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Applied and Environmental Microbiology, November 2005, p. 7092-7098, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7092-7098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Paul A. Colussi and Christopher H. Taron^*

New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938-2723

Received 18 April 2005/ Accepted 16 July 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 87%, 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
For over a decade, the yeast Kluyveromyces lactis has been used for the industrial-scale production of commercially important proteins (14, 16). K. lactis cells rapidly grow to high cell density, efficiently secrete recombinant proteins, and can be easily genetically manipulated, making this organism an attractive eukaryotic host for protein expression. In a typical K. lactis protein expression strategy, a DNA fragment containing the equipment necessary to direct the high-level transcription of a gene of interest is first assembled in Escherichia coli cells prior to its introduction into yeast cells. This fragment typically contains (in 5' to 3' order) (i) a strong yeast promoter, (ii) DNA encoding a secretion leader sequence (if secretion of the protein is desired), (iii) the gene encoding the desired protein, (iv) a transcription terminator sequence, and (v) a yeast-selectable marker gene.

In K. lactis, expression of heterologous genes has been achieved using various promoters isolated from native K. lactis genes (7, 10, 11, 15) or using promoters originating from other yeasts (1, 2, 8, 15, 17). However, the K. lactis LAC4 promoter (P_LAC4) is often used because of its strength and inducible expression (14). K. lactis P_LAC4 drives expression of the LAC4 gene that encodes a native lactase (ß-galactosidase [3, 13]) that is an essential part of the lactose-galactose regulon that allows this organism to utilize lactose as a carbon and energy source (4). Two upstream activating sequences (UAS I and UAS II) located in a 2.6-kb intragenic region between LAC4 and LAC12 regulate the transcription of LAC4, which can be induced 100-fold in the presence of lactose or galactose (5).

In addition to its ability to function as a strong promoter in K. lactis, P_LAC4 constitutively promotes gene expression in E. coli cells. For example, the K. lactis LAC4 gene was originally isolated by screening a genomic DNA library for clones that were able to functionally complement an E. coli ß-galactosidase mutant (3). Bacterial expression of LAC4 was later attributed to nucleotide sequences in P_LAC4 that resemble the Pribnow box transcriptional element of bacterial promoters (4).

The ability of P_LAC4 to promote gene expression in bacteria can be detrimental to the process of assembling and amplifying yeast expression constructs in E. coli prior to their introduction into yeast cells. This is especially problematic if the cloned gene of interest encodes a translated product that is toxic to E. coli cells. In this study, we addressed this issue by introducing site-directed mutations into P_LAC4 to abolish its ability to function in E. coli. We demonstrate that targeted mutagenesis of Pribnow box-like sequences in P_LAC4 inhibits the expression of a reporter protein in E. coli but does not affect the promoter's ability to direct the high-level expression and secretion of proteins in K. lactis. Additionally, we demonstrate the use of a P_LAC4 mutant to express bovine enterokinase, a commercially important protease that has historically been problematic when expressed in E. coli cells. Finally, we present the construction of an E. coli-K. lactis integrative shuttle vector (pKLAC1) that relies upon a mutant form of P_LAC4 to direct protein expression in K. lactis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast strains, transformation, and culturing conditions.
The prototrophic K. lactis strain GG799 (MAT [pGKl1⁺]) is a wild-type industrial isolate (DSM Food Specialties, Delft, The Netherlands) that grows to very high cell density and efficiently secretes heterologous proteins. It was routinely grown and maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30°C. Prior to the transformation of GG799 cells, 1 µg of pGBN1- or pKLAC1-based expression vector containing a gene of interest was linearized by SacII digestion. Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier. Colonies of cells transformed with pGBN1, pGBN1_PGK1, pGBN1_Hyb, pGBN1_PBI, or pGBN1_PBII-PBIII vectors were selected by growth on YPD agar plates containing 200 µg G418 (Sigma, St. Louis, MO) ml^–1 for 2 to 3 days at 30°C. Colonies of cells transformed with pKLAC1-based vectors were selected by growth on agar plates containing 1.17% yeast carbon base (New England Biolabs), 5 mM acetamide, and 30 mM sodium phosphate buffer, pH 7, for 4 to 5 days at 30°C. K. lactis strains expressing heterologous genes were cultured in YP medium containing 2% galactose (YPGal) at 30°C for 48 to 96 h.

PCR.
Primers used in this study are listed in Table 1. Amplification by PCR was performed using high-fidelity Deep Vent DNA polymerase (New England Biolabs). Typical PCR mixtures contained 0.2 mM deoxynucleoside triphosphates, 0.5 µg of each primer, 1x Thermopol buffer, and 100 ng template DNA in a total reaction volume of 100 µl. Thermocycling typically consisted of a "hot start" at 95°C for 5 min, followed by 30 cycles of successive incubations at 94°C for 30 s, 58°C for 30 s, and 72°C (1 min per kb of DNA). After thermocycling, a final extension was performed at 72°C for 10 min.

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TABLE 1. Oligonucleotides used in this study

Construction of K. lactis P_LAC4 variants.
All promoter variants were derived from wild-type P_LAC4 present in the integrative expression vector pGBN1, a K. lactis-E. coli shuttle vector that contains 2,317 bp of P_LAC4 DNA split into 1,663- and 654-bp fragments that are separated by pUC19 plasmid DNA (Fig. 1). Additionally, pGBN1 contains DNA encoding the Saccharomyces cerevisiae -mating factor (-MF) pre-pro domain immediately downstream of P_LAC4 to direct the secretion of heterologously expressed proteins. Finally, pGBN1 carries a Geneticin (G418) resistance gene expressed from the S. cerevisiae ADH2 promoter for dominant selection in yeast.

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FIG. 1. The E. coli-K. lactis integrative expression vector pGBN1. Genes are cloned into the multiple cloning site (MCS) in the same translational reading frame as the S. cerevisiae -mating factor secretion leader sequence (Sc -MF). Transcription is initiated and terminated by the K. lactis LAC4 promoter (PLAC4) and LAC4 transcription terminator sequence (TTLAC4), respectively. The S. cerevisiae ADH2 promoter drives the expression of a bacterial gene conferring resistance to G418 in yeast. E. coli vector sequence has been inserted into a unique SacII site in PLAC4 to allow for propagation in bacteria via an E. coli origin of replication (ORI). The vector is linearized by digestion with SacII or BstXI for integration into the LAC4 promoter locus in the K. lactis genome.

To create plasmid pGBN1_PGK1, a PmlI-HindIII fragment containing 488 base pairs of the S. cerevisiae PGK1 promoter was cloned into the HpaI-HindIII sites of plasmid pGBN1 to replace 1,067 base pairs of native P_LAC4 (Fig. 2B). Primer P1 and primer P2 were used to amplify 283 base pairs of the S. cerevisiae PGK1 promoter using plasmid pGBN1_PGK1 as template. The 283-bp fragment was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1_Hyb. Primer P3 was designed to incorporate mutations into the putative Pribnow box-like sequence (PBI) that lies upstream of the E. coli major transcription start site as detailed in Fig. 2B. Primers P2 and P3 were used to amplify a P_LAC4 fragment containing mutations in PBI using plasmid pGBN1 as template. Amplified DNA from this initial PCR was used as template for a second PCR using primers P2 and P4. The final DNA product was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1_PBI. A PCR-knitting method was used to mutate the PBII and PBIII sequences (Fig. 2B) that lie upstream of the E. coli minor transcription start site using complementary primers P5 and P6. Primers P2 and P5 and primers P4 and P6 were used to amplify 586-bp and 160-bp mutated P_LAC4 DNA fragments, respectively. Each amplified DNA product was purified by QIAquick PCR purification spin column chromatography (QIAGEN, Valencia, CA) and combined as template in a second amplification reaction containing primers P2 and P4. The amplified P_LAC4 DNA fragment containing mutagenized PBII and PBIII sites was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1_PBII-PBIII.

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FIG. 2. Pribnow box-like sequences in PLAC4 and construction of PLAC4 variant expression vectors. (A) Pribnow box-like sequences PBI, PBII, and PBIII are shown relative to the major and minor E. coli transcription start sites associated with PLAC4 and are aligned with the Pribnow box consensus sequence TATAAT. Nucleotides that agree with the consensus sequence are indicated with a vertical line. (B) Expression vectors containing PLAC4 variants. The approximate positions of the E. coli major and minor transcription start sites are shown in the schematic for pGBN1. The approximate positions of the galactose-responsive elements UAS I and II are shown for each construct. Regions of PLAC4 DNA that have been replaced with fragments of the S. cerevisiae PGK1 promoter are shown in black. Mutated bases in the Pribnow box-like sequences in the PLAC4 DNA of plasmids pGBN1PBI and pGBN1PBII-PBIII are indicated with a black dot above each base. All numbered positions are relative to the adenine of the ATG start codon of the S. cerevisiae -mating factor secretion leader (Sc MF) that has been designated position +1.

Cloning and expression analysis of GFP in E. coli.
Green fluorescent protein (GFP) was PCR amplified with primers P7 and P8 using plasmid pGFPuv (Clontech, Palo Alto, CA) as template. Amplified GFP was cloned in frame with the -MF pre-pro domain in the BglII-NotI sites of the various pGBN vectors (see previous section). Lysates of bacteria containing various pGBN-GFP constructs were prepared from 50-ml overnight cultures grown at 30°C in Luria-Bertani medium containing 100 µg ml^–1 ampicillin. Cultures were centrifuged, and the cell pellets were frozen on dry ice, thawed at room temperature, and resuspended in 10 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, containing 50 mM NaCl and 1 mM EDTA). The cells were disrupted with a Sonicator (Heat Systems-Ultrasonics, Plainview, NY) for 15 s on setting 7, and cell debris was removed by centrifugation at 15,000 x g for 10 min. The protein concentration of each lysate was determined by measuring its absorbance at 280 nm. Proteins (100 µg) in each lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10 to 20% polyacrylamide gradient gel, transferred to nitrocellulose, and blocked overnight in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) and 5% nonfat milk (wt/vol) at 4°C. An anti-GFP monoclonal antibody (Clontech) diluted 1:1,000 in PBS-T containing 5% nonfat milk was used to probe the blot, followed by incubation with a horseradish peroxidase-coupled anti-mouse secondary antibody (KPL, Gaithersburg, MD) diluted 1:2,000 in PBS-T containing 5% nonfat milk. Protein-antibody complexes were detected using LumiGlo detection reagents (Cell Signaling Technology, Beverly, MA). The amount of GFP produced in E. coli was measured by densitometry using Molecular Imager FX (Bio-Rad, Hercules, CA) and Quantity One software.

Cloning and expression of human serum albumin and enterokinase in K. lactis.
Primers P9 and P10 were used to amplify the gene encoding human serum albumin (HSA) that was subsequently cloned in frame with the -MF sequence in the XhoI-NotI sites of the various pGBN vectors. Primer P9 was designed to encode the K. lactis Kex1 protease cleavage site (KR) immediately upstream of the HSA open reading frame to ensure correct processing of the protein in the Golgi apparatus. K. lactis strains containing integrated pGBN-HSA DNA were grown in 2-ml cultures of YPGal for 48 h at 30°C. The level of HSA secretion was visually assessed by separation of proteins in 15 µl of spent culture medium by SDS-PAGE on a 10 to 20% polyacrylamide gradient gel followed by Coomassie blue staining.

A DNA fragment encoding the enterokinase catalytic subunit (EK_L) was PCR amplified with primers P11 and P12 and cloned in frame with the -MF pre-pro domain in the XhoI-BglII restriction sites of the various pGBN vectors containing the P_LAC4 variants or in the vector pKLAC1 (see below). The DNA sequence of EK_L in the various pGBN-EK_L or pKLAC1-EK_L vectors was confirmed by nucleotide sequencing. Secretion of enterokinase by K. lactis strains containing integrated pKLAC1-EK_L constructs was assessed by growing cells in 2 ml YPGal for 48 h at 30°C and assaying spent culture medium for enterokinase activity as described below.

Enterokinase activity assay.
Spent culture medium was isolated by microcentrifugation of 1 ml of a saturated culture of pKLAC1-EK_L-integrated K. lactis at 15,800 x g for 1 min to remove cells. Enterokinase activity was measured using the fluorogenic peptide substrate GDDDDK-ß-napthylamide (Bachem, King of Prussia, PA). Spent culture medium (50 µl) was mixed with 50 µl enterokinase assay buffer (124 mM Tris-HCl, pH 8.0, containing 0.88 mM GD₄K-ß-napthylamide and 17.6% dimethyl sulfoxide), and fluorescence intensity (excitation, 337 nm; emission, 420 nm) was measured over time. A comparison of the amount of enzyme activity associated with measured quantities of purified enterokinase (New England Biolabs) to the activity present in spent K. lactis culture medium was used to estimate the amount of active enterokinase secreted by K. lactis strains. To compensate for a mild inhibitory effect that YPGal culture medium has on the enterokinase assay, purified enterokinase was first diluted into spent medium from a culture of untransfected K. lactis cells prior to measuring enterokinase activity as described above.

Construction of vector pKLAC1.
Vector pKLAC1 was created by replacing the S. cerevisiae -MF pre-pro domain and the G418 resistance gene of vector pGBN1_PBI with the K. lactis -MF pre-pro domain and the Aspergillus nidulans acetamidase gene (amdS), respectively. DNA encoding the K. lactis -MF pre-pro domain was PCR amplified from K. lactis genomic DNA using primers 13 and 14 and cloned into the SacI-XhoI sites of pLitmus29 (New England Biolabs). The cloned K. lactis -MF sequence was subsequently excised by HindIII and XhoI digestion and cloned into the HindIII-XhoI sites of plasmid pGBN1_PBI to produce plasmid pGBN1_PBI-KlMF. A 1,520-bp DNA fragment containing all of the A. nidulans amdS gene except the first 128 bp was amplified using primers P15 and P16 and a cloned amdS gene as template (kindly provided by Peter Dekker of DSM Food Specialties, Delft, The Netherlands). This fragment was cloned into the BamH I-SmaI sites of plasmid pGBN1_PBI-KlMF, replacing the G418 resistance gene and producing plasmid pGBN1_{PBI-KlMF-1520}. The remaining 128 bp of the 5' end of the amdS gene was amplified by PCR with primers P16 and P17, digested with BamHI, and cloned into the BamHI site of vector pGBN1_{PBI-KlMF-1520}, and the proper orientation of the fragment was confirmed by DNA sequencing.

Nucleotide sequence accession number.
The resulting vector was named pKLAC1 (GenBank accession no. AY968582).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted mutagenesis of Pribnow box-like sequences in P_LAC4.
The initiation sites of two RNA transcripts (a major and a minor transcript) associated with the E. coli expression of K. lactis P_LAC4 have been previously mapped (4). Three DNA sequences (PBI, PBII, and PBIII) that resemble bacterial Pribnow box transcriptional elements lie approximately 10 bp upstream of the major (PBI) and minor (PBII and PBII) transcription initiation sites (Fig. 2A). We therefore generated a series of four P_LAC4 variants aimed at eliminating the E. coli promoter activity of P_LAC4 by either replacing or introducing point mutations in PBI and PBII/PBIII as shown in Fig. 2B. Vector pGBN1_PGK1 incorporates 485 bp of the S. cerevisiae PGK1 promoter (P_PGK1) in place of 1,067 bp of native P_LAC4, thereby removing both galactose-responsive upstream activating sequences (UAS I and UAS II) and all three Pribnow box-like sequences. Vector pGBN1_Hyb incorporates 283 bp from the 5' end of P_PGK1 in place of 283 bp comprising the 5' end of P_LAC4, resulting in the deletion of all three Pribnow box-like sequences but leaving both UAS intact. Vector pGBN1_PBI contains 6 point mutations that eliminate the Pribnow consensus sequence of PBI between nucleotides –201 and –210 of P_LAC4. Similarly, vector pGBN1_PBII-PBIII contains 5 point mutations that eliminate the Pribnow consensus sequences of PBII and PBIII between nucleotides –139 and –144 of P_LAC4.

Each P_LAC4 variant was tested for its ability to drive the E. coli expression of a reporter gene encoding GFP that was cloned in frame with the S. cerevisiae -mating factor pre-pro domain in each of the pGBN vectors. The presence of GFP produced from P_LAC4 variants in E. coli lysates was analyzed by Western blot analysis. Removal of the PBI sequence by mutation resulted in an 87% decrease in GFP expression (Fig. 3A, lane 5), as determined by densitometry, relative to GFP produced by the wild-type P_LAC4 (Fig. 3A, lane 2). However, mutation of both PBII and PBIII sequences (Fig. 3A, lane 6) did not detectably down-regulate GFP expression. Deletion of all three Pribnow box-like sequences from P_LAC4 by replacement with P_PGK1 DNA (Fig. 3A, lanes 3 and 4) led to a complete loss of detectable GFP expression. These results indicate that the majority of P_LAC4 expression in E. coli is dependent upon the presence of the PBI sequence.

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FIG. 3. PLAC4 variant expression of reporter genes in E. coli and K. lactis. (A) GFP was cloned downstream of each of the various PLAC4 promoter variants shown in Fig. 2B. Proteins from lysates of E. coli carrying each expression construct were separated by SDS-PAGE, and GFP was detected by Western blot analysis as described in Materials and Methods. Lane 1 (pGBN1) is a negative control lysate made from bacteria containing an empty pGBN1 plasmid. (B) HSA was cloned downstream of each PLAC4 promoter variant for expression in K. lactis cells. Secreted proteins in the spent culture medium of K. lactis strains containing the various integrated HSA expression vectors were resolved by SDS-PAGE (4 to 20% acrylamide) and Coomassie blue stained. HSA ran as a single band with an apparent mass of 66 kDa. Lane 1 (pGBN1) shows spent culture medium from a yeast strain containing empty pGBN1 integrated into the chromosome as a negative control.

P_LAC4 variants retain full promoter activity in K. lactis.
To test whether the P_LAC4 variants were able to direct the expression of a heterologous gene in K. lactis, the gene encoding HSA was cloned into each of the pGBN vectors. HSA was chosen as a reporter protein due to its high-level expression and efficient secretion from K. lactis when expressed from wild-type P_LAC4 (7). K. lactis strains containing each of the integrated pGBN1-HSA expression vectors were grown to saturation in YPGal medium, and secreted proteins in the spent culture medium were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie blue staining. HSA migrates as a 66-kDa band that can be readily detected in unconcentrated spent culture medium, and its identity was confirmed by Western blotting with an anti-HSA antibody (data not shown). K. lactis strains containing integrated pGBN1_PBI-HSA, pGBN1_Hyb-HSA, and pGBN1_PBII-PBIII-HSA vectors secreted HSA in amounts comparable to a control strain harboring pGBN1-HSA where HSA is expressed from wild-type P_LAC4 (Fig. 3B, lane 2). These data indicate that mutation or deletion of the PBI, PBII, and PBIII sequences of P_LAC4 does not significantly alter the promoter's ability to function in K. lactis. It is also noteworthy that markedly less HSA was secreted from cells harboring pGBN1_PGK1-HSA (Fig. 3B, lane 3) compared to cells expressing HSA from either wild-type P_LAC4 (Fig. 3B, lane 2) or the other P_LAC4 variants (Fig. 3B, lanes 4 through 6). This is consistent with the notion that HSA expression from P_PGK1 is suppressed in galactose-containing medium because both UAS required for galactose-induced expression have been deleted.

Effects of P_LAC4 variants on the cloning efficiency of bovine enterokinase.
Bovine enterokinase is an important protease that is often used to cleave affinity tags from engineered fusion proteins. Production of enterokinase in E. coli is plagued by low yields that are attributable to the protein's toxicity in bacteria. Therefore, we sought to use the expression of enterokinase in K. lactis as a means to circumvent its poor expression in bacteria. Numerous attempts to assemble K. lactis expression vectors in E. coli, where DNA encoding the enterokinase light chain (EK_L) was placed downstream of wild-type P_LAC4, resulted in widespread isolation of clones containing loss-of-function mutations (e.g., frame shifts or early terminations) within the EK_L coding sequence. We reasoned that the ability of P_LAC4 to promote gene expression in E. coli was likely creating selective pressure against the propagation of intact EK_L-containing clones. We therefore examined whether the P_LAC4 variants that exhibited reduced or abolished expression in E. coli could be used to facilitate cloning of the toxic EK_L gene into K. lactis expression vectors in E. coli prior to their introduction into yeast.

The EK_L gene was PCR amplified using a high-fidelity polymerase and cloned downstream of the various P_LAC4 variants in the pGBN1 vectors (Fig. 2B). The entire EK_L gene (708 bp) of numerous isolated clones was sequenced to determine the presence of loss-of-function mutations. When cloned under the control of wild-type P_LAC4 in pGBN1, 11 of 12 (92%) clones examined contained loss-of-function mutations. However, no mutations were found in EK_L cloned in vectors pGBN1_PGK1 (nine clones sequenced) or pGBN1_Hyb (seven clones sequenced), vectors containing P_LAC4 variants that completely lack E. coli promoter function. Additionally, no mutations were found in EK_L cloned in vector pGBN1_PBI (nine clones sequenced) where E. coli expression is reduced 87% due to mutations in PBI. Additionally, 3 of 10 (30%) EK_L clones in pGBN1_PBII-PBIII contained loss-of-function mutations. Together, these data show that the function of wild-type P_LAC4 in E. coli adversely affects the cloning efficiency of a toxic gene and indicate that P_LAC4 variants that either lack or have severely reduced function in E. coli are better suited for the assembly of K. lactis expression constructs in bacteria.

Construction of pKLAC1, an integrative K. lactis expression vector.
Relying upon the findings of this study, we assembled a novel K. lactis integrative expression vector (pKLAC1) for the secretion of proteins from K. lactis (Fig. 4). This vector is based on the P_LAC4-PBI variant that contains mutations in PBI (Fig. 2B, pGBN1_PBI) and contains (in 5'-to-3' order) a PBI-deficient LAC4 promoter, the K. lactis -mating factor secretion leader sequence, a multiple cloning site, the K. lactis LAC4 transcription terminator, a selectable marker cassette containing the Aspergillus nidulans acetamidase gene (amdS) expressed from the S. cerevisiae ADH2 promoter (P_ADH2), and an E. coli origin of replication and ampicillin resistance gene to allow for its propagation in E. coli. Digestion of this vector with SacII or BstX I generates a linear expression cassette that integrates into the promoter region of the LAC4 locus of the K. lactis chromosome upon its introduction into K. lactis cells. Transformed yeast cells are isolated by nitrogen source selection on yeast carbon base medium containing 5 mM acetamide, which can be converted to a simple nitrogen source only if the expression cassette (containing the amdS gene) has integrated into the chromosome (12).

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FIG. 4. The E. coli-K. lactis integrative expression vector pKLAC1. The pKLAC1 vector (GenBank accession no. AY968582) is organized similarly to pGBN1 with the following modifications. Genes are cloned into the multiple cloning site (MCS) in the same translational reading frame as the native K. lactis -mating factor leader sequence (Kl -MF). Expression in K. lactis is initiated by the PLAC4-PBI promoter variant. The S. cerevisiae ADH2 promoter drives the expression of a fungal acetamidase gene (amdS) for the selection of transformants by growth on acetamide medium. ORI, origin of replication.

Vector pKLAC1 was used to secrete enterokinase from K. lactis cells after successfully assembling the expression vector in E. coli (pKLAC1-EK_L). Strains harboring integrated pKLAC1-EK_L were cultured in YPGal medium for 2 days. Enterokinase proteolytic activity in the spent culture medium was assayed by measuring the rate of cleavage of a fluorogenic peptide. Measurements of activity performed on culture supernatant from seven pKLAC1-EK_L integrated strains showed that all seven secreted active enterokinase (Fig. 5). However, two of the seven strains (KlEK-S1 and KLEK-S4) secreted greater levels of enterokinase activity than the other five. Southern analysis determined that strains KLEK-S1 and KLEK-S4 contained multiple tandem copies of integrated pKLAC1-EK_L (data not shown). The yield of enterokinase secreted from strain KLEK-S1 grown in shake flasks was estimated to be 1.1 mg/liter based on a comparison of secreted enzyme activity to the activity of known quantities of purified enterokinase as described in Materials and Methods.

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FIG. 5. Activity of secreted enterokinase in the spent culture medium of K. lactis cells containing integrated pKLAC1-EKL. Seven K. lactis strains harboring pKLAC1-EKL and wild-type GG799 cells were grown in YPGal medium for 48 h. Cleared spent culture medium was assayed for enterokinase activity by measuring the cleavage of a fluorogenic peptide over time as described in Materials and Methods. KLEK-S1 and KLEK-S4 are two strains that contain multiple copies of integrated pKLAC1-EKL as determined by Southern blot analysis. All other strains contain a single integrated copy of pKLAC1-EKL.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (P_LAC4) can be adversely affected by the serendipitous function of P_LAC4 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 P_LAC4 that resemble the bacterial Pribnow box transcription element. We showed that P_LAC4 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 P_LAC4-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 (4). 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 P_LAC4 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 P_LAC4-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 EK_L gene. This suggests that the cloning efficiency of detrimental genes in E. coli can be dramatically improved even by small decreases in P_LAC4 expression levels. Importantly, none of the point mutations that reduced or eliminated P_LAC4 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, P_LAC4 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 P_LAC4 expression in E. coli (9). 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, P_LAC4 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 P_LAC4-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 P_LAC4 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 P_LAC4-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. 5 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.

 
C.H.T. is grateful to Donald Comb for support. We thank B. Taron, A. Fabre, L. McReynolds, B. Slatko, F. Stewart, and D. Distel for comments on the manuscript.

 
* Corresponding author. Mailing address: New England Biolabs, 240 County Road, Ipswich, MA 01938-2723. Phone: (978) 927-5054. Fax: (978) 921-1350. E-mail: taron{at}neb.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2005, p. 7092-7098, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7092-7098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Read, J. D., Colussi, P. A., Ganatra, M. B., Taron, C. H. (2007). Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains. Appl. Environ. Microbiol. 73: 5088-5096 [Abstract] [Full Text]  

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