Preparation of Cell Extracts for Purification of Proteins Expressed in Pichia pastoris.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. Disruption methods vary depending on the type of cells, the total volume, and the number of samples being processed. For cells enveloped in cell walls (such as yeast), mild techniques such as hypotonic shock are not sufficient to achieve adequate lysis. More vigorous methods are often required. Although the preferred medium- or large-scale method of breaking yeast cells is mechanical shearing, lysis with the aid of glass beads in a BeadBeater is described here.

Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris.

Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.

ER arrival sites associate with ER exit sites to create bidirectional transport portals.

COPI vesicles mediate Golgi-to-ER recycling, but COPI vesicle arrival sites at the ER have been poorly defined. We explored this issue using the yeast Pichia pastoris. ER arrival sites (ERAS) can be visualized by labeling COPI vesicle tethers such as Tip20. Our results place ERAS at the periphery of COPII-labeled ER export sites (ERES). The dynamics of ERES and ERAS are indistinguishable, indicating that these structures are tightly coupled. Displacement or degradation of Tip20 does not alter ERES organization, whereas displacement or degradation of either COPII or COPI components disrupts ERAS organization. We infer that Golgi compartments form at ERES and then produce COPI vesicles to generate ERAS. As a result, ERES and ERAS are functionally linked to create bidirectional transport portals at the ER-Golgi interface. COPI vesicles likely become tethered while they bud, thereby promoting efficient retrograde transport. In mammalian cells, the Tip20 homologue RINT1 associates with ERES, indicating possible conservation of the link between ERES and ERAS.

[Fermentation optimization based on cell self-adaptation to environmental stress - a review].

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.

Influence of Pichia pastoris X-33 produced in industrial residues on productive performance, egg quality, immunity, and intestinal morphometry in quails.

This study was conducted in quails to evaluate the probiotic potential of Pichia pastoris X-33, cultivated in parboiled rice effluent supplemented with biodiesel glycerol or in standard medium Yeast Extract-Peptone-Dextrose (YPD). Forty-days-old female quails were divided into three treatments: T1 (Control) received a basal diet without P. pastoris; T2 (Pichia Effluent) received a basal diet supplemented with P. pastoris grown in parboiled rice effluent and biodiesel glycerol, and T3 (Pichia YPD) received a basal diet supplemented with P. pastoris produced in YPD. The birds were vaccinated against Newcastle Disease (NDV), Avian Infectious Bronchitis (IBV), and Gumboro Disease on days 1 and 28. The following parameters were analyzed: performance, egg quality, humoral immune response to the vaccines, organ weight, and intestinal morphometry. P. pastoris grown in YPD increased egg weight (p < 0.05). The lowest liver weight on day 14 was obtained in Pichia Effluent, whereas both P. pastoris supplemented groups had the lowest duodenum weights on day 14. Besides that, livers and duodenums presented no morphological changes in any of the three treatments. Supplementation of P. pastoris modulated the immune system of the birds, increasing anti-IBV, anti-NDV, and anti-Gumboro antibodies levels compared to the Control (p < 0.05). In conclusion, quail's immune response was improved by Pichia pastoris X-33, either it was grown in YPD or industrial residues, and the egg weight increased with Pichia pastoris X-33 grown in YPD, thereby demonstrating to be a promising probiotic for poultry.

Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.

The golgin PpImh1 mediates reversible cisternal stacking in the Golgi of the budding yeast Pichia pastoris.

The adhesive force for cisternal stacking of Golgi needs to be reversible - to be initiated and undone in a continuous cycle to keep up with the cisternal maturation. Microscopic evidence in support of such a reversible nature of stacking, in the form of 'TGN peeling,' has been reported in various species, suggesting a potential evolutionarily conserved mechanism. However, knowledge of such mechanism has remained sketchy. Here, we have explored this issue in the budding yeast Pichia pastoris which harbors stacked Golgi. We observed that deletion of GRIP domain golgin P. pastoris (Pp)IMH1 increases the peeling of late cisterna, causing unstacking of the Golgi stack. Our results suggest that the PpImh1 dimer mediates reversible stacking through a continuous association-dissociation cycle of its GRIP domain to the middle and late Golgi cisterna under the GTP hydrolysis-based regulation of Arl3-Arl1 GTPase cascade switch. The reversible cisternal stacking function of PpImh1 is independent of its vesicle-capturing function. Since GRIP domain proteins are conserved in plants, animals and fungi, it is plausible that this reversible mechanism of Golgi stacking is evolutionarily conserved.This article has an associated First Person interview with the first author of the paper.

Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells.

Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1 kJ/mol at 80°C, respectively.

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor.

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived "pure concentration" profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison.

BACKGROUND: The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce. RESULTS: A Man5GlcNAc2 glycosylating and a Man8-10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8-10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C. CONCLUSION: This study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

On-chip analysis of carbon dots effect on yeast replicative lifespan.

Carbon dots (CDs) are promising nanomaterials for biosensing, bioimaging, and drug delivery due to their large surface area, excellent optical properties, and thermal and chemical stability. However, biosafety of CDs is still understudied, and there is not a generally accepted standard to evaluate the toxicity of CDs. We present a gradient network generator microfluidic device for dose-dependent testing of toxicity of CDs to a unicellular eukaryotic model organism, yeast Pichia pastoris. We fully characterized the microfluidic model and compare its performance with a conventional method. The gradient generator increased the contact area between the mixing species and enabled a high-throughput testing of CDs in a wide range of concentrations in cell chambers. Real time monitoring of yeast cell proliferation in the presence of CDs showed dose-dependent growth inhibition and various budding cell shape profiles. Comparing the result of microfluidic platform and conventional method revealed statistically significant differences in the proliferation rate of the cells between the two platforms. To understand the toxicity mechanism, we studied binding of CDs to P. pastoris and found increasing interactions of CDs with the cell surface at CDs larger concentrations. This study demonstrated the utility of the gradient generator microfluidic device as a convenient tool for toxicity assessment of nanomaterials at a cellular level.

Protein crystallization in living cells.

Protein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that this phenomenon is also accessible for recombinant proteins. But only recently the advent of high-brilliance synchrotron sources, X-ray free-electron lasers, and improved serial data collection strategies has allowed the use of these micrometer-sized crystals for structural biology. Thus, in cellulo crystallization could offer exciting new possibilities for proteins that do not crystallize applying conventional approaches. In this review, we comprehensively summarize the current knowledge of intracellular protein crystallization. This includes an overview of the cellular functions, the physical properties, and, if known, the mode of regulation of native in cellulo crystal formation, complemented with a discussion of the reported crystallization events of recombinant proteins and the current method developments to successfully collect X-ray diffraction data from in cellulo crystals. Although the intracellular protein self-assembly mechanisms are still poorly understood, regulatory differences between native in cellulo crystallization linked to a specific function and accidently crystallizing proteins, either disease associated or recombinantly introduced, become evident. These insights are important to systematically exploit living cells as protein crystallization chambers in the future.

Comprehending the influence of critical cultivation parameters on the oleaginous behaviour of potent rotten fruit yeast isolates.

AIMS: Oleaginous yeasts were isolated from five different rotten fruits and their oleaginous behaviour was characterized with varying carbon source and concentration (C/N) to identify their possible future applicabilities. METHODS AND RESULTS: Sixteen yeasts were isolated, those accumulating around 20% of their cell dry weights as lipids were screened and identified as Candida tropicalis and Pichia kudriavzevii. Among glucose, maltose and sucrose, glucose was found favourable carbon source in terms of biomass yield and lipid content. Carbon concentration corresponding to C/N ratio 100/2 (C. tropicalis) and 120/2 (P. kudriavzevii) exhibited highest lipid contents-81·2 and 75·7%-and lipid yields-1·1 and 3 g l-1 , respectively. Lipids contained 16-28 types of triacylglycerols, and major fatty acids detected were caprylic, caparate, lauric, stearic palmitic, oleic, linoleic, aracidic and erucic acid with caprylic acid as predominant one. Fatty acid profile also witnessed variations with changing C/N. CONCLUSIONS: Noteworthy lipid contents were achieved in the two isolates higher than any existing reports on them. Pichia kudriavzevii exhibited reasonable overall lipid yield extending the opportunity of improvement upon changing the cultivation conditions. The lipids contained a range of fatty acids with a predominance of caprylic acid having unequivocal biotechnological importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Two potent oleaginous yeast isolates were studied, and this is the only successive report describing oleaginous behaviour of P. kudriavzevii. The cultivation parameters such as C source, concentration and C/N ratio which critically influence the oleaginous behaviour, lipid content, yield and composition have been accessed so as to provide comprehensive understanding on single cell oil production from these isolates. Study progressively contributes to current and upcoming researches in microbial oils and the characteristic fatty acid, and TAG profile provides important leads for their putative applicabilities.

Comprehensive assessment of measurement uncertainty in 13C-based metabolic flux experiments.

In the field of metabolic engineering 13C-based metabolic flux analysis experiments have proven successful in indicating points of action. As every step of this approach is affected by an inherent error, the aim of the present work is the comprehensive evaluation of factors contributing to the uncertainty of nonnaturally distributed C-isotopologue abundances as well as to the absolute flux value calculation. For this purpose, a previously published data set, analyzed in the course of a 13C labeling experiment studying glycolysis and the pentose phosphate pathway in a yeast cell factory, was used. Here, for isotopologue pattern analysis of these highly polar metabolites that occur in multiple isomeric forms, a gas chromatographic separation approach with preceding derivatization was used. This rendered a natural isotope interference correction step essential. Uncertainty estimation of the resulting C-isotopologue distribution was performed according to the EURACHEM guidelines with Monte Carlo simulation. It revealed a significant increase for low-abundance isotopologue fractions after application of the necessary correction step. For absolute flux value estimation, isotopologue fractions of various sugar phosphates, together with the assessed uncertainties, were used in a metabolic model describing the upper part of the central carbon metabolism. The findings pinpointed the influence of small isotopologue fractions as sources of error and highlight the need for improved model curation. Graphical abstract ᅟ.

Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making.

Yeasts are the most important microorganisms in the fermentation of Chinese Baijiu and the interaction of these yeasts could impact the quality of Baijiu. In this study, we initially characterized the Baijiu yeasts and evaluated their fermentation potential. Wickerhamomyces anomalus GZ3 and Saccharomyces cerevisiae G20 were found to generate high yields of ethyl acetate (2.76 g/L) and 2-phenylethanol, respectively. Results also indicated that the use of W. anomalus along with S. cerevisiae increased volatile compounds production, the maximum ethyl acetate production was observed in S. cerevisiae and W. anomalus at 106:106 ratio and have increased by 33 % compared with single culture of W. anomalus. Besides, there was a significant increase of 2-phenylethanol (3.29 g/L) production in single culture of S. cerevisiae with the addition of l-phenylalanine. However, the conversion of l-phenylalanine in mixed culture had significant impact on the yeasts interaction and end flavor of Baijiu. Thus, the present study provided new insights into yeasts interactions in Baijiu fermentation and the effect of some primary metabolites on the end flavor and Baijiu quality.

Structure and function of cytoplasmic serine hydroxymethyltransferase from Pichia pastoris.

Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and glycine, which is crucial for one carbon metabolism. Here, we report the first crystal structure of cytoplasmic SHMT from Pichia pastoris (pcSHMT) diffracted to 2.5 Šresolution in space group C2221. PcSHMT was a contaminant with our target protein expressed in Pichia pastoris and confirmed by mass spectrometry. The overall structure of pcSHMT is similar to Human mitochondrial SHMT and different to E. coli SHMT. Interestingly, the oligomerization of pcSHMT expressed in eukaryotic or prokaryotic system differs significantly and is regulated by pyridoxal-5'-phosphate. Our results revealed a close evolutionary relationship between Pichia pastoris and Human mitochondria.

Analysis of extracellular vesicles produced in the biofilm by the dimorphic yeast Pichia fermentans.

The yeast Pichia fermentans DISAABA 726 strain (P. fermentans) is a dimorphic yeast that under different environmental conditions may switch from a yeast-like to pseudohyphal morphology. We hypothesize that exosomes-like vesicles (EV) could mediate this rapid modification. EV are membrane-derived vesicles carrying lipids, proteins, mRNAs and microRNAs and have been recognized as important mediators of intercellular communication. Although it has been assumed for a long time that fungi release EV, knowledge of their functions is still limited. In this work we analyze P. fermentans EV production during growth in two different media containing urea (YCU) or methionine (YCM) where yeast-like or pseudohyphal morphology are produced. We developed a procedure to extract EV from the neighboring biofilm which is faster and more efficient as compared to the widely used ultracentrifugation method. Differences in morphology and RNA content of EV suggest that they might have an active role during dimorphic transition as response to the growth conditions. Our findings are coherent with a general state of hypoxic stress of the pseudohyphal cells.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017.

Direct Extraction and Purification of Recombinant Membrane Proteins from Pichia pastoris Protoplasts.

In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favoring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography.

Methionine synthase is localized to the nucleus in Pichia pastoris and Candida albicans and to the cytoplasm in Saccharomyces cerevisiae.

Methionine synthase (MS) catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of S-adenosylmethionine. Here, we report that MS is localized to the nucleus of Pichia pastoris and Candida albicans but is cytoplasmic in Saccharomyces cerevisiae The P. pastoris strain carrying a deletion of the MET6 gene encoding MS (Ppmet6) exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of P. pastoris MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation. In silico analysis of the PpMS structure indicated that PpMS may exist in a dimer-like configuration in which Arg-742 of a monomer forms a salt bridge with Asp-113 of another monomer. Biochemical studies indicate that R742A as well as D113R mutations abrogate nuclear localization of PpMS and its ability to reverse methionine auxotrophy of Ppmet6 Thus, association of two PpMS monomers through the interaction of Arg-742 and Asp-113 is essential for catalytic activity and nuclear localization. When PpMS is targeted to the cytoplasm employing a heterologous nuclear export signal, it is expressed at very low levels and is unable to reverse methionine and adenine auxotrophy of Ppmet6 Thus, nuclear localization is essential for the stability and function of MS in P. pastoris. We conclude that nuclear localization of MS is a unique feature of respiratory yeasts such as P. pastoris and C. albicans, and it may have novel moonlighting functions in the nucleus.