Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 1, 2000, pp. 11-15

Bianca M García, Efraín Rodríguez, Tanilo Rivero, Yurima Hidalgo, @ Javier J Menéndez

División de Biotecnología Industrial. Centro de Ingeniería Genética y Biotecnología. AP 6162, CP 10600, Ciudad de La Habana, Cuba. Telf: (53-7) 21 8164; Fax: (53-7) 33 6008; E-mail: javier.menendez@cigb.edu.cu

The expression of dexA gene from Penicillium minioluteum encoding a dextranase in two different strains of the methylotrophic yeast Pichia pastoris has been compared using vectors with two different auxotrophic markers. In both cases the dexA gene was fused to the SUC2 signal peptide coding region from Saccharomyces cerevisiae and inserted in the genome of both strains, replacing the AOX1 gene. Transformants specifying dextranase activity were identified as colonies surrounded by zones of clearing on blue dextran methanol plates. When using the MP36 strain (his3-) all selected transformants carried the expression cassette and secreted an active enzyme to the medium after methanol induction. However, in the case of the GS115 strain (his4-), only 50% of transformants expressed the active enzyme. We also found that the transformants in MP36 expressed higher levels of the enzyme than those in GS115, probably due to differences at the transcriptional level.

Keywords: AOX1 promoter, dextranase, heterologous gene expression, Pichia pastoris

Expresión de la enzima dextranasa en dos sistemas hospedero-vector de la levadura metilotrófica Pichia pastoris. Se comparó la expresión del gen dexA aislado del hongo Penicillium minioluteum y que codifica la enzima dextranasa, en dos cepas diferentes de la levadura metilotrófica Pichia pastoris, mediante el empleo de vectores que portan marcadores auxotróficos diferentes. En ambos casos, el gen dexA se fusionó a la secuencia señal del gen SUC2 de Saccharomyces cerevisiae y se insertó en el genoma de ambas levaduras en el locus AOX1. Los transformantes capaces de secretar la enzima activa, fueron identificados por la aparición de zonas de aclaramiento alrededor de las colonias crecidas en placas que contienen dextrana azul y metanol como inductor. En el caso de la cepa MP36 (his3-), todos los transformantes seleccionados portaron el fragmento de expresión y secretaron dextranasa activa al medio de cultivo. Sin embargo, en el caso de la cepa GS115 (his4-), sólo 50% de los transformantes expresó la enzima activa. Además, los transformantes derivados de la cepa MP36 expresaron la enzima a niveles más altos que los derivados de la cepa GS115, probablemente debido a diferencias a nivel trasncripcional.

Palabras claves: dextranasa, expresión génica heteróloga, Pichia pastoris, promotor AOX1

Introduction

Saccharomyces cerevisiae is one of the best characterized yeasts for the production of heterologous proteins in eukaryotic organisms [1]. However, in recent years, alternative yeast species have gained popularity for this purpose. Among them, Pichia pastoris has become one of the most exploited for academic and commercial purposes [2]. This yeast offers several advantages, such as extremely high yields of intracellular proteins, very high levels of secretion into an almost protein-free medium; ease of fermentation to high cell density, genetic stability and possibilities of scaling-up without yield loss. In P. pastoris foreign genes are typically expressed under the control of the promoter of the yeast alcohol oxidase 1 gene (AOX1). This promoter is fully repressed during growth of the yeast in glucose or glycerol and maximally induced during growth on methanol [3]. This tight regulation is useful in expression of foreign genes whose products may be toxic to the cell. The recent availability of this system as a kit (commercialized by Invitrogen Corp.) has allowed a wide use of this yeast as an expression system in the laboratory [4]. Our group have isolated a histidine auxotrophic mutant of P. pastoris affected in the gene encoding imidazol glycerol-P dehydratase (HIS3). This mutant, named MP36, could be successfully transformed with the HIS3 gene from S. cerevisiae [5] and used as host for the expression of different heterologous proteins [6-11]. We decided to compare the effectiveness of this strain with that of the strain used by Invitrogen Corp. We compared the production of a fungal dextranase expressed in both strains with a vector carrying either HIS3 or HIS4 markers.

Materials and Methods

Strains, plasmids and media

The yeast, bacterial strains and plasmids used in this work are shown in Table 1. The bacterial Escherichia coli strain MC 1061 used for plasmid propagation was grown in LB medium (1% triptona, 0.5% yeast extract, 0.2M NaCl) with 50mg/mL ampicillin. For selection and growth of yeast transformants, YNB minimal medium (Difco Laboratories, USA) supplemented with 2% of glucose was used.

Table 1. Strains and plasmids used in this study.

Recombinant DNA methods

Cloning, DNA manipulations, and E.coli transformation were performed by standard techniques [15]. High specific activity labelling of hybridization probes was carried out by random hexamer priming [16] using [a-32P]dATP (>3000 Ci/mmol, Amersham, UK). For Southern blot analysis [17], DNA was transferred to Hybond-N membranes (Amersham, UK), and treated according to Sambrook et al. [15].

Ten micrograms of total RNA extracted from yeast cells were loaded onto 1% agarose/formaldehyde gels following standard protocols [15], and hybridization was carried out as described by Sambrook et al. [15]. The amount of RNA loaded was normalyzed by etidium bromide staining. DNA sequence analysis was performed by the dideoxy chain termination method of Sanger et al. [18]. Genomic DNA from yeast cells was prepared as described by Rothstein [19].

Construction of the P. pastoris expression vectors

Construction of pPDEX1 plasmid (Figure 1) has been previously described [11]. To express the dextranase enzyme into strain GS115 of P. pastoris, the dextranase-encoding region was amplified by PCR using the plasmid pPDEX1 as template [11]. The primers used in the reaction were 2738 (5'-TTGGATCCA TAATGCT TTTGCAAGCTTTCC-3') and 3079 (5'-GACTGATT ACCGTACACATATCGTCC-3'). The first oligonucleotide contains a BamHI restriction site and hybridized to the 5' region of the SUC2 signal peptide sequence. Primer 3079 hybridized to the 3' of the dextranase encoding region. The PCR product corresponding to dextranase gene plus the SUC2 signal peptide coding region was digested with BamHI and EcoRI, and the resulting 2kb fragment was ligated into pPIC3K [14], which was previously digested with the same enzymes. In the resulting plasmid, named pPICDEX, the PCR product was under the control of the AOX1 promoter and this fusion was checked up by sequencing using the AOX1 5' primer (5'- GACTGGTTCCAATTGACAAGC-3').

Transformation of P. pastoris and selection of dextranase-producing clones

About 10mg of pPDEX1 and pPICDEX1 plasmid DNA were digested with PvuII and BglII, respectively. The digested plasmids were used to transform P.pastoris host strains MP36 and GS115 by electroporation. This procedure was carried out according to Becker and Guarente [20] with cells pulsed in 0.2cm electroporation cuvettes at 1500V, 25mF, 200W, using a BioRad Gene Pulser with Pulse Controller (BioRad, USA). One milliliter of cold 1M sorbitol was added to the cuvettes immediately after pulsing, and His+ transformants were recovered on minimal-agar plates [21] with 2% dextrose. About 100 colonies from each strain were transferred to minimal-agar plates with 0.4% blue dextran (Pharmacia), and dextranase expression was induced with methanol in vapor phase by addition of 0.2mL portions of 100% methanol under the lids of inverted plates. After overnight incubation at 30ºC the formation of clear halos of hydrolysis around the colonies were observed.

Shake-flask dextranase expression studies

Five positive transformants from both MP36 and GS115 strains were selected for dextranase expression studies in shake flasks. The cultures were grown in 50mL of 0.67% YNB medium with 2% glycerol for 22h at 30ºC and 250rpm using an incubator shaker (New Brunswick Scientific Co., USA). The cells were then centrifuged, washed twice with water and resuspended in the same medium with 1% methanol instead of glycerol, to induce the AOX1 promoter and dextranase expression. After 24h of growth in this medium, additional 0.5% methanol pulses were supplied every 12h for 120h. At this time the final dextranase activity in the culture supernatant was measured. The optical density at 530nm (OD530) of the culture samples were determined every 12h. Samples taken at 0 and 42h of methanol induction were used for RNA isolation.

Enzymatic assays

Dextranase activity was determined according to Kosaric et al. [22]. The reducing sugars formed were determined colorimetrically by the dinitrosalicylic acid reagent method [23]. One unit (1 U) is defined as the amount of enzyme that releases 1mmol of glucose equivalents in 1min from dextran T-2000 (Pharmacia, Sweden) at 40ºC and pH 5.5.

Polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [24]. Gels (12.5% acrylamide) were put in a Mighty Small II SE 250 electrophoresis system (Hoefer Scientific Instruments, San Francisco, USA), for protein separation.

Results and Discussion

Transformation of P. pastoris and selection of dextranase-producing clones

To compare effectiveness of different recipients and vectors for heterologous protein production in P.pastoris, the expression levels of P. minioluteum dextranase in strains MP36 and GS115, were measured respectively. The former strain is defective in the his3 gene and can be transformed using plasmids with the HIS3 gene from S. cerevisiae; the latter, which is defective in the his4 gene, is transformed using the homologous HIS4 gene from P. pastoris. To express the dextranase gene in the GS115 strain, the PCR fragment encoding the mature dextranase protein was subcloned into pPIC3K vector [14] to yield the expression plasmid pPICDEX1. This plasmid shares some characteristics in common with pPDEX1 plasmid [11], such as having the dexA gene under the control of the AOX1 promoter and the use of SUC2 signal peptide coding region from S. cerevisiae to direct the secretion of the enzyme. The S. cerevisiae GAP terminator works as efficiently as the AOX1 terminator in P. pastoris, and the Knr gene present in the pPICDEX1 plasmid does not act as an autonomous replication sequence in this yeast. Therefore, the main difference between the two plasmids is the selection marker used (Figure 1). The pPDEX1 and pPICDEX1 plasmids were digested with PvuII and BglII, to generate DNA fragments that were targeted to transplace into the AOX1 locus of MP36 and GS115 strains, respectively. Transformation by electroporation of both strains yielded about 30 HIS+ transformants/mg of DNA. All tested positive transformants (about 80) from MP36 strain secreted an active enzyme. However, in the case of GS115 strain, only 50% of the analyzed transformants formed halos of dextran hydrolysis (Figure 2).

The colonies that did not secrete an active enzyme could arise from gene conversion events, which repair the mutation at the his4 locus without integration of the expression cassette into the yeast genome. This event was likely less frequent in MP36 strain due to the use of the HIS3 gene from S. cerevisiae. This gene product has only a 67% of identity with the HIS3 gene product from P. pastoris [25], which would reduce the frequency of gene conversion.

Analysis of the integration events

To prove the aforementioned hypothesis, which explains the results showed in Figure2, chromosomal DNA was isolated from 12 transformants of each strain and analyzed by Southern blot. The DNAs were digested with EcoRI, and hybridized to a dextranase probe. In MP36-derived transformants, the expression cassette was integrated in all of them, showing a single band of about 3.7kb (Figure 3A). This pattern appeared in a single-copy transplacement clone and produced a Muts (slow methanol utilization) phenotype, meaning that the integration of the expression cassette into the chromosome of the MP36 host disrupted the AOX1 locus.

In vivo circularization of the DNA fragment targeted for transplacement may occur in Pichia cells. Circularization of the PvuII transplacement cassette from the pPDEX1 could allow its insertion into AOX1 without disruption of the gene, since this can occur by single cross-over. Alternatively, circularized fragments could insert themselves wherever they are homologous to the chromosomal DNA; for instance at the AOX2 (due to the fragment of the coding region of the AOX1 gene included in the integrating plasmid) or HIS3 loci. Examples of this type of clones can be seen in lanes 3, 6 and 9 of Figure 3A. This type of integration has been shown and analyzed previously by Clare et al. [26].

We analyzed six transformants from GS115 strain that formed halos in plates containing blue dextran, and six that were not halo producers. In DNA extracted from transformants that expressed an active enzyme, a single band of 3.7kb was observed. As we mentioned above, this pattern corresponds to a single-copy transplacement and a Muts phenotype is obtained (Figure 3B). Southern blot analysis of chromosomal DNA from non expressing transformants showed the absence of the expression cassette in the genome of this type of clones (Figure 3C). The same membrane was hybridized using the AOX1 promoter fragment as probe, and the expected 5.5 kb band was observed (data not shown). These results indicate that the lack of activity in several transformants derived from GS115 strain is due to the absence of the expression cassette in these cells.

The results obtained in the Southern blot analysis of several transformants from both strains showed that the majority of these (75% in MP36 transformants and 83% in GS115 transformants) are Muts, containing a single copy of the transplacement cassette.

Expression of dexA gene in transformants of both P. pastoris strains in shake flasks

Five well-characterized transformants that formed halos of dextran hydrolysis and with the same Muts phenotype (single-copy transplacement of AOX1 locus), were selected. Muts clones grew more efficiently than Mut+ in these conditions, possibly because of the fact that a Mut+ strain is more likely to become oxygen-limited.

The final optical density of the cultures of the GS115 transformants was half the values for the cultures of MP36 clones (Figure 4). In the case of MP36, the levels of dextranase activity varied between 38U/ML and 98U/mL (Table 2). However, the enzymatic activity in GS115 transformants was three order of magnitude lower, even after buffering the medium to pH 6 (data not shown). In these strains the activity was about 0.04U/mL (Table 2).

Table 2. Dextranase induction in 50 mL shake-flask cultures of five transformants derived from MP36 and GS115 strains. The level of secreted dextranase was quantified by measuring the enzymatic activity in the culture supernatant.

Samples of the culture supernatants from both recombinant strains were analyzed by SDS-PAGE. In the case of the MP36 transformants (Figure 5A), a strong protein band with a molecular mass corresponding to the native enzyme from P. minioluteum (lane 6), was observed. On the contrary, in the case of GS115 transformants (Figure 5B), only a weak band could be seen at the same molecular size after a 20-fold concentration of the samples. This result correlates with the low enzymatic activity obtained in the transformants from GS115 strain.

To seek a possible explanation for this result, Northern blot analyses were carried out. RNA isolated at 0and 42h after methanol induction from C2 and I-75 clones, was used. The hybridization signal obtained for the RNA isolated from C2 transformants was stronger than that of I-75 clone, indicating that there are more specific dextranase transcripts in the first case (Figure 6). This suggests that the differences in dextranase expression between these two systems may be due to differences in the steady state of the corresponding mRNA. Assuming a similar half-life for the mRNA in both strains, the most plausible explanation would be a difference in the level of transcription (Figure 6).

A high density fermentation of C2 and I-75 clones was performed and at the end of the run (120 h), the dextranase activity in the culture supernatant of MP36-derived transformat C2 was 980 U/mL, showing over a 10-fold increase in respect to the shake-flask experiments. In the case of GS115 transformants (I-75 clone), we observed a 1000-fold increase in yield, when switching from shake flasks to fermenter. However, dextranase activity remained extremely low about 60 U/mL.

Our results clearly show differences between these two expression systems in the methylotrophic yeast P. pastoris. The most remarkable difference was that MP36 transformants were more efficient in the expression of dextranase gene than GS115 strains. One possible explanation could be that there are differences between the genetic background of these two strains.

Acknowledgments

We would like to thank Dr. Joe Fernández from Invitrogene Corp. for providing P. pastoris GS115 strain and the pPIC3K plasmid, and to José Cremata, Juana María Gancedo and Carlos Gancedo for their critical reading and helpful suggestions regarding this manuscript. This work was supported by the research project 3082-325 from CIGB.

References

1. Gellissen G, Melber K, Janowicz ZA, Dahlems U, Weydemann U, Piontek M, etal. Heterologous gene expression in yeast. Antoinevan Leeuwenhoek Int J Gen Mol Microbiol 1992;62:79-93.

2. Romanos MA. Advances in the use of Pichia pastoris for high-level gene expression. Curr Op in Biot 1995;6:527-33.

3. Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucl Acid Res 1987;15:3859-76.

4. Higgins DR, Cregg JM. Pichia Protocols in "Methods in Molecular Biology", Vol 103. Humana Press Inc., Totowa, NJ; 1998.

5. Yong V, González ME, Herrera L, Delgado J. El gen HIS3 de Saccharomyces cerevisiae complementa una mutación his- de Pichia pastoris. Biotecnología Aplicada 1992;9:55-61.

6. Paifer E, Margolles E, Cremata J, Montesino R, Herrera L, Delgado J. Efficient expression and secretion of recombinant alpha-amylase in Pichia pastoris using two different signal sequences. Yeast 1994;10: 1415-9.

7. Rodríguez L. Estudio en levaduras metilotróficas de la enzima sacarosa invertasa y su aplicación en la obtención de licores de fructosa [dissertation]. Cuba: Universidad de La Habana; 1998.

8. Morales J, Torrens IC, Aguiar J, Ferbeyre G, Sosa A, Martínez V, et al. High level secretion of mucor rennin in the methylotrophic yeast Pichia pastoris. In: Gavilondo JV, Luaces LL, Moya G, Pedraza A, Castro FO, Castro VA, editores. Biotecnología Habana'92. Avances en Biotecnología Moderna; 1992 Jun 8-12; La Habana, Cuba. La Habana: Elfos Scientiae; 1992. p. 22.3

9. Rodríguez M, Rubiera R, Penichet M, Montesinos R, Cremata J, Falcón V, et al. High level expression of the Boophilus microplus Bm86 antigen in the yeast Pichia pastoris forming highly inmu-nogenic particles for cattle. J Biotechnol 1994;33:135-46.

10. Pentón E, Muzio V, González-Griego M. The hepatitis B virus (HBV) infection and its prevention by a recombinant-DNA viral surface antigen (rec-HBsAg) vaccine. Biotecnología Aplicada 1994;11:1-11.

11. Roca H, García B, Rodríguez E, Mateu D, Coroas L, Cremata J, et al. Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris. Yeast 1996;12:1187-1200.

12. Meissner PS, Sisk WP, Berman ML. Bacteriophage l cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 1987;84:4171.

13. Cregg JM, Barringer K, Hessler AY, Madden KR. Pichia as a host system for transformations. Mol Cell Biol 1985;5: 3376-85.

14. Scorer CA, Clare JJ, McCombie WR, Romanos MA, Sreekrishna K. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Bio/Technology 1994;12:181-4.

15. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989.

16. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13.

17. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98: 503-12.

18. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 1977;74: 5463-7.

19. Rothstein R. Cloning in yeast. In: Glover DM editor. DNA Cloning. Vol 2. Oxford: IRL Press; 1985: 45-6.

20. Becker DM, Guarente L. High-efficiency transformation of yeast by electroporation. Meth Enzymol 1991;194:182-7.

21. Galzy P, Slonimski PP. Variation de la levure aucours de la croissance sur l'acide lactique ou sur glucose comme source de carbone. C R Acad Sci Paris 1975;245: 242-37.

22. Kosaric N, Yu K, Zajic JE. Dextranase production from Penicillium funiculosum. Biotech Bioeng 1973;15:729-41.

23. Miller GL. Use of dinitrosalicylic acid reagent fot the determination of reducing sugars. Anal Chem 1959;31:426-8.

24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227: 680-5.

25. Cosano IC, Álvarez P, Molina M, Num-bela C. Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes. Yeast 1998;9:861-8.

26. Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiples gen copies. Gene 1991;105:205-12.

Copyright 2000 Elfos Scientiae

The following images related to this document are available:

Line drawing images