ABSTRACT
As artificial extracellular matrix-like materials, silk-elastin-like protein (SELP) hydrogels, with excellent mechanical properties, high tunability, favorable biocompatibility, and controlled degradability, have become an important candidate in biomedical materials. In this study, SELP is composed of silk-like (GAGAGS) and elastin-like (GXGVP) tandem repeats, in which X residues are set as tyrosine and lysine. Furthermore, SELP polymers are prepared via SpyTag/SpyCatcher. To explore a gentler and more efficient enzymatic crosslinking method, an innovative method was invented to apply laccase to catalyze the formation of SELP hydrogels. Gelation could be successfully achieved in 2-5 min . SELP hydrogels mediated by laccase had the characteristic of low swelling rate, which could maintain a relatively stable shape even when immersed in water, and hence had the potential to be further developed into injectable biomaterials. Additionally, SELP hydrogels cross-linked by laccase showed excellent biocompatibility verified by L929 and HEK 293 T cells with cell viability >93.8 %. SELP hydrogels also exhibit good properties in sustained drug release and cell encapsulation in vitro. This study demonstrates a novel method to construct SELP hydrogels with excellent biocompatibility and expands the possibility of SELP-based material applications in biomedical fields.
Subject(s)
Elastin , Laccase , Humans , Elastin/chemistry , Amino Acid Sequence , Hydrogels/chemistry , HEK293 Cells , Molecular Weight , Silk/chemistry , Biocompatible Materials/chemistryABSTRACT
Despite the fact that yeast Saccharomyces cerevisiae is by far the most commonly used in ethanol fermentation, few have been reported to be resistant to high ethanol concentrations at high temperatures. Hence, in this study, 150 S. cerevisiae strains from the Thailand Bioresource Research Center (TBRC) were screened for ethanol production based on their glucose utilization capability at high temperatures. Four strains, TBRC 12149, 12150, 12151, and 12153, exhibited the most outstanding ethanol production at high temperatures in shaking-flask culture. Among these, strain TBRC 12151 demonstrated a high ethanol tolerance of up to 12% at 40 °C. Compared to industrial and laboratory strains, TBRC 12149 displayed strong sucrose fermentation capacity whereas TBRC 12153 and 12151, respectively, showed the greatest ethanol production from molasses and cassava starch hydrolysate at high temperatures in shaking-flask conditions. In 5-L batch fermentation, similarly to both industrial strains, strain TBRC 12153 yielded an ethanol concentration of 66.5 g L-1 (58.4% theoretical yield) from molasses after 72 h at 40 °C. In contrast, strain TBRC12151 outperformed other industrial strains in cell growth and ethanol production from cassava starch hydrolysis at 40 °C with an ethanol production of 65 g L-1 (77.7% theoretical yield) after 72 h. Thus, the thermotolerant and ethanol-tolerant S. cerevisiae TBRC 12151 displayed great potential and possible uses as an alternative strain for industrial ethanol fermentation using cassava starch hydrolysate. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03436-4.
ABSTRACT
Background: Interleukin-18 (IL-18) is prone to form multimers resulting in inactive aggregates, making this cytokine unstable for clinical use. Therefore, mutations have been introduced into recombinant IL-18 to overcome this issue. Methods: To prevent the formation of disulfide bonds between the IL-18 molecules, multiple mutations targeting surface cysteines (C38, C68, C76, and C127) were introduced into our previously modified human IL-18 double mutant E6K+T63A (IL-18 DM) by direct gene synthesis. The open reading frames of IL-18 wild-type (WT), IL-18 DM, and IL-18 multiple mutant E6K+T63A+C38S+C68S+C76S+C127S (IL-18 DM1234) were inserted in the pET28a expression vector and transformed into Escherichia coli Rosetta2 (DE3) pLysS cells for protein production. The inclusion bodies of WT and mutated IL-18 were extracted by sonication and refolded by stepwise dialysis using 8 M urea as the starting concentration. The refolded IL-18 proteins were tested for aggregation using the ProteoStat protein aggregation assay. Their activity was also investigated by treating NK-92MI cells with each IL-18 at concentrations of 75, 150, and 300 ng/ml with 0.5 ng/ml of human IL-12 and interferon-gamma (IFN-γ) levels in the supernatant were evaluated using ELISA. The structure of modified IL-18 was visualized using molecular dynamics (MD) simulations. Results: IL-18 DM1234 exhibited the lowest aggregation signal, approximately 1.79- and 1.63-fold less than that of the WT and IL-18 DM proteins. Additionally, the IFN-γ inducing activity of IL-18 DM1234 was about 10 and 2.8 times higher than that of the WT and IL-18 DM, respectively. MD simulations revealed that binding site I of IL-18 DM1234 was altered mainly due to surface cysteine replacement with serine (C-to-S substitution). This is the first report showing that C-to-S substitutions in IL-18 improved its activity and stability, suggesting the use of this modified IL-18 for medical purposes in the future.
Subject(s)
Cysteine , Interleukin-18 , Humans , Cysteine/genetics , Escherichia coli/genetics , Interferon-gamma/genetics , Interleukin-18/genetics , Recombinant Proteins/genetics , Renal Dialysis , Serine/geneticsABSTRACT
Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries.
Subject(s)
Biological Products , Circovirus , Saccharomycetales , Animals , Biological Products/metabolism , Capsid Proteins/metabolism , Peptide Hydrolases/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolism , SwineABSTRACT
CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.
Subject(s)
6-Phytase , CRISPR-Cas Systems , Endo-1,4-beta Xylanases , Fungal Proteins , Microorganisms, Genetically-Modified , Saccharomycetales , Secretory Pathway , 6-Phytase/genetics , 6-Phytase/metabolism , Endo-1,4-beta Xylanases/genetics , Endo-1,4-beta Xylanases/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genome, Fungal , Microorganisms, Genetically-Modified/enzymology , Microorganisms, Genetically-Modified/genetics , Saccharomycetales/enzymology , Saccharomycetales/geneticsABSTRACT
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.
Subject(s)
Fungal Proteins/metabolism , Saccharomycetales/genetics , Autophagy , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum Stress , Gene Editing , Genes, Fungal/genetics , Oxidative Stress , Protein Translocation Systems/genetics , Protein Transport/genetics , Real-Time Polymerase Chain Reaction , Saccharomycetales/metabolism , ThermotoleranceABSTRACT
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.
Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Methanol/metabolism , Proteomics/methods , Saccharomycetales/chemistry , Thermotolerance , Fungal Proteins/isolation & purification , Gene Expression Regulation, Fungal , Saccharomycetales/genetics , Saccharomycetales/metabolismABSTRACT
BACKGROUND: Interleukin 18 (IL-18) is an inflammatory cytokine belonging to the interleukin 1 (IL-1) superfamily, and is known for its role in anti-cancer activity by promoting type 1 immune response, and thus may be applied to cancer immunotherapy. Our previous report has showed 16 times higher activity of engineered E6K+T63A IL-18 than of native IL-18 in vitro. However, no data has been acquired for its anti-cancer effect in animal model. OBJECTIVES: To investigate the anti-cancer effect of engineered E6K+T63A IL-18 as an immune stimulant in vivo. MATERIAL AND METHODS: Tumor-bearing mice were treated with native IL-18 or E6K or E6K+T63A IL-18 once a day for 10 days after the tumor reached the volume of 100 mm3. Tumor volume and the number of certain immune cell type in the tumor microenvironment were investigated in this study. RESULTS: The results showed that tumor progression in mice treated with E6K+T63A was slower than in mice treated with E6K and native IL-18. The volume of the tumor was also smaller and the lifespan longer in the E6K+T63A IL-18-treated mice. The proportions of type 1 helper T cell (Th1) and cytotoxic T lymphocyte (CTL) were significantly higher in mice treated with E6K+T63A IL-18. CONCLUSIONS: These results suggest that our engineered IL-18 conferred strong anti-tumor immunity in the animal model.
Subject(s)
Interleukin-18/therapeutic use , Neoplasms , Animals , Interleukin-2 , Mice , Neoplasms/drug therapy , Recombinant Proteins , T-Lymphocytes, Cytotoxic , Th1 Cells , Tumor MicroenvironmentABSTRACT
In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC-MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Fungal Proteins , Proteome , Repressor Proteins/genetics , Saccharomycetales , Fungal Proteins/analysis , Fungal Proteins/genetics , Fungal Proteins/metabolism , Mutation , Proteome/analysis , Proteome/metabolism , Saccharomycetales/metabolism , Saccharomycetales/physiologyABSTRACT
In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.
Subject(s)
6-Phytase/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Industrial Microbiology/methods , Recombinant Proteins/genetics , Saccharomycetales/genetics , Sucrose/pharmacology , 6-Phytase/metabolism , Aspergillus niger/enzymology , Aspergillus niger/genetics , Cell Count , Fermentation , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/drug effects , Methanol/metabolism , Molasses , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Saccharomycetales/drug effects , Saccharomycetales/growth & development , Saccharomycetales/metabolism , Sucrose/metabolism , ThermotoleranceABSTRACT
Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (OthAOX) was covalently immobilized onto barium ferrite (BaFe12O19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of OthAOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized OthAOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde.
Subject(s)
Alcohol Oxidoreductases/chemistry , Barium Compounds/chemistry , Barium Compounds/chemical synthesis , Enzymes, Immobilized/chemistry , Ferric Compounds/chemistry , Ferric Compounds/chemical synthesis , Magnets/chemistry , Microspheres , Saccharomycetales/enzymology , Alcohol Oxidoreductases/metabolism , Biocatalysis , Chemistry Techniques, Synthetic , Enzyme Stability , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Methanol/chemistry , TemperatureABSTRACT
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein expression. In this study, a novel expression system was developed for O. thermomethanolica based on the maltase (mal) gene promoter from this organism. The OtMal promoter function was tested for expression of fungal enzymes as reporter genes. Measurement of xylanase reporter enzyme activity showed that the OtMal promoter was repressed during growth on glucose and was activated by sucrose. When sucrose was used as a carbon source, the OtMal promoter was approximately twice as strong as the constitutive OtGAP promoter. Comparison of the OtMal promoter with the methanol-inducible OtAOX promoter showed that OtMal promoter drove 1.2 and 1.7-fold higher expression of xylanase and phytase reporter, respectively, than OtAOX promoter under inducing conditions at 24 h. Our results indicated that this novel expression system could be useful for the production of heterologous proteins from sucrose in yeast O. thermomethanolica.
Subject(s)
Gene Expression Regulation, Fungal/drug effects , Recombinant Proteins/biosynthesis , Saccharomycetales/metabolism , Sucrose/metabolism , Transcriptional Activation/drug effects , 6-Phytase/analysis , 6-Phytase/genetics , Carbon/metabolism , Culture Media/chemistry , Genes, Reporter , Promoter Regions, Genetic , Recombinant Proteins/genetics , Saccharomycetales/genetics , Saccharomycetales/growth & development , Xylosidases/analysis , Xylosidases/genetics , alpha-Glucosidases/geneticsABSTRACT
In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Repressor Proteins/genetics , Saccharomycetales/genetics , Autophagy/physiology , Basic-Leucine Zipper Transcription Factors/metabolism , Endoplasmic Reticulum/metabolism , Proteomics/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/cytology , Saccharomycetales/metabolism , Thermotolerance/genetics , Thermotolerance/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Unfolded Protein ResponseABSTRACT
Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50⯰C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the Vmax, Km, and kcat of 0.24â¯nmol/min, 0.27â¯mM, and 3628.8 min-1, respectively against methanol. Addition of alginic acid (0.35%) showed a protective effect on enhancing thermal stability of the enzyme, resulting in 72% increase in its half-life at 40⯰C under the operational conditions. This enzyme represents a promising candidate for conversion of bioethanol to acetaldehyde as secondary chemical in biorefinery.
Subject(s)
Alcohol Oxidoreductases , Fungal Proteins , Saccharomycetales/enzymology , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/isolation & purification , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/isolation & purificationABSTRACT
Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9-based tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis. Plasmids were constructed for integrative and episomal expression of CRISPR-Cas9 elements in O. thermomethanolica in which Cas9 and gRNA are transcribed from the alcohol oxidase (AOX) promoter. The expression of these genome-editing elements is controlled by derepression with glycerol and gRNA are flanked by self-cleaving ribozymes. For integrative system, OtHAC1, OtMAL1 and OtMAL2 were disrupted at 63%, 97% and 93%, respectively. In addition, OtMAL1 was also disrupted with episomal system at 92%. These findings indicate that the CRISPR-Cas9 system described herein is thus applicable for studying gene function and strain engineering in yeast O. thermomethanolica.
Subject(s)
CRISPR-Associated Protein 9/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing/methods , Metabolic Engineering/methods , Mutagenesis , Saccharomycetales/genetics , Maltose/metabolism , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sucrose/metabolism , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolismABSTRACT
Designing a tailor-made synergistic system is a promising strategy for developing an effective enzyme for saccharification of lignocellulosic materials. In this study, a cellulolytic enzyme mixture comprising selected core recombinant enzymes for hydrolysis of sugarcane bagasse pretreated by alkaline-catalyzed steam explosion was optimized using a mixture design approach. The optimized enzyme system comprised a cellobiohydrolase (Cel7A) from Talaromyces cellulolyticus, an endo-glucanase (Cel7B) from Thielavia terrestris, a ß-glucosidase (BGL) and an endo-ß1,4-xylanase (XYN) from Aspergillus aculeatus at the ratio of 0.34:0.27:0.14:0.25. The maximum reducing sugar yield of 797 mg/g biomass, comprising 543 and 96.8 mg/g glucose and xylose, respectively were achieved, equivalent to 92.44% and 47.50% recoveries, respectively from the pretreated substrate at the enzyme dosage of 20 mg/g biomass. The sugar yield from the quaternary enzyme mixture was 17.37% higher than that obtained with Accellerase 1500.
Subject(s)
Cellulose/chemistry , Cellulose/metabolism , Glucose/metabolism , Hydrolysis , Saccharum/chemistry , Steam , Xylose/metabolism , Aspergillus/enzymology , Biomass , Cellulose 1,4-beta-Cellobiosidase/metabolism , Endo-1,4-beta Xylanases/metabolism , Sordariales/enzymology , Talaromyces/enzymology , beta-Glucosidase/metabolismABSTRACT
In recent years, cytokine-mediated therapy has emerged as further advance alternative in cancer therapy. Interleukin-18 (IL-18) has exhibited interesting anti-cancer properties especially when combined with IL-12. We engineered IL-18 in order to improve its activity using single point mutagenesis. IL-18 mutants were constructed according to binding residues and polarity which we tried to increase polarity in M33Q and M60Q, enhanced cationicity in E6K, and flexibility in T63A. All IL-18 proteins were expressed in Pichia pastoris, purified, and then measured the activity by treating with the NK-92MI cell line to evaluate interferon-γ (IFN-γ) stimulation. The E6K and T63A mutant forms showed higher activity with respect to native proteins at the concentration of 200 ng mL-1 by inducing the expression of IFN-γ, about factors of 9 and 4, respectively. Meanwhile, M33Q and M60Q had no significant activity to induce IFN-γ. Interestingly, the combination of E6K and T63A mutations could synergize the induction activity of IL-18 to be 16 times at 200 ng mL-1. Furthermore, molecular dynamics studies have elucidated the effect due to mutation on conformation of the binding site of IL-18. The results turn out that E6K provides structural perseverance against mutation, while M33Q and M60Q promote vivid overall change in protein conformation, especially at the binding site. For T63A, mutation yields small difference in structure but clearly increases structural flexibility. However, a small structural change was observed when T63A was combined with E6K. Our research resulted in a novel version of IL-18 which could be a new key candidate for cytokine-mediated therapy.
Subject(s)
Interferon-gamma/biosynthesis , Interleukin-18/chemistry , Killer Cells, Natural/drug effects , Lymphocyte Activation/drug effects , Receptors, Interleukin-18/chemistry , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Cloning, Molecular , Gene Expression , Humans , Interferon-gamma/metabolism , Interleukin-18/genetics , Interleukin-18/immunology , Interleukin-18/pharmacology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Kinetics , Models, Molecular , Molecular Weight , Pichia/genetics , Pichia/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Engineering , Protein Interaction Domains and Motifs , Receptors, Interleukin-18/genetics , Receptors, Interleukin-18/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.
Subject(s)
6-Phytase/biosynthesis , Adaptation, Physiological , Biotechnology/methods , Carbon/pharmacology , Dietary Sucrose/pharmacology , Recombinant Proteins/biosynthesis , Saccharomycetales/enzymology , Batch Cell Culture Techniques , Cell Count , Feasibility Studies , Fermentation/drug effects , Oxygen/analysis , Saccharomycetales/drug effects , Saccharomycetales/growth & development , TemperatureABSTRACT
The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous protein expression via secretion to the culture medium. Efficient secretion is a major bottleneck for heterologous protein production in this strain. To improve protein secretion, we explored whether the use of a native signal peptide sequence for directing heterologous protein secretion and overexpression of native ER-resident chaperone genes could improve heterologous protein secretion in O. thermomethanolica. We cloned and characterized genes encoding α-mating factor (Otα-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Otα-MF were shown to promote higher secretion of heterologous endoxylanase comparing with the classical pre-pro sequence of Saccharomyces cerevisiae. However, in the case of heterologous glycosylated phytase, only the Otα-MF pre-pro sequence significantly enhanced protein secretion. The effect of chaperone overexpression on heterologous protein secretion was tested in cotransformant cells of O. thermomethanolica. Overexpression of ER-resident chaperones improved protein secretion depending on heterologous protein. Overexpression of OtBiP, OtCNE1, and OtPDI significantly increased unglycosylated endoxylanase secretion at both 30 and 37 °C while only OtBiP overexpression enhanced glycosylated phytase secretion at 30 °C. These observations suggested the possibility to improve heterologous protein secretion in O. thermomethanolica.
Subject(s)
Endoplasmic Reticulum/metabolism , Fungal Proteins/metabolism , Molecular Chaperones/metabolism , Protein Sorting Signals , Saccharomycetales/metabolism , Adaptation, Physiological , Amino Acid Sequence , Fungal Proteins/chemistry , Genetic Vectors , Hot Temperature , Molecular Sequence Data , Plasmids , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomycetales/physiologyABSTRACT
Scheffersomyces stipitis strain BCC15191 is considered as a biotechnologically valuable yeast for its ability to ferment glucose and xylose, the main sugar components in plant biomass, to ethanol. However, the wild strain lacks of endogenous cellulases and hemicellulases that limited biomass utilization. In order to improve biomass degrading ability of S. stipitis BCC15191, new integrative plasmids harboring constitutive TEF1 promoter and codon-optimized zeocin or hygromycin antibiotic resistance genes were developed. Aspergillus niger endoxylanase and Aspergillus aculeatus endoglucanase activities were demonstrated in transformant cells expressing codon-optimized genes. S. stipitis co-expressing endoxylanase and endoglucanase was able to grow in medium containing xylan and ß-glucan as carbon sources and directly produced ethanol with yields of 2.7 g/L. It could also use pretreated corncob as a carbon source for ethanol production. These results suggested that recombinant S. stipilis is possible for consolidated bioprocessing of biomass.