Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.     

Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.     

Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin

Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated.     

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.     

The conformation of mature human alpha-amylase conditions its secretion from yeast

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.     

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.     

Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element

A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a 'universal' fungal vector appears to be feasible.     

Role of gamma-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.     

Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha

The methylotrophic yeast, Hansenula polymorpha, has been developed as a host system for the synthesis of heterologous proteins. The middle surface antigen of hepatitis B virus (preS2-HBsAg) has been synthesized under the control of a methanol-regulated promoter derived from the methanol oxidase-encoding gene. The synthesized preS2-HBsAg protein was found to be secreted outside the cell membrane into the periplasm and further excreted into the culture medium following permeabilization of the cell wall with beta-1,3-glucanase (beta Glu). Cell cultures treated with beta Glu were able to continuously synthesize and secrete 22-nm particles of preS2-HBsAg into the medium for several days. The overall yield of antigen from treated cultures was found to be over threefold greater than that of untreated controls. The observation that complex supramolecular structures, such as the 22-nm particles of preS2-HBsAg, can be secreted by H. polymorpha and released into the medium, suggests the potential for these yeasts to be an alternative secretory host.     

Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.

We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.     

Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway

In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.     

Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.     

Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae

Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.     

Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.     

Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.     

Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha

We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios.     

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.