Pichia Pastoris For Production Research Articles

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies.

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

Effect and mechanism of signal peptide and maltose on recombinant type III collagen production in Pichia pastoris.

Recombinant type III collagen plays an important role in cosmetics, wound healing, and tissue engineering. Thus, increasing its production is necessary. After an initial increase in output by modifying the signal peptide, we showed that adding 1% maltose directly to the medium increased the yield and reduced the degradation of recombinant type III collagen. We initially verified that Pichia pastoris GS115 can metabolize and utilize maltose. Interestingly, maltose metabolism-associated proteins in Pichia pastoris GS115 have not yet been identified. RNA sequencing and transmission electron microscopy were performed to clarify the specific mechanism of maltose influence. The results showed that maltose significantly improved the metabolism of methanol, thiamine, riboflavin, arginine, and proline. After adding maltose, the cell microstructures tended more toward the normal. Adding maltose also contributed to yeast homeostasis and methanol tolerance. Finally, adding maltose resulted in the downregulation of aspartic protease YPS1 and a decrease in yeast mortality, thereby slowing down recombinant type III collagen degradation. KEY POINTS: • Co-feeding of maltose improves recombinant type III collagen production. • Maltose incorporation enhances methanol metabolism and antioxidant capacity. • Maltose addition contributes to Pichia pastoris GS115 homeostasis.

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems.

Systematic modulating carbon metabolism to improve α-farnesene production in Pichia pastoris

α-farnesene, a simple acyclic sesquiterpenes, has significant economic value in medicine, condiments, cosmetics and bioenergy. In this study, we increased α-farnesene production through modifying three module pathways: the main MVA biosynthesis module, the farnesyl pyrophosphate (FPP) branch module and the cytosolic acetyl-CoA supply module. To do these, the appropriate promoters were used to control the expression level of the key enzymes to strengthen the main MVA biosynthesis module and the cytosolic acetyl-CoA supply module as well as to weaken FPP branch module in P. pastoris X33 with expression of α-farnesene synthase (αFS) from Malus domestica. As a result, a α-farnesene high-producing strain P. pastoris XF22 was obtained, which produced 17.92 ± 1.35 g/L of α-farnesene with a productivity of 0.107 g/(L·h) and a yield of 0.078 g/(g glucose) after 168 h in fed-batch fermentation. Although the titer of α-farnesene is not the highest, the productivity and the yield in this study are the highest among the reported literatures, demonstrating that strain XF22 is a competitive platform strain for α-farnesene production.

Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris.

d-lactic acid, a chiral organic acid, can enhance the thermal stability of polylactic acid plastics. Microorganisms such as the yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of d-lactic acid, have been metabolically engineered to produce it in high titers. However, tolerance to d-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to d-lactic acid and increases d-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high d-lactic acid concentrations. Furthermore, integrating a d-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that could produce d-lactic acid at a titer of 5.12 ± 0.35 g/L in 48 h, a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to d-lactic acid, including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of d-lactic acid by manipulating yeast flocculation.

Applicability of the heterologous yeast promoters for recombinant protein production in Pichia pastoris.

Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters (Kluyveromyces marxianus TPI, Hansenula polymorpha PMA, Candida tropicalis ICL, and Saccharomyces cerevisiae CUP) in Pichia pastoris. The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI, glucose for CUP, and ethanol for ICL. The DNA region showing the highest activity was determined as 1000bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters. KEY POINTS: • Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host. • HpPMA has the highest promoter activity among the heterologous promoters tested. • Reporter gene expression with ScCUP is responsive to elevating Cu2+in P. pastoris.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production.ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Recombinant expression of hen egg white lysozyme with the assistance of xylanase fusion partner in Pichia pastoris

ABSTRACT Due to its bacteriolytic activity, hen egg white lysozyme (HEWL) is widely used in the feed, food, and pharmaceutical industries. However, its application is hindered by low protein expression levels in microbial expression systems. In this work, a novel fusion protein expression strategy was proposed for increasing the expression level of HEWL. First, HEWL, fused with a highly expressed fusion protein partner xylanase XynCDBFV, is expressed in Pichia pastoris. Secondly, a linker including endogenous protease cleavage sites was introduced between two fusion proteins in order to separate them directly during the secretion process. Finally, the results show that the supernatant of XynCDBFV-HEWL has a higher HEWL expression level and activity compared with HEWL only. It should be noted that the expression of HEWL reaches to about 3.5 g/L, and the activity of HEWL against Micrococcus lysodeikticus reaches to 1.50 × 105 U/mL in a fed-batch fermentation, which is currently the highest level of recombinant expression of an egg white-derived lysozyme. Taken together, we acquired bioactive HEWL for large-scale recombinant production in Pichia pastoris using a novel fusion protein expression strategy, which could then be used for a variety of applications.

Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation Spectroscopy

Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K.

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.

Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

BackgroundPichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions.ResultsIn this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris.ConclusionsThis study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.

Two Fusarium copper radical oxidases with high activity on aryl alcohols

BackgroundBiomass valorization has been suggested as a sustainable alternative to petroleum-based energy and commodities. In this context, the copper radical oxidases (CROs) from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts for the selective oxidation of primary alcohols to aldehydes. Originally defined by the archetypal galactose 6-oxidase from Fusarium graminearum, fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of native CROs for applications. However, only 10% of the annotated AA5_2 members have been characterized to date.ResultsHere, we define two homologues from the filamentous fungi Fusarium graminearum and F. oxysporum as predominant aryl alcohol oxidases (AAOs) through recombinant production in Pichia pastoris, detailed kinetic characterization, and enzyme product analysis. Despite possessing generally similar active-site architectures to the archetypal FgrGalOx, FgrAAO and FoxAAO have weak activity on carbohydrates, but instead efficiently oxidize specific aryl alcohols. Notably, both FgrAAO and FoxAAO oxidize hydroxymethyl furfural (HMF) directly to 5-formyl-2-furoic acid (FFCA), and desymmetrize the bioproduct glycerol to the uncommon L-isomer of glyceraldehyde.ConclusionsThis work expands understanding of the catalytic diversity of CRO from AA5_2 to include unique representatives from Fusarium species that depart from the well-known galactose 6-oxidase activity of this family. Detailed enzymological analysis highlights the potential biotechnological applications of these orthologs in the production of renewable plastic polymer precursors and other chemicals.

Dual Regulation of Cytoplasm and Peroxisomes for Improved Α-Farnesene Production in Recombinant Pichia pastoris.

Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.

Optimization of a Recombinant Lectin Production in Pichia pastoris Using Crude Glycerol in a Fed-Batch System

The production of heterologous proteins for medical use is an important area of interest. The optimization of the bioprocesses includes the improvement of time, costs, and unit operations. Our study shows that a lectin fraction from Tepary bean (Phaseolus acutifolius) (TBLF) has cytotoxic effects on colon cancer cells and in vivo antitumorigenic activity. However, the low-yield, time-consuming, and expensive process made us focus on the development of a strategy to obtain a recombinant lectin using engineered Pichia pastoris yeast. Pure glycerol is one of the most expensive supplies; therefore, we worked on process optimization using crude glycerol from biodiesel production. Recombinant lectin (rTBL-1) production and purification were evaluated for the first time by an experimental design where crude glycerol (G65) was used and compared against pure glycerol (G99) in a controlled stirred-tank bioreactor with a fed-batch system. The recombinant lectin was detected and identified by SDS-PAGE, Western blot, and UHPLC–ESI–QTOF/MS analysis. The results show that the recombinant lectin can be produced from G65 with no significant differences with respect to G99: the reaction rates were 2.04 and 1.43 mg L−1 h−1, and the yields were 264.95 and 274.67 mgL−1, respectively. The current low cost of crude glycerol and our results show the possibility of producing heterologous proteins using this substrate with high productivity.

Integration of GC-MS in identification of possible final metabolites from phytase production in Pichia Pastoris based on sorbitol induction optimization

The isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction technique was investigated. The biological activity of extracellular phytase after optimization with co-substrates induction in 4 liters fermentor (NBS) increased to 13250 U/ml. This led to a 509 fold increases in comparison to the other type of phytase. This effect was studied via induction with sorbitol/methanol in fermentation by Pichia Pastoris GS115 (Mut+) at 20 °C. The interference of by products; methylal, hexamine and (S)-(+)-1,2-propanediol with release of phytase in Pichia Pastoris under methanol induction were detected and cannot be repressed by methanol induction alone. The TLC was used for glycerin analysis under methanol/sorbitol induction and the results were lesser compare to that obtained during phytase production under methanol induction alone. This work showed the higher expression of heterologous proteins and by fed batch fermentation; the expression identified an advantage of producing a significant activity of phytase. Practical applications Plant derived products including sorbitol have been used as alternative medicines for the therapeutic treatment of various diseases, food supplements and could be used in many manufacturing processes. It serves as a culture media for bacteria, and helps to distinguish the pathogenic E. coli O157:H7 from its most other strains. Cells growing on methanol require high oxygen consumption. Sorbitol was used as an alternative cheap co-feeding for the production of proteins and is a non-repressing carbon source for AOX1 promoter with no effect on the level of r-protein at its induction phase. This report describes the isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction techniques, and sorbitol showed to be a promising co-substrate, as it could enhance both cell growth and targeted protein productivity. This co-feeding and fed-batch induction technique was used for recombinant phytase production in a small and large scale production and the metabolites were analyzed.

A novel thermophile β-galactosidase from Thermothielavioides terrestris producing galactooligosaccharides from acid whey

β-Galactosidases are key enzymes in the food industry. Apart from the hydrolysis of the saccharide bond of lactose, they also catalyze transgalactosylation reactions, producing galactooligosaccharides (GOS) with prebiotic activity. Here we report the heterologous production in Pichia pastoris of a novel β-galactosidase from the fungus Thermothielavioides terrestris. The enzyme (TtbGal1) was purified and characterized, showing optimal activity at 60 °C and pH 4. TtbGal1 is thermostable, retaining almost full activity for 24 h at 50 °C. It was applied to the production of GOS from defined lactose solutions and acid whey, a liquid waste from the Greek yoghurt industry, reaching yields of 19.4 % and 14.8 %, respectively. HILIC–ESI-QTOF-MS analysis revealed the production of GOS with up to 4 saccharide monomers. The results demonstrate efficient GOS production catalyzed by TtbGal1, valorizing acid whey, a waste with a heavy polluting load from the dairy industry.

Process development of recombinant Aspergillus flavus urate oxidase production in Pichia pastoris intracellularly and its characterization as a potential biosimilar

Recombinant A. flavus Urate Oxidase (rUOX), which catalyzes uric acid to allantoin, is highly effective in treating tumor lysis syndrome (TLS). However, its production process is quite complicated, and its use as a therapy is often prohibitive because of the high cost. In this work, we demonstrate, for the first time, a new bioprocess for the production of rUOX expressed in P. pastoris GS115 intracellularly in a soluble and active form, which aims to develop biosimilars that address the current limitations. For the downstream process, the homogenate clarification and intermediate purification were rationally designed with regards to protein characteristics, including polyethyleneimine precipitation, ammonium sulfate precipitation, and flow-through module anion-exchange chromatography. Response surface methodology (RSM) was conducted to optimize the cation exchanger polishing step to determine the appropriate conditions that satisfy the purity and yield requirements. Finally, 2.2 g rUOX (99% purity) was obtained from the 1 l fermentation broth. Various characterizations were carried out to confirm rUOX’s similarity to its commercial counterpart with regards to both physicochemical and enzymatic aspects. Overall, the rUOX derived from P. pastoris has the potential to be used in therapeutic applications.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

BackgroundPichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.ResultsPotential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.DiscussionThe microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.