Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

OBJECTIVES: Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). RESULTS: The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1 mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05 U l-1, which is more than ten-fold higher relative to standard growth conditions (BMM10 medium, pH 6 and 30 °C). CONCLUSION: Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lower cultivation temperature.

Impacts of Magnetic Immobilization on the Growth and Metabolic Status of Recombinant Pichia pastoris.

Downstream processing is an expensive step for industrial production of recombinant proteins. Cell immobilization is known as one of the ideal solutions in regard to process intensification. In recent years, magnetic immobilization was introduced as a new technique for cell immobilization. This technique was successfully employed to harvest many bacterial and eukaryotic cells. But there are no data about the influence of magnetic immobilization on the eukaryotic inducted recombinant cells. In this study, impacts of magnetic immobilization on the growth and metabolic status of induced recombinant Pichia pastoris as a valuable eukaryotic model cells were investigated. Results based on colony-forming unit, OD600, and trypan blue assay indicated that magnetic immobilization had no adverse effect on the growth and viability of P. pastoris cells. Also, about 20-40% increase in metabolic activity was recorded in immobilized cells that were decorated with 0.5-2 mg/mL nanoparticles. Total protein and carbohydrate of the cells were also measured as main indicatives for cell function and no significant changes were observed in the immobilized cells. Current data show magnetic immobilization as a biocompatible technique for application in eukaryotic expression systems. Results can be considered for further developments in P. pastoris-based expression systems.

Improved Production of Recombinant Myrosinase in Pichia pastoris.

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

Expression of recombinant protease MarP from Mycobacterium tuberculosis in Pichia pastoris and its effect on human monocytes.

OBJECTIVE: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. RESULTS: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. CONCLUSION: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.

Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris.

We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% ß-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of ~16% from N-terminal region. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. In vitro digestibility of the protein using SGF and SIF showed the disappearance of ~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. Further in vivo digestibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases.

Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris.

BACKGROUND: Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. RESULTS: Through an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the 'compensatory mechanism', which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity. CONCLUSIONS: This GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions.

A novel fermented soybean, inoculated with selected Bacillus, Lactobacillus and Hansenula strains, showed strong antioxidant and anti-fatigue potential activity.

The aim of this study was to develop a novel fermented soybean food (FSF) using selected Bacillus subtilis GD1, Bacillus subtilis N4, Bacillus velezensis GZ1, Lactobacillus delbrueckii subsp. bulgaricus and Hansenula anomala, as well as to assess its antioxidant and anti-fatigue activity. These Bacillus strains had excellent enzyme producing and soybean transformation capacity. FSF showed the highest peptide, total phenol, total flavonoid content, antioxidant activity, and suitable organic acid and biological amine content. In intense exercise mice, FSF treatment markedly increased hepatic glycogen level, decreased metabolite accumulation, improved the activities of antioxidant enzymes and decreased malondialdehyde (MDA) level in serum and liver, respectively. Furthermore, FSF treatment increased nuclear factor-erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE)-dependent gene expression. Together, the selection of microbial starter culture and mixed culture fermentation are essential for the effective enrichment of bioactive compounds, and FSF has stronger antioxidant and anti-fatigue activity.

Valorization of Pichia spent medium via one-pot synthesis of biocompatible silver nanoparticles with potent antioxidant, antimicrobial, tyrosinase inhibitory and reusable catalytic activities.

The purpose of this study was to determine the feasibility of a novel biogenic synthesis of silver nanoparticles using Pichia pastoris spent medium - a major waste product from heterologous protein expression as the sole reducing and capping agent, with potential biological, antimicrobial and wastewater remediation applications. Using UV-vis spectroscopy, TEM, XRD, EDX, FTIR, SEM we demonstrated the successful bio-fabrication of Pichia spent medium encapsulated silver nanoparticles (PSM-AgNPs). PSM-AgNPs displayed substantial antiradical activity against DPPH and ABTS. The antiradical activity against ABTS was similar to that of the control, ascorbic acid. PSM-AgNPs also revealed potent anti-tyrosinase and antibacterial activity against some common foodborne pathogenic microbes. Human erythrocyte hemolytic and embryonic colon Caco-2 cell viability assays suggest that PSM-AgNPs was biocompatible. In addition, PSM-AgNPs, was also effective in the catalytic degradation of methyl orange and Congo red dyes with pseudo first order rate constants of 0.2301 min-1 and 4.7 × 10-3 s-1, respectively. These results present a clean, convenient, and inexpensive approach for the biosynthesis of silver nanoparticles with potential implications in the eco-friendly, safe and effective utilization of waste culture media, mitigation of pathogenic bacteria and management of industrial effluents.

Increased glycosidase activities improved the production of wine varietal odorants in mixed fermentation of P. fermentans and high antagonistic S. cerevisiae.

A selected Pichia fermentans strain was simultaneously and sequentially inoculated in synthetic and real juice with S. cerevisiae strains of different antagonistic activities in a ratio 1:1 to observe the correlation between varietal odorants and glycosidase activities. Fermentations using pure S. cerevisiae strains were used for comparison. Yeast biomass and glycosidase activities were monitored, varietal odorants were detected using HS-SPME-GC/MS during fermentation. The final wine aroma attributes were analyzed by trained panelists. Results showed that co-inoculation with high antagonistic S. cerevisiae resulted in higher glycosidase activities than others. Pearson correlation analysis indicated that yeast biomass was positively related to glycosidase activities during fermentation. The increase in glycosidase activities was the main reason for the higher production of terpenes and C13-norisoprenoids, and for the lower C6 compound content, which lead to superior fruity and floral aromas in the final wine samples of the high antagonistic S. cerevisiae group.

Glycan Recognition and Application of P-Type Lectins.

Cation-dependent mannose 6-phosphate receptor (CD-MPR) and cation-independent MPR (CI-MPR) belong to the P-type lectin family. Both intracellular and cell surface MPRs can recognize and bind with the terminal mannose 6-phospahte (M6P) residues of N-glycans attached to the mammalian lysosomal enzymes and the related co-factors. Domain9 (Dom9), which is one of the extracytoplasmic region of the CI-MPR, has relatively higher affinity for M6P residues. Here we describe the production of recombinant Dom9-His protein by Pichia pastris, purification, and application as a probe for lectin blotting.

Compound isolation and biological activities of Piptadeniastrum africanum (hook.f.) Brennan roots.

ETHNOPHARMACOLOGICAL RELEVANCE: The dicotyledonous plant Piptadeniastrum africanum (hook.f.) Brennan (Fabaceae) is used in traditional medicine to treat various human complaints including bronchitis, coughing, urino-genital ailments, meningitis, abdominal pain, treatment of wounds, malaria and gastrointestinal ailments, and is used as a purgative and worm expeller. AIM OF THE STUDY: The present study describes the phytochemical investigation and the determination of the antimicrobial, antiplasmodial and antitrypanosomal activities of crude extract, fractions and compounds extracted from Piptadeniastrum africanum roots. MATERIALS AND METHODS: Isolated compounds were obtained using several chromatographic techniques. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR) and by comparing their NMR data with those found in literature. In vitro antimicrobial activity of samples was evaluated using the microdilution method on bacterial (Escherichia coli, Proteus mirabilis, Staphylococcus aureus) and fungal (Candida krusei) strains, while in vitro cell-growth inhibition activities were assessed against two parasites (Trypanosoma brucei brucei and Plasmodium falciparum strain 3D7). The cytotoxicity properties of samples were assayed against HeLa human cervical carcinoma. RESULTS: Five compounds were isolated and identified as: tricosanol 1, 5α-stigmasta-7,22-dien-3-ß-ol 2, betulinic acid 3, oleanolic acid 4 and piptadenamide 5. This is the first report of the isolation of these five compounds from the roots of P. africanum. The (Hex:EtOAc 50:50) fraction exhibited moderate antibacterial activity against P. mirabilis (MIC 250 µg/mL), while the other fractions and isolated compounds had weak antimicrobial activities. Only the EtOAc fraction presented a moderate antimalarial activity with an IC50 of 16.5 µg/mL. The MeOH crude extract and three fractions (Hexane, Hexane-EtOAc 25% and EtOAc-MeOH 25%) exhibited significant trypanocidal activity with IC50 values of 3.0, 37.5, 3.8 and 9.5 µg/mL, respectively. CONCLUSION: These results demonstrated a scientific rational of the traditional uses of P. africanum and indicate that this plant should be further investigated to identify some of the chemical components that exhibited the activities reported in this study and therefore may constitute new lead candidates in parasiticidal drug discovery.

Enantiomeric effect of paclobutrazol on the microorganism composition during wine fermentation.

Pesticide residues in food can bring potential risks to human health and has been widely concerned in recent years. In the current study, the influence of paclobutrazol, which resided in raw material (grape) on wine fermentation process, were investigated. The degradation kinetic results indicated that the enantiomers of paclobutrazol not be degraded during 30 days of fermentation process. In order to achieve the fermented microorganism information of diversity, community composition, and function, the analysis of 16S rRNA and ITS sequencing were performed. Results demonstrated that the dominant microorganisms multiplied and the microbial diversity in the samples decreased as the fermentation process progresses. Furthermore, the paclobutrazol stimulated the growth of Pichia, which was observed during wine fermentation and which may have an underlying impact on the quality of the wine. The above results inferred that paclobutrazol residue could disturb the microbial community stability during wine fermentation, and the stable existence of paclobutrazol will cause potential risks to food safety and human health. In this work, we have successfully devised a method to investigate the influences of pesticide residues in raw materials during food processing and conclusions from this study could provide basis for dietary risk assessment.

Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis.

OBJECTIVE: To provide an alternative therapeutic modality for rheumatoid arthritis (RA), a novel bispecific antibody (BsAb) targeting human tumor necrosis factor α (TNF-α) and human complement component C5a was constructed. RESULTS: BsAb was expressed in Pichia pastoris and secreted into the culture medium as a functional protein. In vitro functional study demonstrated that BsAb could simultaneously bind to TNF-α and C5a and neutralize their biological actions. Furthermore, BsAb showed significant improvements in both the antigen-binding affinity and the neutralizing ability as compared to its original antibodies produced in E. coli. It was also found that TNF-α and C5a had an additive/synergistic effect on promoting the production of inflammatory cytokines and chemokines and C5a receptor (C5aR) expression in human macrophages. Compared to single inhibition of TNF-α or C5a with respective antibody, BsAb showed a superior efficacy in blocking inflammatory cytokines, chemokines, and C5aR response, as well as in lowering the C5a-mediated chemotaxis of macrophages via C5aR in vitro. CONCLUSIONS: With improved production processing and the ability to simultaneously block TNF-α and C5a action, BsAb has a great potential to be developed into a therapeutic agent and may offer a better therapeutic index for RA.

Characterization of heavy metal toxicity in some plants and microorganisms-A preliminary approach for environmental bioremediation.

In situ bioremediation processes are important for control of pollution and clean-up of contaminated sites. The study and implementation of such processes can be designed through investigations on natural mechanisms of absorption, biotransformation, bioaccumulation and toxicity of pollutants in plants and microorganisms. Here, the phytotoxic effects of Cr(VI) and Cd(II) on seed germination and plant growth of Lepidium sativum have been examined at various concentrations (30-300 mg/L) in single ion solutions. The studies also addressed the ecotoxicity of metal ions on Azotobacter chroococcum and Pichia sp. isolated from soil. Microbial growth was estimated by weighing the dry biomass and determining the enzymatic activities of dehydrogenase and catalase. The results showed that Cr(VI) and Cd(II) can inhibit L. sativum seed germination and root development, depending on the metal ion and its concentration. The phytotoxic effect of heavy metals was also confirmed by the reduced amounts of dried biomass. Toxicity assays demonstrated the adverse effect of Cr(VI) and Cd(II) on growth of Azotobacter sp. and Pichia sp., manifested by a biomass decrease of more than 50 % at heavy metal concentrations of 150-300 mg/L. The results confirmed close links between phytotoxicity of metals and their bioavailability for phytoextraction. Studies on the bioremediation potential of soils contaminated with Cr(VI) and Cd(II) using microbial strains focusing on Azotobacter sp. and Pichia sp. showed that the microbes can only tolerate heavy metal stress at low concentrations. These investigations on plants and microorganisms revealed their ability to withstand metal toxicity and develop tolerance to heavy metals.

Identification of major ADH genes in ethanol metabolism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h-1. The specific growth rate was further improved to 0.018 h-1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.

Genetically modified Pichia pastoris, a powerful resistant factory for gold and palladium bioleaching and nanostructure heavy metal biosynthesis.

A metal-resistant engineered Pichia pastoris was developed here to fulfil the metal bioleaching in aqueous conditions. Parent and recombinant yeasts were grown in YPD medium containing different concentrations of ion metals. XRD, electron microscopy and particle size analyser were used for the characterisation and the nanoparticle analyses. The nanoparticle production kinetics were studied by ICP-OES. The cytotoxicity of nanoparticles was assayed against human cell lines. Media colours changed to a range from purplish-brown to grey during early fermentation stages. The maximum biosorption capacities were recorded 81.23 and 493.35 mg/g for gold and palladium in batch conditions, respectively. Various physical investigations proved monodispersed spherical nanoparticles around 100 nm in size. Pure palladium nanoparticles and PdCl2 represented the least cytotoxic potency towards T47D and EPG85.257 cells. The results demonstrated that the genetically modified yeast is a cost-effective, high-throughput, robust, and facile system for metal biosorption.

Deletion of Gcw13 represses autophagy in Pichia pastoris cells grown in methanol medium with sufficient amino acids.

OBJECTIVE: The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation. RESULTS: Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells. CONCLUSIONS: Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.

Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris.

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α-factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates.

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into D-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.