Pichia Pastoris Strain GS115 Research Articles

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing.

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50°C) and lower stability over alkaline pH range, but better thermal stability at 60°C to 70°C and resistance to feed pelleting inactivation (80°C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.

Production of recombinant great sturgeon (Huso huso, Linnaeus, 1758) growth hormone (GH) by Pichia pastoris (Guillierm, 1956)

In this research, the encoding cDNA of growth hormone (GH) was cloned from the pituitary gland of great sturgeon Huso huso (three adults: two females and one male, 7–9 years old, 70–90 kg, reared in concrete ponds). In order to obtain the great sturgeon recombinant GH expression in Pichia pastoris, the mature encoding cDNA was first cloned in TA vector PTZ57R and then sequenced. After confirmation of the correct GH sequence, the GH coding sequence was subcloned into pHILS1 expression vector. The yeast Pichia pastoris GS115 strain was transformed with the expression plasmid. Results obtained from this study showed that great sturgeon GH recombinants were expressed upon induction with methanol and exported into the medium. The level of expression was examined using RNA analysis, SDS-PAGE, and western blot analysis. RNA analysis of the recombinant strains showed a sharp, specific band in 800 bp. The specific band in transformants indicated the presence of GH RNA in the yeast. SDS-PAGE and western blot analysis showed a specific 21 kDa band for the growth hormone. Culture conditions were optimized for pH = 6 and incubation time (after 24 h induction, peaking at 72 h) for maximal protein production. The results provide useful information for the future production of recombinant growth hormones in other sturgeon species.

Heterologous expression and enzymatic characterization of fructosyltransferase from Aspergillus niger in Pichia pastoris

In this work, the cDNA encoding fructosyltransferase (FTase) from Aspergillus niger YZ59 (CICIM F0901) was obtained and expressed in the methylotrophic yeast Pichia pastoris strain GS115. The yield of recombinant FTase in a 5-L fermentor reached 1020.0 U/mL after 96 h of induction, which was 1160.4 times higher that of native FTase from A. niger YZ59. The specific activity of recombinant FTase was 6.8×10(4) U/mg. The optimum temperature and pH of the recombinant FTase were 55 °C and 5.5, respectively. The recombinant FTase was stable below 40 °C and at pH from 3.0 to 10.0. Using sucrose as the substrate, the Km and Vmax values of recombinant FTase were 159.8 g/L and 0.66 g/(L min), respectively. The turnover number (kcat) and catalytic efficiency (kcat/Km) of recombinant FTase was 1.1×10(4) min(-1) and 68.8 L/(g min), respectively. The recombinant FTase was slightly activated by 5mM Ni(2+), Mg(2+), K(+), Fe(3+), or Mn(2+), but inhibited by all other metal ions (Na(+), Li(+), Ba(2+), Ca(2+), Zn(2+), and Cu(2+)). The highest yield of fructooligosaccharides for purified FTase reached approximately 343.3 g/L (w/v). This is the first study reporting the heterologous expression of FTases from A. niger in P. pastoris. This study plays an important role in the fructooligosaccharide synthesis industry by recombinant FTases.

Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter

A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01h−1. By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37°C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.

Gene cloning and characterization of a novel laccase from the tropical white-rot fungus Ganoderma weberianum TZC-1

The novel laccase gene GwLac1 was cloned from Ganoderma weberianum TZC-1 by reverse transcriptase PCR with degenerate primers on the basis of conserved copper-binding regions of known laccases and the rapid amplification of cDNA ends technique. GwLac1 contains 9 introns and its open reading frame is 1566 bp long. The coding domain sequence of GwLac1 was cloned into the vector pPICZB and expressed in Pichia pastoris strain GS115, resulting in the highest yield of laccase, 2.26 U/mL, when the transformants had been cultivated at 20°C for 8 days. The molecular mass of recombinant GwLAC1 was a little more than 50 kDa as estimated by SDS-PAGE and exhibited catalytic properties with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate. rGwLAC1 was stable at pH of 5.0–7.0 and at temperatures <45°C. The pH and temperature optima and Km of the enzyme for ABTS oxidation were 2.2, 35°C and 85.5 μM, respectively.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

BackgroundWe previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.ResultsSeveral new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%.ConclusionsOur experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-6834-7-111) contains supplementary material, which is available to authorized users.

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

Abstract A study of the Mut+ phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L−1 Dry Cell Weight (DCW) and 26.07 mg L−1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with MutS expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut+ phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut+ phenotype. The results from this study demonstrated that expression of HBsAg with Mut+ AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut+ phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.

Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse

The primary plant cell wall is composed of cellulose, hemicellulose, lignin and protein in a stable matrix. The concomitant depolymerization of lignin by laccase and of hemicelluloses by xylanase can improve lignocellulose degradation in the production of second generation biofuels. A thermophilic variant of xylanase A (XynAG3) and the thermostable laccase (CotA), both from Bacillus subtilis, were produced in co-transformed Pichia pastoris strain GS115. Mobility changes in SDS-PAGE after Endo H digestion indicated that both enzymes were glycosylated. The maximum catalytic activity of the XynAG3Pp and the CotAPp was observed at 58°C and 75°C, respectively, and both enzymes presented high activity at pH 5.0. The half-life at 60°C of XynAG3Pp and CotAPp was 150min and 540min, respectively. The relative levels of CotAPp and XynAG3Pp in culture broths were altered by the concentration of methanol used for induction, and CotAPp:XynAG3Pp ratios of 1:1.5 and 1:2 were evaluated against milled sugar-cane bagasse. The highest activity was observed at a 1:2 ratio of CotAPp:XynAG3Pp, and was 44% higher as compared to the sum of the activities of the individual enzymes in the same assay conditions. These results demonstrate the synergistic action between an endoxylanase and a laccase against the natural lignocellulosic substrate.

Genetically engineered Pichia pastoris yeast for conversion of glucose to xylitol by a single-fermentation process

Xylitol is industrially synthesized by chemical reduction of D-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce D-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of D-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the D-xylulose-forming D-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a single-fermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078g xylitol/g glucose and productivity of 0.29g xylitol/Lh. This was the first report to convert xylitol from glucose by the pathway of glucose-D-arabitol-D-xylulose-xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris.

Human cystatin C (CysC) is a cysteine proteinase inhibitor with many potential applications. To facilitate further studies of the functions and applications of CysC, we improved the heterologous expression of CysC using a basic codon optimization method. In this study, we cloned the high-GC content wild-type sequence of the CysC gene and also designed a slightly AT-biased sequence, with codons optimized for expression in the Pichia pastoris GS115 strain. Our results showed that the optimized coding sequence of human CysC increased the expression and secretion of the CysC protein by approximately 3- to 5-fold (90-96 mg CysC/L) in yeast, compared with the expression levels of the native CysC gene (17.9-18.4 mg CysC/L). We designed, constructed, and applied an optimized version of the CysC gene for the Pichia expression system. Our results demonstrate that the optimized coding sequence provides a higher yield of secreted CysC than that produced using the wild-type gene. Our data also serve as a practical example demonstrating a rational design strategy for the heterologous expression of secreted proteins.

A novel mono- and diacylglycerol lipase highly expressed in Pichia pastoris and its application for food emulsifier preparation

A mono- and diacylglycerol lipase (MDL) was cloned from Penicillium cyclopium and expressed in Pichia pastoris strain GS115. The recombinant enzyme was named Lipase GH1. High cell density fermentation was performed by culture in a 7.5-L fermenter using BSMG medium, in which the phosphate in basal salt medium was replaced by sodium glycerophosphate (Na2GP). The maximal lipase activity detected was 18,000U per mL, and total protein content in the fermentation supernatant was 3.94g per L. The activity of the liquid enzyme remained stable under alkaline conditions at 4°C for 6 months and was 50% after one year. Lipase GH1 was used for the synthesis of mono- and diacylglycerols (MAGs and DAGs), which are commonly used emulsifiers for industrial applications. A conversion rate of 84% after 24h of reaction was obtained using glycerol/oleic acid molar ratio 11:1, water content 1.5wt%, enzyme dosage 80U per g, and reaction temperature 35°C. Lipase GH1 was more efficient for the synthesis of MAGs and DAGs than was Lipase G50 (a similar, commercially available lipase derived from Penicillium camemberti) when oleic acid was used as an acyl donor. Lipase GH1 has potential for food emulsifier preparation.

High expression of recombinant human catalase and its immunomodulatory effects on H1N1 influenza virus infection

Catalase is a key antioxidant enzyme and has been implicated in many pathophysiological processes of human diseases. In a previous study, we developed a yeast-based expression system for recombinant human catalase (rhCAT), and proved its therapeutic effects for treating H1N1 virus-induced pneumonia. However, the preparation of rhCAT was insufficient for further research applications. Here, we describe a much more convenient construction strategy for rhCAT based on the Pichia pastoris GS115 strain in a 14L bioreactor. Quantitative-PCR, Western blotting, and activity assay were used to demonstrate the stable and efficient high-level expression (1500U/mL; 77% recovery) achieved by this newly developed simple two-step purification procedure. The rhCAT synthesized by this new procedure was applied to an H1N1-infected mouse model (doses of 50 and 100kU/mice/day) to assess its capabilities for inducing immunomodulatory effects. The results showed that the rhCAT could restore the impaired phagocytosis, alleviate the induced reductions in spleen and thymus organ weights, and markedly reduce the lung tissue viral load, all in a dose-dependent manner. Thus, this study provides not only a simple method for large-scale preparation of active rhCAT, but also in vivo evidence of the recombinant protein's immunomodulatory activity which may have clinical applications in treating H1N1 or other viral infections.

Expression and purification of enterovirus type 71 polyprotein P1 using Pichia pastoris system

Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.

Production of ethanol from cellobiose by recombinant β-glucosidase-expressing Pichia pastoris: Submerged shake flask fermentationr

Pichia pastoris strain GS115 was engineered to express a synthetic gene encoding β-glucosidase for its use in ethanol production. β-glucosidase was expressed and secreted either as mature enzyme or N- hexahistidine (His6)-tagged fusion protein, under the control of the strong methanol inducible promoter AOX1. The recombinant β-glucosidase was partially characterized and showed properties comparable to native enzyme. The transformed yeast acquired the ability to assimilate and ferment 1% cellobiose in non aerated batch cultures. Ethanol concentrations were 2.56 g/l and 1.35 g/l which corresponded to 84% and 45% for the non-His-tagged and His-tagged transformants respectively compared to glucose fermentation. A 2.4 fold increase in ethanol production was obtained upon methanol induction of yeast cells in the fermentation medium. Key words: Pichia pastoris; Beta glucosidase; Gene cloning; Cellobiose; Submerged Fermentation; Ethanol.

Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

ABSTACT: The objective of this work was to isolate the polygalacturonase genes of Galactomyces citri-aurantii IJ-1 harvested from rotten citrus peels and to heterologously express these genes in Pichia pastoris. Two polygalacturonase (PG) genes from G. citri-aurantii IJ-1 were obtained and tentatively named PG1 and PG2. The genes were cloned into pPICZαC, and expressed in Pichia pastoris strain GS115 with a native signal peptide or the α-factor secretion signal peptide of Saccharomyces cerevisiae. All of the recombinant proteins were successfully secreted into the culture media and confirmed as a single band with a molecular weight of 35 to 38 kDa by SDS-PAGE. The specific enzyme activities of recombinant PG1 and PG2 purified by His-tag affinity resin were 4,749 and 6,719 U/mg, respectively, with an optimal pH and temperature of pH 4.0 and 50°C. The Michaelis-Menten kinetic constants for PG1 and PG2, K (m), were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn(2+), the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu(2+) and Fe(3+) acted as strong inhibitors to the PGs.

The effect of carbon sources on the expression level of thermostable L2 lipase in Pichia pastoris

Thermostable lipases are enzymes that are particularly pleasing for industrial purposes such as in the production of detergents, animal skin-based industry and food processing endeavours. Thermostable L2 lipase gene fished out from Bacillus sp. L2 was cloned into Pichia pastoris strain GS115 under constitutive expression system of pGAPZαA. Study performed on various carbon sources revealed that glucose and glycerol support the growth of Pichia pastoris and expression of L2 lipase. In addition, the by-product of sugar refinery processes (molasses) was found to be a potential carbon source for the growth of P. pastoris and L2 lipase expression. Since the thermostable L2 lipase will be extracellularly secreted into the medium, the use of P. pastoris expression system is seen as an alternative to the conventional expression system of Escherichia coli that applies intracellular expression of the L2 lipase gene encoded. Key words : Thermostable lipase, Pichia pastoris, constitutive expression, carbon source.

A Novel Targeted Multi-Functional Fusion Protein Possesses Inhibitory Activities Against Bacteria, Thrombin and Platelet Aggregation

In the present study, we designed a novel targeted multi-functional fusion protein LHAD composed of LL-37, FXa recognition peptide, hirudin-12-residue, AAP, and RGD peptide. It was expressed in the Pichia pastoris GS115 strain and purified by affinity chromatography. The in vitro studies suggested that the novel designed protein exhibited antibacterial activity, anti-platelet aggregation and anti-thrombin activities. Moreover, the capability of anti-thrombin was significantly increased compared to that of natural hirudin. Our study may provide a potential approach to design multi-functional drugs for the prevention and management of thrombosis.

Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid

The delta 6-desaturase gene was cloned from Rhizopus stolonifer, which could accumulate up to 49% of gamma-linolenic acid (GLA, C18:3 Δ(6,9,12)) to the total fatty acids. The cloned DNA contains a 1,380 bp open reading frame encoding a protein of 460 amino acids, which showed high similarity to those of fungal delta 6-desaturases with three conserved histidine-rich motifs and HPGG motif. Notably, this deduced sequence had a shorter C-terminus. Results demonstrated that the cDNA sequence exhibited delta 6-desaturase activity by accumulation of about 22.4 % of GLA to the total fatty acids in the recombinant Pichia pastoris strain GS115.

Cloning of a gene encoding glycosyltransferase from Pueraria lobata (Wild.) Ohwi and its expression in Pichia pastoris

The key enzyme of puerarin biosynthesis in Pueraria lobata (Willd.) Ohwi was unclear but may involve glycosylation. To investigate the regulation of puerarin biosynthesis, a putative UDP-dependent glycosyltransferase (UGT) gene, PlUGT1 was isolated from P. lobata root, which contained abundant puerarin. PlUGT1encoded 480 deduced amino acid residues with a conserved UDP-glucose-binding domain, which has 61 to 84% similarity to homologues from other plant species. SDS polyacrylamide gel electrophoresis and western blotting results showed that, fusion protein migrated as a single protein band with a molecular weight of 55 kDa. A yeast expression vector pPICZA-PlUGT1 was constructed and was transformed into Pichia pastoris strain GS115. Several recombinants containing multi-copy expression cassettes were obtained on the zeocin-YPD plate and confirmed by southern dot blotting. The yield of PlUGT1 attained 0.05 g/l when recombinant cells were cultured at pH 5.5, 30°C and induced with 0.5% methanol for 72 h. The expression of PlUGT1 protein correlates positively with the copy numbers of PlUGT1 in transformed yeast cells. These results suggest that, the PlUGT1 protein can be expressed efficiently in the P. pastoris expression system and may supply a new economic and convenient way for the production of PlUGT1 protein. Key words: Pueraria lobata (Willd.) Ohwi, glycosyltransferase, cloning, expression,Pichia pastoris.

Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full‐Length Nonstructural Gene in Pichia pastoris

Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).