiently clone the toxic protease enterokinase in E. coli
and secrete it from K. lactis
cells. Additionally, the use of pKLAC1 is applicable to the expression of other recombinant proteins that are problematic in E. coli
due to their toxicity or other detrimental effects on bacterial cells. For example, we have recently utilized pKLAC1 to successfully clone and express in K.lactis
the gene encoding mouse transthyretin following numerous unsuccessful attempts using various prokaryote-based systems (J. Ingram, P. A. Colussi, C. H. Taron, and B. Slatko, unpublished data). Additionally, pKLAC1 has been used to clone and express in K.lactis
toxic glue proteins from marine organisms (J. Platko, personal communication) and a multifunctional bacterial cellulase (D. Distel, personal communication) that were unable to be expressed using various prokaryotic expression systems.