Methanol-Inducible Promoter of Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica BCC16875 Potential for Production of Heterologous Protein at High Temperatures

Methanol-utilizing metabolism is generally found in methylotrophic yeasts. Several potential promoters regulating enzymes in this pathway have been extensively studied, especially alcohol oxidase. Here, we characterized the alcohol oxidase gene promoter from thermotolerant Ogataea thermomethanolica (OthAOX). This promoter can be induced by methanol, and was shown to regulate expression of phytase up to 45 °C. The pattern of heterologous phytase N-glycosylation depends on the induction temperature. Unlike the AOX promoter from Pichia pastoris, this OthAOX initially turns on the expression of the heterologous protein at the de-repression stage in the presence of glycerol. Full induction of protein is observed when methanol is present. With this methanol-inducible promoter, target protein can be initially produced prior to the induction phase, which would help shorten the time for protein production. Being able to drive protein expression at various temperatures prompts this newly identified AOX promoter to be potential tool for heterologous protein production in high temperature conditions.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

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

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

Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation

A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml^?1 and enzyme activity reached 1,030 U ml^?1. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg^?1 with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4–5 °C increases in the melting temperatures (T_m). The K_m and K_cat of appA-Q258N/Q349N were 0.43 mM and 3,058 s^?1, respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.     

Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression

A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH₄)₂SO₄ precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.     

Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm,Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5–2.0 and 5.5–6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84 % of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.     

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein

This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P.?thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis

Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8–9 % of the total cell protein.     

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.     

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

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

Definition of culture conditions for Arxula adeninivorans,a rational basis for studying heterologous gene expression in this dimorphic yeast

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l^?1 was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l^?1. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h^?1 and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.     

High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris

A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca²⁺ and Mg²⁺ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K _m) of 1.89 mg/mL and a maximum reaction rate (V _max) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.     

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25–60 °C and pH 3.0–9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL^?1. In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.