Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris

The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml^?1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml^?1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation.     

Production of Aspergillus niger β-mannosidase in Pichia pastoris

β-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various sugar chains. cDNA corresponding to the β-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The β-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44mmol/l for p-nitrophenyl β-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).     

Secretory expression of the α-subunit of human coagulation factor XIII in the yeast Pichia pastoris

Pro-FXIIIa (the -subunit of FXIII with activation peptide, which must be removed to produce the active form of FXIIIa), cloned from human placenta cDNA library, was overexpressed in the methylotrophic yeast Pichia pastoris GS115 (his4) and secreted into the culture medium to yield the recombinant pro-FXIIIa subunit with a predicted molecular mass of approximately 83 kDa. The gene was located immediately downstream of the strong yeast alcohol oxidase promoter (AOX1). In shake flask culture, recombinant pro-FXIIIa (rFXIIIa) was secreted into the culture medium at above 50 mg l^–1. The fibrin-stabilizing activity of the recombinant pro-FXIIIa, after thrombin activation, was confirmed using fibrin cross-linking patterns, and analyzed by SDS-PAGE.     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast

α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.     

Production of high activity Aspergillus niger BCC4525 β-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis

A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high k_cat/K_m (461 mg^?1 s^?1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant β-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.     

Improved expression of Rhizopus oryzae α-amylase in the methylotrophic yeast Pichia pastoris

In our previous study, the α-amylase from Rhizopus oryzae (RoAmy) was expressed in Escherichia coli and Saccharomyces cerevisiae but the obtained recombinant RoAmy (rRoAmy) yields were too low. The aim of the present research was to obtain high-level expressions of RoAmy in the methylotrophic yeast Pichia pastoris. To this end, we constructed P. pastoris strains with the capability to express recombinant α-amylase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) and methanol-inducible alcohol oxidase 1 promoters. The levels of inducibly expressed rRoAmy were higher than those of constitutively expressed. The maximal inducible rRoAmy expression levels for the Mut(+) strains (41.1mg/l) were approximately eight times higher than those for the Mut(s) strains and 24 times higher than those expressed under the control of the GAP promoter. For both inducible and constitutive expressions, the S. cerevisiae α-prepro sequence and the native signal sequence of RoAmy were used separately to direct the secretion of rRoAmy into the culture medium of P. pastoris. Low levels of intracellular amylase activities that had been detected after shake-flask fermentation indicated that both signal sequences could effectively direct the secretion of rRoAmy under all studied conditions. In addition, the secretion levels of rRoAmy directed with its own signal peptide were 7-10% higher than those directed by the α-prepro sequence.     

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.     

Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70°C and pH 5.0.     

Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

Cloning of the Aspergillus niger gene encoding α-l-arabinofuranosidase A

Using l-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of -l-arabinofuranosidase A by an Aspergillus niger d-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an -l-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding -l-arabinpfuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.     

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.α-Galactosidase (αGal) (EC 3.2.1.22) is of particular interest in view of its biotechnological applications. αGal from coffee beans demonstrates a relatively broad substrate specificity, cleaving a variety of terminal α-galactosyl residues, including blood group B antigens on the erythrocyte surface. Treatment of type B erythrocytes with coffee bean αGal results in specific removal of the terminal α-galactosyl residues, thus generating serological type O erythrocytes (8). Cyamopsis tetragonoloba (guar) αGal effectively liberates the α-galactosyl residue of galactomannan. Removal of a quantitative proportion of galactose moieties from guar gum by αGal improves the gelling properties of the polysaccharide and makes them comparable to those of locust bean gum (18). In the sugar beet industry, αGal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (28). Raffinose and stachyose in beans are known to cause flatulence. αGal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (16).αGals are also known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). Dey et al. showed that αGals are classified into two groups based on their substrate specificity. One group is specific for low-M_r α-galactosides such as pNPGal (p-nitrophenyl-α-d-galactopyranoside), melibiose, and the raffinose family of oligosaccharides. The other group of αGals acts on galactomannans and also hydrolyzes low-M_r substrates to various extents (6).We have studied the substrate specificity of αGals by using galactomanno-oligosaccharides such as Gal³Man₃ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotriose) and Gal³Man₄ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotetraose). The structures of these galactomanno-oligosaccharides are shown in Fig. ​Fig.1.1. Mortierella vinacea αGal I (11) and yeast αGals (29) are specific for the Gal³Man₃ having an α-galactosyl residue (designated the terminal α-galactosyl residue) attached to the O-6 position of the nonreducing end mannose of β-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 αGal (12) and Penicillium purpurogenum αGal (25) show a preference for the Gal³Man₄ having an α-galactosyl residue (designated the stubbed α-galactosyl residue) attached to the O-6 position of the third mannose from the reducing end of β-1,4-mannotetraose. The M. vinacea αGal II (26) acts on both substrates to almost equal extents. The difference in specificity may be ascribed to the tertiary structures of these enzymes. Open in a separate windowFIG. 1Structures of galactomanno-oligosaccharides.Genes encoding αGals have been cloned from various sources, including humans (3), plants (20, 32), yeasts (27), filamentous fungi (4, 17, 24, 26), and bacteria (1, 2, 15). αGals from eukaryotes show a considerable degree of similarity and are grouped into family 27 (10).Here we describe the cloning of P. purpurogenum αGal cDNA, its expression in Saccharomyces cerevisiae, and the purification and characterization of the recombinant enzyme.     

Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59–97 above kDa glycoprotein, 52–57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.     

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS ₄ locus (38 of Mut⁺ clone) or into theAOX1 locus (45 of Mut^s clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of the recombinantS. cerevisiae.