Translation elongation factor 1-α gene from <Emphasis Type="Italic">Pichia pastoris</Emphasis>: molecular cloning,sequence, and use of its promoter

The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P _TEF1 ) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P _GAP ) by using a bacterial lipase gene as a reporter gene. P _TEF1 demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P _GAP . The sequence of the gene was deposited in GenBank under accession no. EF014948.     

Expression, purification, and immobilization of His-tagged d-amino acid oxidase of Trigonopsis variabilis in Pichia pastoris

High-level expression of d-amino acid oxidase (DAO) has been reported in Pichia pastoris by integrating the DAO gene under the control of the alcohol oxidase promoter (P_AOX1). However, the time taken to reach peak product concentration is usually long (∼43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (P_GAP). The maximal level of HDAO expression using the P_GAP integrant is attained in 13 h and is equal to that obtained using the P_AOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiae α-factor secretion signal under a P_GAP or P_AOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under P_GAP was purified by agar-based affinity support and then immobilized on Amberzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U g⁻¹ (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under P_GAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application.     

A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5′ end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61–65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using β-lactamase as a reporter, we show that the FLD1 promoter (P_FLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of β-lactamase produced under control of P_FLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (P_AOX1). Thus, P_FLD1 is an attractive alternative to P_AOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.     

Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast,Pichia thermomethanolica,for heterologous gene expression,especially at elevated temperature

The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 °C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 °C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.     

Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P_PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P_AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P_PPLCC1 was compared with those of P_AOX1 and P_CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P_PPLCC1 and P_CUP1, respectively, after induction with 0.2 mM CuSO₄ at OD₆₀₀ = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For P_AOX1 activity, yeast cells harboring P_AOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P_PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P_PPLCC1 is additionally superior to P_AOX1 since it does not require laborious feeding with a carbon source.     

Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

The ORF encoding the Debaryomyces castellii CBS 2923 phytase was isolated. The deduced 461-amino-acid sequence corresponded to a 51.2 kDa protein and contained the consensus motif (RHGXRXP) which is conserved among phytases. No signal sequence cleavage site was detected. Nine potential N-glycosylation sites have been predicted. The protein shared 21–69% sequence identities with various phytases of yeast or fungal origin. Heterologous expression of the D. castellii CBS 2923 phytase in the methylotrophic yeast Pichia pastoris was tested under both the P. pastoris inducible alcohol oxidase (AOX1) promoter and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Maximum production levels obtained were 476 U ml⁻¹, with the AOX1 expression system and 16.5 U ml⁻¹ with the GAP one. These productions corresponded to a 320-fold and a 10-fold overexpression of the protein, respectively as compared to the homologous production. The biochemical characteristics of the recombinant phytase were identical to those of the native enzyme.     

Characteristics and applications of recombinant thermostable amylopullulanase of Geobacillus thermoleovorans secreted by Pichia pastoris

The 3′-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuΔC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuΔC) was higher under GAP promoter (36.2 U ml⁻¹, α-amylase; 33.5 U ml⁻¹, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml⁻¹). The heavily glycosylated Gt-apuΔC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.

     

Expression of the xylan-degrading genes of Bacillus pumilus IPO in Saccharomyces cerevisiae

Xylanase (xynA) and β-xylosidase (xynB) genes of Bacillus pumilus were expressed in Saccharomyces cerevisiae by using the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae. Yeast cells harboring a plasmid pNAX2 containing xynA produced xylanase in the cytoplasm of the cell to an extent as much as 5% of the total soluble protein in the cell extract. Xylanase produced in yeast had an extra methionine at the N-terminus, but had the same specific activity as that produced by B. pumilus IPO. The xylanase in the yeast was not glycosylated and was immunologically identical to that of B. pumilus IPO. Yeast cells harboring a plasmid pYXB containing xynB produced β-xylosidase in the cytoplasm of the cell (3% of the total soluble protein). β-Xylosidase purified from the yeast strain exhibited specific activity nearly equal to the value of enzyme purified from B. pumilus, and had an N-terminal sequence identical to the sequence of the enzyme from B. pumilus.     

Enhancing the efficiency of the <Emphasis Type="Italic">Pichia pastoris AOX1</Emphasis> promoter via the synthetic positive feedback circuit of transcription factor Mxr1

Background

The methanol-regulated AOX1 promoter (P_AOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of P_AOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of P_AOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal.

Results

The characteristics of P_AOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (P_AOX2). This novel approach enhanced P_AOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow P_AOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on P_AOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction.

Conclusions

We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

     

Cloning and sequencing of the putative glyceraldehyde-3-phosphate dehydrogenase gene from Cephalosporium acremonium and its application to heterologous gene expression

A candidate for Cephalosporium acremonium glyceraldehyde-3-phosphate dehydrogenase gene (GAP) was cloned using the corresponding Saccharomyces cerevisiae gene as a probe. It encoded peptides showing marked similarity with those from S. cerevisiae and Aspergillus nidulans. A fused gene, GAP-HPT, consisting of the putative GAP promoter and the open reading frame of the bacterial hygromycin B phosphotransferase gene (HPT) was constructed, and it was successfully used to transform C. acremonium to a hygromycin B-resistant phenotype.     

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

Identification and characterization of P GCW14 : a novel, strong constitutive promoter of Pichia pastoris

The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P _GCW14 , and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P _GCW14 were cloned upstream of the EGFP gene and promoter activity was analyzed by measuring fluorescence intensity. P _GCW14 exhibited significantly stronger promoter activity than the classic strong constitutive promoters P _TEF1 and P _GAP under various carbon sources, suggesting that P _GCW14 is a strong and constitutive promoter. Hence, P _GCW14 can be used as a promoter for high-level expression of heterologous proteins.     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.