Acetamide selection increases multicopy integration frequency.
To determine if the selection method influences the frequency of targeted tandem integration of a vector into the chromosome of K. lactis
cells, two integrative expression vectors that differed only in their selectable marker genes were compared. Vectors pKLAC1 and pGBN19 both contain a variant of the strong K. lactis
lactase promoter (PLAC4-PBI
), which drives expression of a heterologous gene (2
) and directs targeted vector insertion into the promoter region of the chromosomal LAC4
locus (Fig. ). However, pKLAC1 contains the A. nidulans
acetamidase gene (amdS
), which permits growth of transformed strains on nitrogen-free medium containing acetamide, whereas pGBN19 contains a bacterial neomycin gene that renders transformed strains resistant to the antibiotic G418.
FIG. 1. Targeted vector integration at the LAC4 chromosomal locus. (A) A SacII- or BstXI-linearized expression vector containing a gene of interest (GOI) is targeted for insertion into the LAC4 promoter region of the K. lactis chromosome by the 3′ and (more ...)
Two whole-cell PCR strategies were devised to detect targeted integration of pKLAC1 or pGBN19 into the K. lactis chromosome. The first strategy used a forward primer (primer 1) that anneals to chromosomal LAC4 promoter DNA lying upstream of the vector integration site and a reverse primer (primer 2) that anneals to either expression vector. A 2.4-kb amplicon is generated only if targeted integration of pKLAC1 or pGBN19 constructs has occurred correctly at the LAC4 locus (Fig. ). The second PCR strategy exploited a unique genomic architecture that is created when tandem integration of two or more pKLAC1 or pGBN19 vector copies (termed “multicopy integration”) occurs. In this strategy, only strains having multiple vector integrations produce a 2.3-kb amplicon (Fig. ). However, this analysis does not indicate the number of integrated vector copies.
Used in combination, these whole-cell PCR strategies allow analysis of vector integration patterns in populations of cells transformed with pKLAC1 or pGBN19 constructs. For example, transformed strains that do not generate the 2.4-kb amplicon have integrated the vector ectopically at a locus other than the LAC4 target locus. Thus, the frequency of correctly targeted vector integration at LAC4 (termed the “targeting efficiency”) can be assessed by determining the percentage of a sample transformant population that successfully amplifies the 2.4-kb amplicon. Additionally, generation of the 2.4-kb amplicon, but not the 2.3-kb amplicon, indicates that a strain contains only a single vector copy integrated at LAC4 (Fig. ), whereas production of both amplicons confirms that a strain contains at least two tandem vector integrations at the LAC4 locus (Fig. ). The frequency of tandem vector integration in a transformant population (termed the “multicopy integration frequency”) can be determined by first identifying a sample population of strains that produce the 2.4-kb amplicon and then determining the percentage of that population that also produce the 2.3-kb amplicon.
The multicopy integration frequencies were determined for sample populations of strains transformed with various pKLAC1 and pGBN19 constructs. Four heterologous genes encoding HSA, MBP, Gluc, and EKL were separately cloned into pKLAC1 and pGBN19 and used to individually transform K. lactis GG799 cells. Transformants were selected by growth on medium containing either 5 mM acetamide (for pKLAC1 constructs) or 200 μg G418 ml−1 (for pGBN19 constructs). For each construct, the multicopy integration frequency (see above) was determined for a sample population of >80 transformants. For transformed strains isolated using growth on medium containing acetamide (pKLAC1-based constructs), the multicopy integration frequencies were 98 to 100%, indicating that nearly the entire sample population was comprised of strains having multiple vector integrations (Fig. ). The targeting efficiencies of pKLAC1-HSA, pKLAC1-MBP, pKLAC1-Gluc, and pKLAC1-EKL were 99%, 98%, 97%, and 91%, respectively, indicating that ectopic integration of these vectors occurred infrequently. In contrast, only 7 to 23% of strains transformed with pGBN19-based constructs contained multiple vector copies when selected on 200 μg G418 ml−1 (Fig. ). The targeting efficiencies of vectors pGBN19-HSA, pGBN19-MBP, pGBN19-Gluc, and pGBN19-EKL were 89%, 97%, 85%, and 99%, respectively.
FIG. 2. 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 (more ...)
To determine if transformant selection with higher concentrations of G418 could increase the frequency of formation of multicopy strains, cells transformed with pGBN19-HSA were plated on growth media containing 200, 300, 400, or 1,000 μg G418 ml−1. Transformation efficiency decreased about fivefold, from 165 ± 5.0 CFU μg of vector−1 on medium containing 200 μg G418 ml−1 to 30 ± 8.0 CFU μg of vector−1 on medium containing 1,000 μg G418 ml−1, suggesting that a more stringent selection was being imposed on the transformant population at higher G418 concentrations. The multicopy integration frequency was determined for sample populations of at least 80 transformants selected on media containing 200, 300, 400, and 1,000 μg G418 ml−1. Only 7 to 29% of these sample populations were multiply integrated (Fig. ), indicating that increasing the G418 concentration during transformant selection did not increase the frequency of formation of multiply integrated strains.
These data demonstrate that both methods of selection permit highly efficient vector integration at the target LAC4 locus. However, transformant selection by growth on medium containing acetamide dramatically enriches transformant populations for strains having multiple vector integrations compared to G418 selection. Additionally, this phenomenon occurs irrespective of the heterologous gene present in the expression vector.
Distribution of copy numbers in multicopy strains.
The average integrated vector copy numbers were determined for populations of transformants formed by growth on medium containing 5 mM acetamide (for pKLAC1-HSA) or 200 μg ml G418−1 (for pGBN19-HSA). Transformants were screened by whole-cell PCR to identify sample populations of strains having multiple vector integrations at the LAC4 locus (generation of both 2.4-kb and 2.3-kb amplicons). Of 55 randomly chosen acetamide-selected pKLAC1-HSA transformants tested in this manner, all 55 were multiply integrated and were further subjected to copy number determination (see below). Due to the low multicopy integration frequency observed with G418 selection, numerous pGBN19-HSA transformants had to be tested by PCR to identify a statistically significant sample population of multiply integrated strains. Of 288 pGBN19-HSA transformants, all generated the 2.4-kb amplicon and therefore had at least one vector copy integrated at LAC4. However, of these, only 54 strains were multicopy strains that also produced the 2.3-kb amplicon. The remaining 234 strains were deemed to be single-copy pGBN19-HSA integrants. All 54 multicopy pGBN19-HSA strains were further subjected to copy number determination.
To determine the number of vector integrations in multicopy strains, a strategy was used that involved digestion of genomic DNA with restriction endonucleases that flank the vector integration site (in the LAC4 promoter), followed by separation of large digested DNA fragments by pulsed-field gel electrophoresis and Southern analysis (Fig. ). In this method, the vector copy number is indicated by the size of the hybridized DNA fragment (Fig. ). The populations of 55 acetamide-selected multicopy transformants and 54 G418-selected multicopy transformants were analyzed in parallel. The distribution of copy numbers within these populations is shown in Fig. . The mean copy numbers were 3.8 ± 0.45 and 1.5 ± 0.16 at the 95% confidence interval for acetamide- and G418-selected sample populations, respectively. The difference in average copy numbers was confirmed with a t test (α = 0.05). Thus, the average copy number doubled in transformant populations selected by growth on medium containing 5 mM acetamide.
FIG. 3. 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 (more ...) Simultaneous coexpression of multiple heterologous proteins.
The high multicopy integration frequency observed with acetamide selection was exploited to construct strains that simultaneously secrete multiple heterologous proteins. In this approach, two or more pKLAC1-based expression constructs, each harboring a different heterologous gene, were linearized and simultaneously introduced into K. lactis cells by cotransformation, followed by colony formation on growth medium containing acetamide. The high multicopy integration frequency associated with acetamide selection increases the probability that two different expression vectors will become tandemly integrated in the same cell, leading to coexpression of the two heterologous proteins.
Expression vectors that direct high-level production of HSA (pKLAC1-HSA) and MBP (pKLAC1-MBP) were each linearized and used to cotransform K. lactis cells using growth on medium containing acetamide for transformant selection. A sample population of 93 multiply integrated transformants was identified by whole-cell PCR, and each transformant was tested for its ability to secrete HSA and MBP by Western dot blotting. Surprisingly, 70% of the multicopy strains (65 of 93) in the sample population secreted both proteins (Fig. ). Similar data were obtained by cotransforming cells with three pKLAC1-based vectors containing DNA encoding HSA, MBP, or luciferase. A sample population of 96 multiply integrated transformants was identified by whole-cell PCR and tested for secretion of HSA and MBP by Western dot blotting and for secretion of luciferase activity into the growth medium. The majority of multicopy integrants tested (60 of 96, or 63%) showed secretion of all three heterologous proteins (Fig. ). The remaining 37% of transformants (35 of 96) produced all pairwise combinations of two proteins, and only one strain produced a single protein (MBP). However, when four expression constructs were used to cotransform cells, a significant drop in the percentage of multicopy transformants secreting all four proteins was observed (25 of 95, or 26%), although strains producing all four proteins were still easily identified (Fig. ).
FIG. 4. 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. (more ...)
The yields of two cosecreted proteins were also compared. Cleared spent culture media of nine transformants cosecreting MBP and HSA were examined by SDS-polyacrylamide gel electrophoresis separation and Coomassie staining. Qualitatively, all nine transformants produced both proteins (Fig. , lanes 4 to 12) in quantities comparable to those obtained with characterized reference strains that secrete HSA (YCT384) (Fig. , lane 2) or MBP (YCT463) (Fig. , lane 3) to approximately 75 mg liter−1 and 65 mg liter−1, respectively.
FIG. 5. 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 (more ...) Expression of multisubunit proteins.
Numerous eukaryotic secretory proteins are comprised of more than one polypeptide. Some examples are antibodies; cell surface receptors, like the major histocompatibility complexes; and various proteases (e.g., enterokinase and blood-clotting factors). From a biotechnology standpoint, antibodies represent an important class of two-subunit proteins that are often expressed in yeasts. Therefore, we sought to determine if strains producing active antibody Fab fragments could be easily constructed using cotransformation and acetamide selection.
The genes encoding Fab fragments of monoclonal antibodies that recognize E. coli MBP and human transferrin were each amplified from RNA isolated from mouse hybridoma cell lines using reverse transcription-PCR and subsequently subcloned into separate pKLAC1 vectors. For each antibody, vector pairs containing DNA encoding a Fab heavy chain and a light chain were linearized and used to cotransform K. lactis cells using growth on medium containing acetamide for transformant selection. For both anti-MBP and anti-transferrin, sample populations of 95 multiply integrated transformants were identified by PCR. These strains were microcultured in 96-deep-well microtiter plates, and spent culture medium from each well was assayed for the presence of an active antibody fragment, using ELISA. A large percentage of each sample transformant population expressed a Fab antibody fragment (82% and 93% of anti-MBP and anti-transferrin transformants, respectively). Additionally, both secreted Fab antibodies specifically recognized their respective antigens, indicating that the secreted proteins were properly assembled (Fig. ).
FIG. 6. Specificities of two secreted Fab antibody fragments. Two representative transformed strains that secrete either anti-MBP (open bars) or anti-transferrin (black bars) Fabs were grown, and cleared medium from each was culture incubated in the presence (more ...)