Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.     

Structural comparison of the Pichia pastoris alcohol oxidase genes

In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway. The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids. The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively. In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5′ or 3′ non-coding regions. Although alcohol oxidase is found in peroxisomes of P. pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes.     

Disruption of the KEX1 gene in Pichia pastoris allows expression of full‐length murine and human endostatin

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright © 1999 John Wiley & Sons, Ltd.     

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein

An alkaline serine protease (SF1) from Kocuria kristinae F7 was purified. The molecular mass of the SF1 protease was estimated to be 57 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The N-terminal amino acid sequence of the first 14 amino acids of the SF1 protease showed low homology with bacterial proteases, suggesting that the enzyme had not been described previously. The SF1 protease exhibited maximal activity at pH 9.0 and 60 °C. The activity of the SF1 protease was enhanced by the presence of Ca²⁺ and Mg²⁺ ions. It showed high stability in the presence of NaCl and ethanol. Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses indicated that soybean protein isolates treated with the SF1 protease generated four principal new hydrophilic peptides. Mass spectrometry analyses indicate that the distribution of molecular weight of these peptides was from 0.705 to 1.305 kDa. Hydrolysis of soybean protein isolates with the SF1 protease increased the level of total free amino acids, essential amino acids and flavor amino acids. The SF1 protease may decrease the bitterness of soy protein hydrolysates. The results showed that the SF1 protease of Kocuria kristinae F7 appears to be good candidate enzyme for potential application in acceleration of fermented soybean food ripening.     

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19‐1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19‐1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. The GenBank Accession Nos of the sequences are AF255332 (K.lactis GDI1) and AY007574 (P. pastoris GDI1). Copyright © 2001 John Wiley & Sons, Ltd.     

Identification of major ADH genes in ethanol metabolism of Pichia pastoris

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.     

Pichia pastoris protease-deficient and auxotrophic strains generated by a novel,user-friendly vector toolbox for gene deletion

Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.     

High‐yield secretion of recombinant gelatins by Pichia pastoris

Recombinant non‐hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae α‐mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3·0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono‐arginylic sites, by a putative Kex2‐like protease, could be successfully abolished by site‐directed mutagenesis of these sites. Production levels as high as 14·8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, B and C vectors with the Tn903 kan^R marker from pFA6a KanMX6, which confers G‐418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa‐histidine‐tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production. Copyright © 2009 John Wiley & Sons, Ltd.     

The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg^–1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K_m and V_max values were 5.6 and 4.8 mg mL^–1, and 2785 and 1608 μmol min^–1 mg^–1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g^–1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.     

ACYLATION OF FOOD PROTEINS AND HYDROLYSIS BY DIGESTIVE ENZYMES: A REVIEW

The acylation of proteins involves the covalent attachment of acyl groups such as fatty acids and amino acids to both α- and ε-amino groups of the polypeptide chain. Chemical methods are currently used to covalently attach amino acids to casein via isopeptide bonds. By modifying food proteins with acylation, it is possible to increase the nutritional value of the protein, judging from growth tests on rats. Enzymatic methods via transglutaminase and human placental factor XIII for preparing protein derivatives have also been tested. In vitro hydrolysis studies using intestinal homogenates have indicated that the intestinal mucosal hydrolases efficiently cleave acylated proteins as well as ε-peptide bonds. Hog intestinal aminopeptidase N, as well as N-acylpeptide hydrolase and acylase I have been found to be responsible for the biological utilization of N-acylated food proteins.     

Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.     

Cloning,nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.     

A quantitative analysis of amino acid accumulation in developing grain of normal and Opaque-2 maizes

From previous observations concerned with the qualitative and quantitative evolution of protein groups in developing maize (Zea mays L.) grain, it may be deduced that the amount per grain of any amino acid incorporated into the proteins is linearly related to the amount of true protein in the grain. In this study, such a linear relationship is characterised quantitatively from experimental data by regressing the individual amino acid against the total protein accumulated in the developing grain of both normal (+) and opaque-2 (o2) maize. The linear least squares regression on total nitrogen content in the grain is also used for specifying the quantitative variations of any free and protein-incorporated amino acid in the grain. The slope B_j of the regression line represents the relative rate of accumulation of amino acid (j) in grain protein (or nitrogen) and its limiting content in protein (or nitrogen) of a grain which accumulates a large amount of it. B_j, which is compared to the amount of amino acid (j) present in the protein of immature grains, or of mature endosperm, or germ, is close to the amount of amino acid (j) present in the protein of mature endosperm. The same holds for the B_j value determined from opaque-2 maize and which differs from the amount of B_j determined using the normal variety for many amino acids. Using experimental data reported in the literature, a linear relationship has also been found in describing quantitative variations of amino acid in the developing grain of normal and Hiproly barley.     

Cloning of a thermostable xylanase from Actinomadura sp. S14 and its expression in Escherichia coli and Pichia pastoris

A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-β-xylanase, β-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-β-xylanase activity on xylan and xylooligosaccharides than on xylotriose.     

Studies to analyse the relationship between IFNα2b gene dosage and its expression,using a Pichia pastoris‐based expression system

Human interferon α2b (hIFNα2b) is the most important member of the interferon family. Escherichia coli, yeasts, mammalian cell cultures and baculovirus‐infected insect cells have been used for expressing recombinant human interferon. Recently a Pichia pastoris‐based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b‐expressing cassettes were created via an in vitro multimerization approach. P. pastoris host strain X‐33 was transformed using these expression cassettes. Groups of P. pastoris clones transformed with different copies of the IFNα2b expression cassette were screened for intrachromosomal integration. The IFNα2b expression level of stable transformants was checked. The copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest. Copyright © 2013 John Wiley & Sons, Ltd.