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.     

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Mut^s) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.     

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris

Human α-defensin 5 (HD₅), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD₅ through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD₅ mature peptide (rmHD₅) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD₅ by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD₅ into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD₅ vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD₅ nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD₅, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD₅. The results showed that about 165.0 mg/l of rmHD₅ with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD₅ was recovered after purification. The in vitro experiments revealed that rmHD₅ exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD₅ in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.     

Immunolocalization of inhibin α-subunit in the human testis

Summary The localization of inhibin -subunit in the human testis was studied at the light- and electron-microscope level with immunostaining techniques. Antibodies against specific fragments of porcine and human inhibin -subunits were utilized. At light microscopy, inhibin -subunit immunoreactivity was detected in Sertoli cells, spermatocytes and in some Leydig cells. At electron microscopy, gold labeling was found in the cisternae of the Golgi apparatus and in the endoplasmic reticulum of Sertoli and Leydig cells. Gold labeling for inhibin was also found in coated vesicles in the cytoplasm of Sertoli cells as well as in coated pits and coated vesicles in the cytoplasm of some spermatocytes. The results of the present study suggest that, in the human testis, inhibin is produced by Sertoli and Leydig cells and is taken up by spermatocytes, on which it might act in a paracrine manner.     

STAT5 signaling in expression of the α-subunit of interleukin-2 receptor in human blood lymphocytes

A comparative study of STAT3 and STAT5 activity (assessed by tyrosine phosphorylation level) and the expression of an α-subunit of the interleukin-2 receptor (examined by cytophotometric evaluation of CD25 cell number) during phytohemaglutinin (PHA)-induced proliferation of human blood lymphocytes (HBLs) has been carried out. It was found that the level of STAT3 phosphorylation was high both in resting and competent HBLs and remained unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen either in resting or competent HBL. In the presence of PHA, STAT5 phosphorylation was observed no earlier than in 2–5 h; maximal phosphorylation was detected after 24 h. In competent HBLs, exogenous IL-2 induced high phosphorylation of STAT5 in 30 min that was retained for the next 24–48 h. Alterations in the level of tyrosine phosphorylation of STAT5 correlated with CD25 expression. WHI-P131, a JAK3 kinase inhibitor, prevents STAT5 activation, CD25 surface expression, and lymphocyte proliferation. It is concluded that JAK3/STAT5 signaling via an IL-2 receptor is necessary to support the long-term expression of a high-affinity αβγ_c-receptor of IL-2 and HBL optimal proliferation.     

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.     

Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture

Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h⁻¹ for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y _P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l⁻¹ h⁻¹.     

Constitutive expression of alkaline β-mannanase in recombinant Pichia pastoris

Alkaline β-mannanase has important applications for specific industrial processes like pulp bleaching and the detergent industry. The low yield of alkaline β-mannanase produced from native microbes such as alkaliphilic Bacillus limits its applications. Pichia pastoris is the most efficient heterologous system to produce alkaline mannanase. However, the previous use of the AOX system required large amount of methanol and sophisticated operation strategy, which are undesirable in large scale production. In this study, we established a safe and simple constitutive expression process for mannanase production in P. pastoris. The mannanase gene was successfully expressed under the control of GAP promoter. Sequential optimization of the constructed strains was also performed including the copy number optimization and co-expression of chaperone genes. A two-stage feeding strategy was then applied for the finally optimized strain. After 96 h fermentation, a production level of 2980 U/mL was finally reached, illustrating the potential of the GAP constitutive expression system for industrial scale preparation of alkaline β-mannanase.     

Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.     

Characterization of the reciprocal binding sites on human α-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-a-thrombin interactions. -Thrombin activated Factor XIII more efficiently (Km = 0.83 ± 0.08 × 10^-7 M; V/K = 14.90 ± 3.20 × 10^-3 min^-1) than -thrombin (Km = 6.14 ± 1.26 × 10^-7 M; V/K = 3.30 ± 1.00 × 10^-3 min^-1) or -thrombin (Km = 6.25 ± 1.15 × 10^-7 M; V/K = 3.00 ± 0.80 × 10^-3 min^-1). Immobilized FPR--thrombin bound plasma Factor XIII (Kd = 0.17 ± 0.04 × 10^-7 M) > Factor XIIIa (Kd = 0.69 ± 0.18 × 10^-7 M) > liver transglutaminase (Kd = 4.73 ± 1.01 × 10^-7 M) > Factor XIII A-chain (Kd = 49.00 ± 9.40 × 10^-7 M). FPR--thrombin and -thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 × 10^-7 M and 13.6 × 10^-7 M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR--thrombin: IC₅₀ = 1.0 × 10^-7 M, 3.0 × 10^-6 M and 1.52 × 10^-4 M, respectively. Transglutaminase also inhibited Factor XIII binding to ×-thrombin (IC₅₀ = 2.0 × 10^-6 M). Thrombin-binding site was localized to G^-38-M^-731 fragment of Factor XIII A-chain, probably within homologous regions (N^-72-A^-493) of transglutaminase. R^-320-E^-579 of -thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in -thrombin were essential for binding but not catalytic H^-363 or residues R^-382-N^-394 and R^-443-G^-475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris

β-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l^?1. Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 °C and pH 7. It showed not only a β-xylosidase activity with a K_m of 8 mM and a V_max of 54 μmol min^?1 mg^?1 for hydrolysis of p-nitrophenyl β-d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg^?1) on p-nitrophenyl arabinofuranoside.     

Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6×His tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni²⁺-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni²⁺-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.     

High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc

Bioprocess and Biosystems Engineering - β-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important...     

Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris

A β-glucosidase gene (bgl) from Aspergillus oryzae GIF-10 was cloned, sequenced and expressed. Its full-length DNA sequence was 2,903 bp and included three introns. The full-length cDNA sequence contained an open reading frame of 2,586 nucleotides, encoding 862 amino acids with a potential secretion signal. The A. oryzae GIF-10 bgl was functionally expressed in Pichia pastoris. After 7-day induction, protein yield reached 321 mg/mL. Using salicin as the substrate, the specific activity of the purified enzyme reached 215 U/mg. The purified recombinant β-glucosidase was a 110-kDa glycoprotein with optimum catalytic activity at pH 5.0 and 50 °C. The enzyme was stable between 20 and 60 °C, and retained 65 % of its activity after being held at 60 °C for 30 min. The recombinant β-glucosidase was relatively stable in a broad range of pHs, from 4.0 to 6.5. It showed broad specific activity, hydrolyzing a range of (1-4)-β-diglycosides and (1-4)-α-diglycosides, and Mn²⁺ stimulated its activity significantly.     

Production of the stable human histamine H₁ receptor in Pichia pastoris for structural determination

G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.     

A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 (III) chain in Pichia pastoris

Recombinant human full-length mature collagen α1 (III) chain (rhCOL3A1) was secreted by Pichia pastoris GS115, using the Saccharmyces cerevisiae á-mating factor prepro signal, and the theoretical molecular weight of rhCOL3A1 was 95.344 kDa. The gene cloned from human placenta, was designed and cloned into expression vector pPIC9K under the control of a strong inducible promoter AOX1.The expression stage of rhCOL3A1 was sensitive to different carbon ratios through mixed fermentation. LCMS/ MS analysis and western blotting demonstrated that the recombinant human full-length mature collagen a1 (III) gene was successfully expressed in P. pastoris GS115 during the methanol induction stage. Furthermore, an effective strategy of mixed fermentation was established to express rhCOL3A1 in shake flash. Compared to single carbon induction, when induced with mixed carbon at the ration of 0.8 (glycerol/methanol), the time corresponding to the highest yield of rhCOL3A1 (1.27 g/L) was drastically reduced by 50%. The same conclusion was observed from RT-qPCR. Consequently, a new strategy which was more time-saving and effective was provided for the large-scale producing the full-length mature rhCOL3A1.     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

β-glucanases of the yeast Pichia polymorpha

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl--d-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A₅₀ has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different -glucanases located in the periplasmic space is presented.     

Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.