Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis

This study describes the expression in Pichia pastoris of hepatitis B surface antigens (HBsAg) corresponding to the S region of the four major subtypes: adr, adw2, ayr and ayw3 and to the preS2-S region of the two subtypes adr and adw2. The recombinant yeast strains have been selected amongst methanol utilization positive (Mut⁺) or sensitive strains (Mut^s) and cultivated to high cell density in bioreactor using a short protocol. Our results prove the efficiency of P. pastoris to produce all the major HBsAg subtypes and confirm the ability of the methanol regulated promoter of alcohol oxidase I gene (AOX) to express heterologous protein through phenotype Mut⁺ or Mut^s strains.All these recombinant HBsAg proteins, including subtype ayr, whose production has never been presented, have been highly purified using a short original sequence of steps which includes high-pressure cell disruption associated with detergent treatment, ultrafiltration and immunopurification chromatography using a mAb anti-HBs. The whole process avoids possible alterations of antigenic properties and allows to obtain with high yield, high quality reagents for in vitro diagnosis.     

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut⁻ phenotype cannot grow on methanol as a single carbon source. We assessed the Mut⁻ phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (P_AOX1) was active in the Mut⁻ phenotype and showed adequate eGFP fluorescence levels and protein yields (Y_P/X) in small-scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using P_AOX1HSA and P_AOX1vHH expression constructs. Scenario B comprising a glucose-methanol phase and a 72-hr-long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut⁻ phenotype, the oxygen demand, heat output, and specific methanol uptake (q_methanol) in the methanol phase were reduced by more than 80% compared with the Mut^S phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut⁻ strain for current and future applications in the field of recombinant protein production.     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.     

Production of a sterol esterase from Ophiostoma piceae in batch and fed‐batch bioprocesses using different Pichia pastoris phenotypes as cell factory

The potential biotechnological applications for the Ophiostoma piceae sterol esterase (OPE) are conditioned to the availability of high enzyme amounts at low prices. This enzyme is a versatile biocatalyst with different biotechnological applications. In this work a systematic study on its heterologous production in different Pichia pastoris strains and operational strategies is presented. The best results were obtained using an AOX1 defective yeast strain in a fed‐batch bioprocess using methanol as inducer substrate at a set point of 2.5 g L^?1 and sorbitol as cosubstrate by means of a preprogramed exponential feeding rate at a μ = 0.02 h^?1, reaching 30 U mL^?1 of enzyme and a volumetric productivity of 403.5 U L^?1 h^?1. These values are twofold higher than those obtained with a Mut⁺ phenotype using methanol a sole carbon source. OPE was the main protein secreted by the yeast, 55% for Mut^s versus 25% for Mut^+. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1012–1020, 2014     

Expression of HBsAg and preS2-S protein in different yeast based system: A comparative analysis

We have expressed both S and preS2-S genes coding for the hepatitis B small (S) and medium (M) proteins, respectively, in different yeast based expression systems and compared the production level of the recombinant proteins. In Saccharomyces cerevisiae, viral genes were expressed under the inducible Gal10/cyc1 and the constitutive PGK promoters using 2 μ replicating vectors. We showed that the yield of S protein was higher than M protein under both inducible (14.27 vs 10.9 mg/l) and constitutive (9.18 vs 6.39 mg/l) conditions, respectively. In the methylotrophic yeast Pichia pastoris, the viral genes were expressed in GS115 (Mut⁺: Methanol Utilizing) and KM71 (Mut^S: Methanol Utilizing Slow) under the control of the alcohol oxidase promoter (AOX1). In Mut^S background, both S and preS2-S genes were expressed at higher levels than in Mut⁺. In attempt to increase the yield of recombinant viral proteins in S. cerevisiae, we have co-expressed both inducible and constitutive vectors harboring the S or preS2-S genes leading to recombinant strains called UTS (containing pDP/S + pYePIT/S) and UTP (containing pDP/preS2-S + pYePIT/preS2-S). We showed that the recombinant S and preS2-S proteins were successfully detected and the production level reached 18.31 mg/l for the S and 13.22 mg/l for the M proteins.Our comparative study provides evidence that in small scale, S. cerevisiae is more suitable for HBsAg and preS2-S proteins production than P. pastoris under inducible rather than constitutive condition.     

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD₆₀₀ 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H₂O₂ inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.     

High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris

The porcine circovirus type 2 (PCV2) capsid protein (Cap) is an important antigen for the development of vaccines. To achieve high-level expression of recombinant PCV2 Cap in Pichia pastoris, the wild-type Cap (wt-Cap) and optimized Cap (opti-Cap) gene fragments encoding the same amino acid sequence of PCV2 were amplified by PCR using DNA from lymph nodes of postweaning multisystemic wasting syndrome-suffered pigs and synthesized based on the codon bias of the methylotrophic yeast P. pastoris, respectively. The wt-Cap and opti-Cap gene fragments were inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the alcohol oxidase 1 (AOX1) promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmids, designated as pPIC9K-wt-Cap and pPIC9K-opti-Cap, were linearized using SacI and transformed into P. pastoris GS115 by electroporation. The expressed intracellular soluble opti-Cap reached 174 μg/mL without concentration in a shake flask and kept good reactivity to PCV2-specific positive sera, whereas the wt-Cap could not be detectable throughout three times electroporation. Strong specific PCV2-Cap antibodies were elicited from piglets immunized with vaccine based on opti-Cap. To the best of our knowledge, the achieved opti-Cap yield is the highest ever reported. Our results demonstrated that codon optimization play an important role on the high-level expression of a codon-optimized PCV2-Cap gene in P. pastoris, and the vaccine based on opti-Cap may be a potential subunit vaccine candidate.     

Production and Purification of Human Fibroblast Collagenase (MMP-1) Expressed in the Methylotrophic YeastPichia pastoris

The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeastPichia pastoris.The proMMP-1 encoding DNA was fused to theSaccharomyces cerevisiaepre-pro α-mating factor secretion signal in theP. pastorispPIC9 expression plasmid, transformed into strain GS115 (His⁻), and His⁺Mut^s(slow methanol utilization) transformants were selected. Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations. The protein was purified to greater than 95% homogeneity. The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS–PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity. These data suggest that theP. pastorisexpression system offers a convenient and efficient means to produce and purify MMP-1.     

Combined use of <Emphasis Type="Italic">GAP</Emphasis> and <Emphasis Type="Italic">AOX1</Emphasis> promoters and optimization of culture conditions to enhance expression of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> lipase

Rhizo mucor miehei lipase (RML) is an industrially important enzyme, but its application is limited due to its high cost. In this study, a series of measures such as codon optimization, propeptide addition, combined use of GAP and AOX1 promoters, and optimization of culture conditions were employed to increase the expression of RML. Three transformants of the constitutive-inducible combined Pichia pastoris strains were generated by transforming the pGAPZαA-rml vector into the pPIC9K-rml/GS115 strain, which resulted in high-expression yields of RML. Using the shake flask method, highest enzyme activity corresponding to 140 U/mL was observed in the strain 3-17, which was about sixfold higher than that of pPIC9K-rml/GS115 or pGAPZαA-rml/GS115. After optimization of culture conditions by response surface methodology, the lipolytic activity of strain 3-17 reached 175 U/mL in shake flasks. An increase in the copy number simultaneously with the synergistic effect provided by two promoters led to enhanced degree of protein expression.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Scale-up from shake flasks to pilot-scale production of the plant growth-promoting bacterium Azospirillum brasilense for preparing a liquid inoculant formulation

Azospirillum brasilense has industrial significance as a growth promoter in plants of commercial interest. However, there is no report in the literature disclosing a liquid product produced in pilot-scale bioreactors and is able to be stored at room temperature for more than 2 years. The aim of this work was to scale up a process from a shake flask to a 10-L lab-scale and 1,000-L pilot-scale bioreactor for the production of plant growth-promoting bacterium A. brasilense for a liquid inoculant formulation. Furthermore, this work aimed to determine the shelf life of the liquid formulation stored at room temperature and to increase maize crops yield in greenhouses. Under a constant oxygen mass transfer coefficient (K _L a), a fermentation process was successfully scaled up from shake flasks to 10- and 1,000-L bioreactors. A concentration ranging from 3.5 to 7.5?×?10⁸ CFU/mL was obtained in shake flasks and bioreactors, and after 2 years stored at room temperature, the liquid formulation showed one order of magnitude decrease. Applications of the cultured bacteria in maize yields resulted in increases of up to 95 % in corncobs and 70 % in aboveground biomass.     

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.     

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris

Cost effective ¹³C/¹⁵N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The ¹³C/¹⁵N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut⁺) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (Mut^S) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for ¹³C-isotope labeling can be substantially reduced using the Mut⁺ strain compared to the Mut^S strain, as no ¹³C₃-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for ¹⁵N-labeling by an optimal supply of 10 g/L (¹⁵NH₄)₂SO₄ every 24 h.     

Alleviating Redox Imbalance Enhances 7-Dehydrocholesterol Production in Engineered Saccharomyces cerevisiae

Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D₃) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD⁺ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD⁺ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance.     

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.     

Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in Arabidopsis

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide‐resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide‐resistant respiration was also accompanied by the increase of plastid NADPH/NADP⁺ ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin–Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX‐derived plastidial NADPH/NADP⁺ ratio change. Further, our results thus revealed a molecular mechanism of chloroplast–mitochondria interactions.