Phosphate-Responsive Promoter of a Pichia pastoris Sodium ...

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Dec 22, 2008 - PHO89, which encodes a putative sodium (Na )-coupled phosphate ... the glyceraldehyde-3-phosphate dehydrogenase promoter, PPHO89.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3528–3534 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.02913-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 11

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter䌤† Jungoh Ahn,1 Jiyeon Hong,1 Myongsoo Park,1 Hyeokweon Lee,1 Eungyo Lee,1 Chunsuk Kim,1 Joohwan Lee,1 Eui-sung Choi,2 Joon-ki Jung,1 and Hongweon Lee1* Biotechnology Process Engineering Center1 and Systems Microbiology Research Center,2 KRIBB, Daejeon 305-806, Republic of Korea Received 22 December 2008/Accepted 20 March 2009

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Naⴙ)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (PPHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. PPHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1␣ and the glyceraldehyde-3-phosphate dehydrogenase promoter, PPHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple PPHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of PPHO89 for controlled production of recombinant proteins in P. pastoris. Pichia pastoris is a methylotrophic yeast species that is increasingly used as a host system for heterologous protein expression for both academic and industrial purposes. To date, more than 500 proteins have been cloned and expressed using this system (19; http://faculty.kgi.edu/cregg/index htm), and it has provided the protein production platform for several structural genomics programs (25, 35). In this system, most recombinant proteins have been produced using the alcohol oxidase I promoter (PAOX1), which is completely repressed when cells are grown on glucose and is induced in the presence of methanol (31). However, there are circumstances in which the promoter may not be suitable; the principal problem is that the highly volatile and inflammable compound methanol is required for transcription (29). Use of methanol for the induction of gene expression is often not permitted in the production of food products since methanol is a petroleum by-product (13, 33). Also, PAOX1-based fermentation requires relatively long times and sophisticated feeding strategies for maintenance of the induction phase. These properties hamper the industrial applicability of this approach. Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to PAOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (PFLD1) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter

(PTEF1), with more highly growth-associated expression characteristics, as an alternative to PGAP (2). The current study describes the use of a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expression of a heterologous gene in P. pastoris. When microorganisms are starved for an essential nutrient, they respond by increasing the expression of genes that restrict the metabolic consequences of the nutrient limitation (5, 21, 24). Substrate limitation especially affects metabolic activities expressed by the specific rate of carbon or oxygen uptake, yet under phosphate limitation conditions the specific rate of oxygen uptake can remain high even at a very low rate of growth (30). These results imply that the metabolic activities and cell viability are maintained even in stationary phase in a phosphate-limited environment, which could be exploited for the production of recombinant proteins. Also, it was reported previously that transcriptional activities of the promoters of phosphate-responsive genes, such as the genes encoding alkaline phosphatase in Escherichia coli (6, 18) and acid phosphatase in Saccharomyces cerevisiae (32), are regulated by phosphate and are highly activated by the simple and cheap technique of lowering the phosphate concentration in the culture medium. We previously analyzed phosphate-responsive genes in P. pastoris under steady-state phosphate-limited chemostat growth conditions (12). One of the genes detected was the PHO89 gene encoding the putative Na⫹-coupled phosphate symporter Pho89, a high-affinity transporter of phosphate (12). In the present study, the PHO89 gene from P. pastoris and its promoter region were isolated and sequenced. Furthermore, the fermentation process was optimized for industrial application of this promoter.

* Corresponding author. Mailing address: Biotechnology Process Engineering Center, KRIBB, Daejeon 305-600, Republic of Korea. Phone: 82-42-860-4520. Fax: 82-42-860-4516. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 27 March 2009.

MATERIALS AND METHODS Microorganisms. E. coli DH5␣ [[F⫺ endA1 hsdR17(rK⫺ mK⫺) supE44 thi-1 ␭⫺ recA1 gyrA96 ␾80dlacZ⌬M15] was used as the host strain for cloning and main-

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FIG. 1. Expression vectors used in this study. CLLip, gene encoding lipase from Bacillus stearothermophilus fused to a CBD and linker of endoglucanase II from T. harzianum; AMss, ␣-amylase signal sequence from A. oryzae; Ampr, ampicillin resistance gene; PPHO89, PHO89 promoter of P. pastoris sodium-coupled phosphate symporter; pBR322 ori, E. coli replication origin; HIS4, histidinol dehydrogenase gene from P. pastoris; TT, transcription termination and polyadenylation signal from the AOX1 gene; 3⬘AOX1, 3⬘ AOX1 downstream sequence AvrII, BglII, StuI, SalI, and NotI, restriction enzyme sites.

tenance of plasmids. P. pastoris GS115 (his4) (Invitrogen, Carlsbad, CA) was used as the host strain for expression of heterologous genes. Cloning of an ORF of PHO89 and its upstream region. A promoter region regulating the PHO89 gene was cloned using inverse PCR with the synthetic primers 5⬘-CCAAGAATGCAAACATCATGGCG-3⬘ and 5⬘-GGTGAGATCC ACTGGGGATGGAGTGG-3⬘ and with the previously cloned PHO89 gene as a probe (12). The genomic DNA of P. pastoris was used as the template for the PCR after it was digested with various restriction enzymes. One of the genomic DNA fragments obtained after digestion with KpnI, an amplified DNA fragment containing part of the PHO89 open reading frame (ORF) and its upstream region, a 1,044-bp promoter, was amplified by PCR. The full-length PHO89 ORF was obtained by PCR using synthetic primers (forward primer 5⬘-GAATTCAT GAGTTTGGTTGCACTTCATCAATTCG-3⬘ and primer 5⬘-GGATCCCTAA GGAGTCAATTCGTATTGACCTCCCC-3⬘) based on the partially cloned PHO89 gene with genomic DNA of P. pastoris as the template. Construction of a lipase expression vector using PPHO89 of P. pastoris. A lipase gene was used as a reporter gene to verify the activity of the cloned PHO89 promoter (PPHO89). In detail, lipase from Bacillus stearothermophilus L1 fused to a cellulose binding domain (CBD) from Trichoderma harzianum was used as a reporter, as previously described for PTEF1 (2). A recombinant expression vector carrying a lipase gene and PPHO89 was based on a pPIC9 vector (Invitrogen) (Fig. 1). The signal sequence from Aspergillus oryzae ␣-amylase and an L1 lipase fragment fused to the CBD were amplified by PCR using plasmid pYEGA-AMCLLip (1) as the template and synthetic primers (forward primer 5⬘-GCAGCT AGGATGATGGTCGCGTGGTGG-3⬘ and reverse primer 5⬘-GCAGCGGCC GCAAGCTTTTAAGGCCGCAAACTCGC-3⬘; the underlined nucleotides are the added sequences required for addition of the AvrII and NotI sites at the 5⬘ and 3⬘ ends, respectively). The PPHO89 region was amplified by PCR using forward primer 5⬘-AGATCTGGAGCCAGCACAGGAATCGGTGG-3⬘) containing a BglII site (underlined nucleotides) and reverse primer 5⬘-GCAGCTA GGTGTGAATGATTATAAGATGAGTCATC-3⬘) containing an AvrII site (underlined nucleotides). The amplified fragment containing ␣-amylase signal sequence-CBD-L1 lipase sequence was digested with AvrII and NotI, while the amplified fragment containing PPHO89 was digested with BglII and AvrII. Subsequently, both digested fragments were ligated to the pPIC9 vector prior to digestion with BglII (partial digestion) and NotI. The resultant plasmid, in which CBD-L1 lipase sequence was controlled by PPHO89, was designated pNPS-AMCLLip. P. pastoris transformation. P. pastoris GS115 was transformed with the expression vectors that were constructed. For insertion into a his4 gene in the genome of P. pastoris GS115, the expression vectors were digested with BspEI, a site for which is present in the his4 gene, and then transformed into P. pastoris by a lithium chloride transformation method (Invitrogen). Selection of transformants was performed using His⫺ agar plates containing (per liter) 20 g of glucose, 6.7 g

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of yeast nitrogen base without amino acids, 0.77 g of His⫺ DO supplement (BD Biosciences, Franklin Lakes, NJ), and 20 g of agar. Primary screening for positive transformants was carried out on halo-forming plates with 1% tributyrin for detection of lipase activity. From the positive transformants obtained, a single transformant that had only a single copy of the recombinant expression vector inserted into the genome was selected using the relative and quantitative real-time PCR methods with a 2⫺⌬⌬CT calculation (3, 4), as suggested in previous work (2). Cultivation. A single colony of the transformant grown on His⫺ agar was inoculated into 20 ml of YPD medium containing (per liter) 20 g of glucose, 10 g of yeast extract, and 20 g of Bacto peptone and incubated overnight at 30°C. The culture was transferred to a 1-liter Erlenmeyer flask containing 200 ml of YPD broth and incubated overnight at 30°C. This culture was used as a seed culture for all the cultures in this study. Thirty milliliters of the culture was inoculated into 5-liter jar fermentors (KoBiotech, Incheon, Korea) with 1,970 ml of various media. To examine the suitability of gene expression under the control of PPHO89 according to different initial phosphate concentrations, three batch cultures were grown with defined medium containing (per liter) 50 g of glucose, 3 g of MgSO4 䡠 7H2O, 15 g of (NH4)2SO4, 4.55 g of K2SO4, 1.03 g of KOH, 0.3 g of CaCl2, 2 ml of trace elements, and different initial phosphate concentrations (adjusted using 0.025, 0.25, or 2.5 g/liter of NaH2PO4 䡠 2H2O as the only phosphate source). To examine the stable production phase, four batch cultures with various initial glucose concentrations (50, 110, 200, and 320 g/liter) in the defined media described above were grown with NaH2PO4 䡠 2H2O as the sole phosphate source (fixed concentration for all four media, 0.25 g/liter). To investigate gene expression under the control of PPHO89 in complex media, a fed-batch culture was generated with medium containing (per liter) 30 g of glucose, 3 g of MgSO4 䡠 7H2O, 15 g of (NH4)2SO4, 4.55 g of K2SO4, 1.03 g of KOH, 0.3 g of CaCl2, 5 g of yeast extract, 20 g of Bacto peptone, and 1 ml of trace elements, and glucose was intermittently supplied to prevent depletion. All the cultures in this study were grown at 30°C and pH 6.0 controlled with 24% NH4OH. Also, to maintain the dissolved oxygen level above 30%, agitation and aeration were controlled using 400 to 1,000 rpm and 1 to 1.5 liters of air per liter of medium per min, respectively. Analytical methods. The growth of yeast cells was monitored by measuring the optical density at 600 nm using a UVICON930 spectrometer (Kontron Analytical, Lucerne, Switzerland). The cell dry weight was estimated by using a predetermined conversion factor, 0.27 g (dry weight)/liter/unit of optical density at 600 nm. To determine residual glucose concentrations, 1 ml of culture broth was centrifuged, and the glucose concentration in the supernatant was measured using a YSI model 2000 glucose and lactate analyzer (YSI, Yellow Springs, OH). The ethanol concentration in the supernatant was measured by high-pressure liquid chromatography (Gilson, Middleton, WI) using an HPX 87H column (Bio-Rad, Hercules, CA) and an ERC-7515A refractive index detector (ERC, Tokyo, Japan). Lipase activity was determined by pH-stat analysis using a 718 STAT Titrino (Metrohm, Westbury, NY) (2). One unit was defined as the amount of enzyme required for release of 1 ␮mol of fatty acid per min under the assay conditions at 50°C. The phosphate concentration was quantitatively assayed as previously described (8). Western blotting. Proteins were resolved by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The separated proteins were stained with Coomassie brilliant blue. Subsequently, the proteins were transferred electrophoretically onto a nitrocellulose filter. Polyclonal L1 lipase antibody from a rabbit and alkaline phosphatase-conjugated goat anti-rabbit antibody were used as primary and secondary antibodies, respectively, for detection of L1 lipase protein as a reporter. Nucleotide sequence accession number. The sequences of the ORF consisting of the PHO89 gene and its upstream region determined in this study have been deposited in the GenBank database under accession no. EU938135.

RESULTS ⴙ

Cloning of an Na -coupled phosphate symporter gene and promoter. Genes that are upregulated in P. pastoris under phosphate-limited conditions have been screened by growth of the yeast in steady-state, phosphate-limited chemostat environments at different dilution rates (16). In this study, their mRNAs were used as templates for reverse transcription-PCR to construct a cDNA library. Among the various genes detected in the cDNA library, the gene encoding the putative

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FIG. 2. Nucleotide sequence of the P. pastoris-derived PHO89 gene with 5⬘ and 3⬘ flanking regions. The TATA box that is discussed in the text is enclosed in a box. The potential consensus sites (CACGTG/T) recognized by Pho4p are underlined, as is a stretch of seven amino acid residues (G-A-N-D-V-A-N) that is highly conserved in other sodium-coupled phosphate transporters.

Na⫹-coupled phosphate symporter Pho89 was identified using nucleotide sequencing and homology analysis (12). An ORF consisting of the PHO89 gene and its upstream region were cloned using inverse PCR with synthetic primers and with the previously cloned PHO89 gene as the template. Sequence analysis of the PHO89 gene and promoter (PPHO89). The PHO89 gene ORF consisted of 1,731 nucleotides encoding a 576-amino-acid polypeptide with a predicted molecular mass of 63.3 kDa (Fig. 2). A putative TATA box was identified at position ⫺102, and the sequence of the putative translation initiation site (CACATGAG; putative start codon underlined) agreed well with the Kozak consensus sequence (CA[C/A][A/C]ATGNC; start codon underlined) (15). Also,

two CACGTG/T motifs for the binding site of Pho4p (10, 21) were detected at positions ⫺583 and ⫺414 of ATG (underlined nucleotides in Fig. 2). This suggests that the P. pastorisderived PHO89 gene is under the control of the PHO regulatory system (17, 21, 24). The deduced amino acid sequences showed similarity with the corresponding proteins of Pichia stipitis (GenBank accession no. EAZ63542; 61% sequence identity), S. cerevisiae (GenBank accession no. P3836; 57% sequence identity), and Candida albicans (GenBank accession no. EAL03934; 59% sequence identity). Also, a stretch of seven amino acid residues (G-A-N-D-V-A-N) (Fig. 2) that is highly conserved in other sodium-coupled phosphate transporters was present. A membrane-spanning domain in the

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FIG. 4. Western blot for batch cultures of P. pastoris GS115/pNPSAM-CLLip described in the legend to Fig. 3. Lane M contained the standard marker. The numbers above the lanes indicate the culture times (in hours). The arrow indicates the position of the expressed CLLip.

FIG. 3. Results obtained with batch cultures of P. pastoris GS115/ pNPS-AM-CLLip with different initial phosphate concentrations. Batch cultures A (squares), B (triangles), and C (circles) contained initial concentrations of NaH2PO4 䡠 2H2O of 2.5, 0.25, and 0.025 g/liter (as the sole phosphate source), respectively.

deduced peptide was predicted using the transmembrane (TM) prediction program (7; http://www.ch.embnet.org), and 12 TM regions (TM1 to TM12) were evident (see Fig. S1 in the supplemental material), with a large hydrophilic loop between TM7 and TM8. Construction of expression vector using PPHO89. The potential use of PPHO89 in P. pastoris was investigated using the lipase gene from B. stearothermophilus L1 fused to the CBD (CLLip) (1) as a reporter gene. The ␣-amylase signal sequence from A. oryzae was fused to CLLip for extracellular production of CLLip, and the fusion construct was placed under the control of PPHO89 (Fig. 1). Effect of phosphate concentration on gene expression under the control of PPHO89. Batch cultivations using a 5-liter fermentor jar system were carried out to examine gene expression under the control of PPHO89 under phosphate-limited conditions. P. pastoris transformed with pNPS-AM-CLLip was grown in batch cultures A, B, and C with initial concentrations of NaH2PO4 䡠 2H2O of 2.5, 0.25, and 0.025 g/liter as the only phosphate source, respectively (Fig. 3). Cell growth was significantly influenced by the initial phosphate concentration. Culture A had a low concentration of phosphate only at the end of log-phase growth, suggesting that there was no phosphate-

limited growth. Cells were grown to a concentration of 18.9 g (dry weight)/liter with complete depletion of the initial glucose (50 g/liter) within 22 h after inoculation. In culture B, phosphate was depleted from the medium within 15 h after inoculation, and the growth rate of the cells was less than that in culture A. The cells grew to a concentration of 16.5 g (dry weight)/liter, and the initial glucose was completely consumed at 37.5 h postinoculation. In culture C containing a very small amount of phosphate, depletion of phosphate was observed at the start of culture growth, which resulted in only minimal growth of the cells; 43 g/liter of glucose was detected after 40 h, and the cell concentration reached only 4.1 g (dry weight)/liter. The lipase activities that resulted from expression of the foreign gene in cultures A and B were detected when the phosphate concentration in culture media decreased to less than 0.02 g/liter as the cells consumed phosphate for growth. However, although depletion of phosphate was observed immediately, no lipase activity was detected in culture C, likely due to the inhibited cell growth in the near-absence of phosphate. These observations were corroborated by Western blotting (Fig. 4) and are consistent with the notion that the “promoteron” transcriptional status and the “promoter-off” transcriptional status of PPHO89 are related to phosphate limitation and phosphate excess, respectively. The tight phosphate-mediated control of PPHO89, in turn, regulates the level of expression of the recombinant proteins under the control of PPHO89. Gene expression under the control of PPHO89 at a high glucose concentration. Because the increase in lipase activity ceased when the glucose in culture B was exhausted (Fig. 3), we supposed that a ready supply of glucose might prolong the production phase for expression of foreign genes under the transcriptional control of PPHO89. To examine the stable production phase, four batch cultures with various initial glucose concentrations (50, 110, 200, and 320 g/liter) were grown (Fig. 5) with NaH2PO4 䡠 2H2O at a fixed concentration of 0.25 g/liter as the sole phosphate source in all four media. As judged by the increase in the initial glucose concentration, inhibition of cell growth was related to increased osmolarity and ethanol

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FIG. 6. Results obtained with a fed-batch culture of P. pastoris GS115/pNPS-AM-CLLip with intermittent addition of glucose in complex medium.

FIG. 5. Results obtained with batch cultures of P. pastoris GS115/ pNPS-AM-CLLip with different initial concentrations of glucose. Circles, triangles, squares, and diamonds indicate batch cultures with initial glucose concentrations of 50, 110, 200 and 320 g/liter, respectively.

production. However, a glucose concentration below 200 g/liter did not significantly affect cell growth, while growth was inhibited by 320 g/liter glucose. In all cultures, ethanol was produced at concentrations below 8 g/liter and was subsequently consumed. The production of lipase increased in all cultures until the glucose was exhausted and showed similar patterns except for the culture with an initial glucose concentration of 320 g/liter. Also, the amount of protein produced per cell, expressed as the lipase activity per cell, increased continuously until the glucose was exhausted (122.5 h in the culture with 200 g/liter glucose), which indicates that the long-lasting

phase of foreign protein production using PPHO89 can be stably maintained even in stationary phase. Gene expression under the control of PPHO89 in complex media. As shown in Fig. 6, the expression of a foreign gene under the control of PPHO89 in complex media instead of defined media was studied using a fed-batch culture containing yeast extract and Bacto peptone as sources of phosphate (phosphate contents, 3.7% and 0.4%, respectively, according to the supplier’s manual) (36). The initial phosphate concentration in the complex media was calculated to 0.265 g/liter, but the actual initial phosphate concentration was determined by a phosphate assay to be 0.11 g/liter. The initial glucose concentration was 30 g/liter, and glucose was supplied intermittently to prevent exhaustion while the glucose concentration was maintained at a noninhibitory level. Lipase activity was observed by 12 h postinoculation, corresponding to phosphate depletion. Compared to the results for the culture with defined medium containing glucose at an initial concentration of 200 g/liter of (Table 1), the lipase activity per cell, productivity (amount of product/h), and specific productivity (amount of product/cell/h) were 1.27-, 2.95-, and 3.74-fold greater, respectively This result demonstrates that PPHO89 works effectively in complex media, and the PPHO89-based fermentation process was optimized by using complex media.

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TABLE 1. Summary of the main results obtained for 5-liter jar fermentations Initial Cell wt (g Lipase activity Lipase activity/g Productivity Specific productivity Culture phosphate (U/ml) (U/g 关dry wt兴 cells) (U/liter/h) (U/g 关dry wt兴 cells/h) time (h)b 关dry wt兴/liter) concn (g/liter)

Culture

Batch 1 (50 g/liter glucose) Batch 2 (50 g/liter glucose) Batch 3 (110 g/liter glucose) Batch 4 (200 g/liter glucose) Fed batch with complex medium Fed batch with complex medium using PTEFa Fed batch with complex medium using PGAPa

Cell yield (g 关dry wt兴 cells/g phosphate)

1.84 0.18 0.18 0.18 0.11

25.0 37.5 72.5 122.5 41.5

21.2 16.8 25.1 31.3 24.7

3 54 101 226 225

141 3,205 4,022 7,216 9,137

120 1,440 1,390 1,840 5,430

5.7 85.5 55.4 58.7 219.6

11.5 93.3 139.4 173.9 224.5

NAc

70.0

107.2

226

2,108

3,230

30.1

NCd

NA

70.0

106.1

100

942

1,430

13.5

NC

a

Data from reference 2. Culture time for maximum lipase activity. NA, not assayed. d NC, not calculated. b c

DISCUSSION Commercial production of recombinant proteins for industrial and medical uses has increased significantly in recent years (22). For this reason, development of efficient processes for the production of recombinant proteins using microorganisms has become important. A key step in this production is selection of a promoter that directs expression of the target gene in the host strain selected (11). Hence, promoter strength and tight regulation (a low basal level of expression yet a high conditional level of expression) that can be achieved easily and cost-effectively are key goals (14). Furthermore, taking into account the stable production of recombinant proteins under large-scale fermentation conditions, the promoter must be capable of maintaining high and long-lasting (durable) metabolic activity and cell viability in a mechanistically simple and economically reasonable manner. In this study, use of a P. pastoris phosphate-responsive promoter was examined. Phosphate starvation forces microorganisms to respond by upregulating genes involved in scavenging and specific uptake of phosphate from an external source (5, 21, 24). Consistent with this physiological response, a cDNA library of phosphate-responsive genes in P. pastoris grown in phosphate-limited chemostat cultures was previously found to have a putative PHO89 gene responsible for the high-affinity phosphate transporter (12). The PHO89 gene and its promoter region were successfully isolated in the present study. Analysis of the PHO89 gene from P. pastoris revealed an 1,731-bp ORF with the potential to encode a 576-amino-acid polypeptide. Based on hydropathy analysis of the primary amino acid sequence, the proposed secondary structure prediction includes 12 putative TM domains with a large hydrophilic loop between TM7 and TM8, similar to the TM domains found in S. cerevisiae (20). Analysis of the PHO89 promoter region revealed two putative Pho4p binding sites for the PHO regulatory system. However, it was reported that a promoter region of P. pastoris acid phosphatase does not possess equivalent Pho4p binding sites (23). These contrasting results may reflect the fact that the previous analysis involved only a small promoter region 313 bp upstream from the initial ATG codon of acid phosphatase. The major objective of this study was to examine whether

PPHO89 contains properties desirable for the production of a recombinant protein. First, it was demonstrated by examination of its activation depending on the external phosphate concentration that PPHO89 is tightly regulated by phosphate (Fig. 4). Thus, activity coincides with a low phosphate concentration. Second, to compare the strength of PPHO89 with that of PTEF1 or PGAP, which are strong promoters in P. pastoris, we used the results of a study in which a reporter gene in PPHO89 was expressed under the control of PTEF1 or PGAP (2). The specific productivity for PPHO89 in the complex media was considerably higher than that for PTEF1 or PGAP (7.4- and 14.8-fold higher for comparisons with PTEF1 and PGAP, respectively) (Table 1), indicating that the transcriptional activity of PPHO89 is high enough to allow recombinant protein to accumulate to a high level. Third, phosphate derepression of PPHO89 represents a simple and cost-effective approach. Thus, when the phosphate in culture media was exhausted as the cells consumed phosphate for growth, PPHO89 was derepressed. Therefore, selection of a proper initial phosphate concentration is a major consideration for the activation of PPHO89. Also, monitoring of phosphate by the spectrometric assay method (8) is relatively easy and rapid. From a physiological standpoint, cells must be phosphate limited for activation of PPHO89. The absence of a nutritional component often causes stress responses that result in growth arrest or cell death (5, 9, 34). However, the cells continued to grow under phosphate starvation conditions, although the growth rate was reduced. The continued growth likely reflects liberation of phosphate from organic phosphate sources and concomitant incorporation of the phosphate into nucleic acids for cell growth (30), as shown in this study by the increase in the cell yield per gram of phosphate based on the increase in the carbon/phosphate ratio in culture media (Table 1). This metabolic adaptation may make it possible to stably maintain metabolic activities and cell viability even at stationary phase under phosphate starvation conditions (30). Consequently, especially as demonstrated by the culture with an initial glucose concentration of 200 g/liter (Fig. 4), the long phase of production of foreign proteins using PPHO89 can be stably maintained even in stationary phase.

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Based on the information described above, the phosphateresponsive promoter PPHO89 and the process involving it are extremely attractive for industrial applications. Phosphate-responsive promoters have been exploited in other microorganisms, such as E. coli (6, 18) and S. cerevisiae (27). However, a growth limitation other than sugar limitation in E. coli and S. cerevisiae triggers acetic or alcoholic fermentation, leading to arrest of cell growth and lower productivity (11, 28). In addition to this problem, Hensing et al. (11) indicated that difficulties in maintaining phosphate limitation in complex media and switching to a phosphate-free medium in large-scale fermentation severely limited the applicability of phosphate-responsive promoters. The results of this study showing that P. pastoris does not produce ethanol at a level that may cause arrest of cell growth or lower productivity even with a high glucose concentration offer hope that the roadblocks may be overcome. Adding to this optimism, the fermentation processes described here were developed using complex media and phosphate depletion by cell growth instead of by switching to a different medium. In conclusion, the current study provides valuable information on the isolation of the high-affinity phosphate transporter gene from P. pastoris and the potential use of its promoter as a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expression of heterologous genes in P. pastoris. To our knowledge, this study is the first study to describe the use of a phosphate-responsive promoter in P. pastoris. Our results confirm that the PPHO89 promoter is an ideal promoter in terms of comparative response strength, tight regulation by phosphate, simplicity, and costeffectiveness. REFERENCES 1. Ahn, J. O., E. S. Choi, H. W. Lee, S. H. Hwang, C. S. Kim, H. W. Jang, S. J. Haam, and J. K. Jung. 2004. Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulosebinding domain. Appl. Microbiol. Biotechnol. 64:833–839. 2. Ahn, J. O., J. Y. Hong, H.-W. Lee, M. S. Park, E. G. Lee, C. S. Kim, E. S. Choi, J. K. Jung, and H. W. Lee. 2007. Translation elongation factor 1-a gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Appl. Microbiol. Biotechnol. 74:601–608. 3. Arocho, A., B. Chen, M. Ladanyi, and Q. Pan. 2006. Validation of the 2-⌬⌬Ct calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts. Diagn. Mol. Pathol. 15:56–61. 4. Bieche, I., M. Olivi, M. H. Champeme, D. Vidaud, R. Lidereau, and M. Vidaud. 1998. Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer. Int. J. Cancer 78:661–666. 5. Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. W. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265–3274. 6. Boidol, W., M. Simonis, M. Topert, and G. Siewert. 1982. Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of E. coli. Mol. Gen. Genet. 185:510–512. 7. Claros, M. G., and J. J. Heijnen. 1994. TopPredII: an improved software for membrane protein structure prediction. Comput. Appl. Biosci. 10: 685–686. 8. Fiske, C. H., and Y. Subbarow. 1927. The colorimetric determination of phosphorous. J. Biol. Chem. 66:375–400. 9. Gasch, A. P., and M. Wernet-Washburne. 2002. The genomics of yeast responses to environmental stress and starvation. Funct. Integr. Genomics 2:181–192. 10. Hayashi, N., and Y. Oshima. 1991. Specific cis-acting seqeunce for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:785–794. 11. Hensing, M. C. M., R. J. Rouwenhorst, J. J. Heijnen, J. P. van Dijken, and

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