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Pex23 family proteins localize to the endoplasmic reticulum and play a role in peroxisome and lipid body formation. The yeast Hansenula polymorpha contains four members: Pex23, Pex24, Pex29 and Pex32. We previously showed that loss of Pex24 or Pex32 results in severe peroxisomal defects, caused by reduced peroxisome-endoplasmic reticulum contact sites. We now analyzed the effect of the absence of all four Pex23 family proteins on other cell organelles. Vacuoles were normal in all four deletion strains. The number of lipid droplets was reduced in pex23 and pex29, but not in pex24 and pex32 cells, indicating that peroxisome and lipid droplet formation require different Pex23 family proteins in H. polymorpha. In pex23 and pex29 cells mitochondria were fragmented and clustered accompanied by reduced levels of the fusion protein Fzo1. Deletion of DNM1 suppressed the morphological phenotype of pex23 and pex29 cells, suggesting that mitochondrial fusion is affected. pex23 and pex29 cells showed retarded growth and reduced mitochondrial activities. The growth defect was partially suppressed by DNM1 deletion as well as by an artificial mitochondrion-endoplasmic reticulum tether. Hence, the absence of Pex23 family proteins may influence mitochondrion-endoplasmic reticulum contact sites., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2021 Cold Spring Harbor Laboratory Press.)

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., (© 2020 Roy Chowdhury et al.)

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

We report cloning and expressing of recombinant human VEGF-A 165 , 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-A 165 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-A 165 production. Supplementing the Pichia pastoris cell culture medium with Mg 2+ , 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-A 165 production was with the addition of PBA, PS 20, and MgSO 4 . 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-A 165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Pp Imh1 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 Pp Imh1 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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., (© 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 Man 5 GlcNAc 2 glycosylating and a Man 8-10 GlcNAc 2 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 Man 5 GlcNAc 2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man 8-10 GlcNAc 2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man 5 GlcNAc 2 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 Man 5 GlcNAc 2 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. Man 5 GlcNAc 2 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.

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., (© 2018 The Society for Applied Microbiology.)

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 10 6 :10 6 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., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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.

In the field of metabolic engineering 13 C-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 13 C 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.

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., (© 2017 Wiley Periodicals, Inc.)

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 Å resolution in space group C222 1 . 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2017 American Institute of Chemical Engineers.)

Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris are the standard platforms for biopharmaceutical production with 40% of all between 2010 to 2014 approved protein drugs produced in those microbial hosts. Typically, products overexpressed E. coli and S. cerevisiae remain in the cytosol or are secreted into the periplasm. Consequently, efficient cell disruption is essential for high product recovery during microbial production. Process development platforms at microscale are essential to shorten time to market. While high-pressure homogenization is the industry standard for cell disruption at large scale this method is not practicable for experiments in microscale. This review describes microscale methods for cell disruption at scales as low as 200 µL. Strategies for automation, parallelization and miniaturization, as well as comparability of the results at this scale to high pressure homogenization are considered as those criteria decide which methods are most suited for scale down. Those aspects are discussed in detail for protein overexpression in E. coli and yeast but also the relevance for alternative products and host such as microalgae are taken into account. The authors conclude that bead milling is the best comparable microscale method to large scale high-pressure homogenization and therefore the most suitable technique for automated process development of microbial hosts with the exception of pDNA production., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity., Results: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones., Conclusions: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Background: Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-D-xylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. Therefore, it is necessary to develop a high amount of effective thermoalkaliphilic xylanases. This study aims to enhance thermoalkaliphilic xylanase production in Pichia pastoris through fermentation parameters optimization and novel efficient fed-batch strategy in high cell-density fermentation., Results: Recombinant xylanase activity increased 12.2%, 7.4%, 12.0% and 9.9% by supplementing the Pichia pastoris culture with 20 g/L wheat bran, 5 mg/L L-histidine, 10 mg/L L-tryptophan and 10 mg/L L-methionine in shake flasks, respectively. Investigation of nutritional fermentation parameters, non-nutritional fermentation parameters and feeding strategies in 1 L bioreactor and 1 L shake flask revealed that glycerol and methanol feeding strategies were the critical factors for high cell density and xylanase activity. In 50 L bioreactor, a novel glycerol feeding strategy and a four-stage methanol feeding strategy with a stepwise increase in feeding rate were developed to enhance recombinant xylanase production. In the initial 72 h of methanol induction, the linear dependence of xylanase activity on methanol intake was observed (R 2  = 0.9726). The maximum xylanase activity was predicted to be 591.2 U/mL, while the actual maximum xylanase activity was 560.7 U/mL, which was 7.05 times of that in shake flask. Recombinant xylanase retained 82.5% of its initial activity after pre-incubation at 80 °C for 50 min (pH 8.0), and it exhibited excellent stability in the broad temperature (60-80 °C) and pH (pH 8.0-11.0) ranges., Conclusions: Efficient glycerol and methanol fed-batch strategies resulting in desired cell density and xylanase activity should be applied in other P. pastoris fermentation for other recombinant proteins production. Recombinant xylanases with high pH- and thermal-stability showed potential in various industrial applications.

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110-380 mg L -1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.

Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy., (© 2017 Elsevier Inc. All rights reserved.)

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.

Background: Microtiter plates (MTP) are often applied as culture vessels in high-throughput screening programs. If online measuring techniques are available, MTPs can also be applied in the first steps of process development. For such small-scale bioreactors dipping probes are usually too large; therefore, optical measurements are often used. For example, the BioLector technology allows for the online monitoring of scattered light and fluorescence in each well of a continuously orbitally shaken MTP. Although this system provides valuable data, these measurements are mainly of a semi-quantitative nature. Therefore, signal calibration is required to obtain absolute values. With the µRAMOS technology it became possible for the first time to quantify the oxygen transfer rate (OTR) separately in each well of an MTP. In this work, a device is presented that combines both techniques, to provide a hitherto unparalleled high amount of information from each single well., Results: Because both systems (BioLector and µRAMOS) are based on optical measurements, the measurements need to be synchronized to avoid interferences with the optical signals. The new experimental setup was applied for online monitoring in cultures of Escherichia coli and Hansenula polymorpha. It has been demonstrated that the well-to-well reproducibility is very high, and that the monitored signals provide reliable and valuable information about the process. With varying filling volumes, different maximum oxygen transfer capacities (OTR max ) were adjusted in oxygen-limited cultures. The different degrees of stress during the culture due to oxygen limitation affected microbial growth and also impacted reproducibility from culture to culture. Furthermore, it was demonstrated that this new device significantly simplifies the experimental efforts: instead of parallel cultures in a shake flask and MTP, just one single experiment in MTP needs to be conducted to measure the OTR, dissolved oxygen tension (DOT), scattered light and fluorescence., Conclusions: The new device is a very suitable system for the online monitoring of cultures in continuously orbitally shaken MTPs. Due to the high number of parameters that can simultaneously be measured with this small-scale device, deeper insight into the investigated microbial system can be achieved. Furthermore, the experimental efforts to obtain OTR, DOT, scattered light and fluorescence signals during a culture are decreased. Ultimately, this new technology and the resulting high amount of collected data will eliminate the currently existing separation between screening and process development. Graphical abstract Picture of the combined μRAMOS and BioLector setup which allows for measurements of the oxygen transfer rate (OTR), dissolved oxygen tension (DOT), scattered light and fluorescence in each single well of an orbitally shaken microtiter plate.

In this work, the evolution of different biogenic fluorophores involved in the metabolism of Pichia pastoris was determined at four different single-wavelength pairs (excitation/emission) during batch culture in microwell plates and used for effective and reliable biomass estimation by means of chemometric tools. The chemometric tools for biomass estimation were multiple linear regression (MLR), partial least squares regression (PLSR), and principal component regression (PCR). Variable importance in the projection (VIP) scores were used to rate the importance of model input variables, indicating tryptophan as the most important variable for biomass estimation. A direct correlation between the single fluorescence signals of tryptophan and biomass was additionally set up. Results indicate a successful fitting of the MLR, PLSR, PCR, and direct tryptophan correlation models for the present case and confirm the relevance of biogenic fluorophores for bioprocess state variables monitoring. The root mean squared error of prediction (RMSEP) between the predicted and measured values for the validation batches was 0.017, 0.023, 0.025, and 0.049 g L(-1) dry cell weight for MLR, PLSR, PCR, and direct tryptophan correlation, respectively. The presented approach of indirectly measuring biomass based on combined single-wavelength fluorescence measurements can be used for the development of a low-cost alternative to 2D fluorescence spectroscopy. Biotechnol. Bioeng. 2016;113: 2394-2402. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required., (Copyright © 2016 Elsevier B.V. All rights reserved.)

RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.

The wide applications of titanium dioxide nanoparticles (TiO2 NPs) increase the possibility of their exposure to ecosystems, and therefore an improved understanding of their effects to organisms is required. However, their potential toxicity on eukaryotes, especially fungi, needs further detailed investigation. Here, we investigated the effects of anatase TiO2 NPs on the reactive oxygen species (ROS)-scavenging system in the model fungal organism, Pichia pastoris. Results showed that the NPs entered cells and had toxicity to this fungus, and their toxicity was attributed to cell wall damage, cell membrane damage, and ROS accumulation, but not apoptosis or autophagy. Interestingly, the synthesized TiO2 NPs impaired but not activated the ROS-scavenging system, which contributes to the cytotoxicity. Moreover, this impairment was associated with down-regulation of antioxidant-related genes, especially those genes involved in GSH regulation. Hence, GSH may play a key role in the interaction between TiO2 NPs and yeast cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics. The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel. The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells. The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing.

An accurate measurement or estimation of the volumetric mass transfer coefficient kL a is crucial for the design, operation, and scale up of bioreactors. Among different physical and chemical methods, the classical dynamic method is the most widely applied method to simultaneously estimate both kL a and cell's oxygen utilization rate. Despite several important follow-up articles to improve the original dynamic method, some limitations exist that make the classical dynamic method less effective under certain conditions. For example, for the case of high cell density with moderate agitation, the dissolved oxygen concentration barely increases during the re-gassing step of the classical dynamic method, which makes kL a estimation impossible. To address these limitations, in this work we present an improved dynamic method that consists of both an improved model and an improved procedure. The improved model takes into account the mass transfer between the headspace and the broth; in addition, nitrogen is bubbled through the broth when air is shut off. The improved method not only enables a faster and more accurate estimation of kL a, but also allows the measurement of kL a for high cell density with medium/low agitation that is impossible with the classical dynamic method. Scheffersomyces stipitis was used as the model system to demonstrate the effectiveness of the improved method; in addition, experiments were conducted to examine the effect of cell density and agitation speed on kL a., (© 2014 Wiley Periodicals, Inc.)

Bioinformatic analysis of the genome of the methylotrophic yeast Hansenula polymorpha revealed 39 putative glycosylphosphatidylinositol-anchored proteins (GPI-proteins). Notably, dibasic motifs in the proximal ω-site, that has been reported as a plasma membrane retention signal in Saccharomyces cerevisiae GPI-proteins, were not found in any of the predicted GPI-proteins of H. polymorpha. To evaluate the in silico prediction, C-terminal peptides of 40 amino acids derived from ten H. polymorpha GPI-proteins were fused to the Aspergillus saitoi α-1,2-mannosidase (msdS). Cell wall fraction analysis showed that nine of the ten msdS-GPI fusion proteins were mostly localized at the cell wall. Surface expression of functional msdS was further confirmed by in vitro enzyme activity assay and by glycan structure analysis of cell wall mannoproteins. The recombinant H. polymorpha strains expressing surface-displayed msdS have the potential as useful hosts to produce glycoproteins with decreased mannosylation.

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, β-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on β-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.

In the methylotrophic yeast, Pichia pastoris, Gss1 protein is a glucose sensor involved in pexophagy, glucose utilization and glucose catabolite repression. This study identifies that deletion of 150 residues of Gss1 affects slightly glucose catabolite repression and pexophagy, while maintaining signalling function of Gss1. Substitution of one conserved amino acid R180K of Gss1 protein has no visible phenotype, in contrast to corresponding changes in glucose sensors from other yeast species. We suggest that C-terminal cytoplasmic extension of PpGss1plays different role to that of its homologs in Saccharomyces cerevisiae and Hansenula polymorpha., (© 2013 International Federation for Cell Biology.)

A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.

Traditional biochemical approaches, as well as the complementary methods of living cell fluorescence microscopy and immunofluorescence microscopy, can serve to characterize the subcellular localization of proteins and organelles. This chapter describes methods for isolation of crude organelle fractions from methanol- or oleate-grown Pichia pastoris, followed by protease protection and carbonate extraction assays to dissect the subcellular localization of peroxisomal matrix and membrane proteins. These biochemical tools can be used to analyze the targeting efficiency of proteins to the peroxisome membrane and matrix, as well as the topology of membrane proteins.

Metabolic flux analysis based on tracing patterns of stable isotopes, particularly (13)C, comprises a set of methodologies to experimentally quantify intracellular biochemical reaction rates, i.e., to measure carbon flux distributions through a metabolic network. This allows quantifying the response of a metabolic network to an environmental or genetic perturbation (i.e., the metabolic phenotype). Here, we describe a protocol based on growing yeast on a (13)C-labelled substrate and subsequent NMR detection of (13)C-patterns in proteinogenic amino acids. To calculate metabolic fluxes, we describe two complementary mathematical approaches using available software; namely, an approach based on the estimation of local ratios in network nodes, and a method based on a global iterative fitting approach. Furthermore, we consider specificities of these protocols for their application to the yeast Pichia pastoris growing on multicarbon substrates other than glucose (glycerol), as well as the case when methanol is used as co-substrate in combination with glucose or glycerol.

A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040 × 10(6) IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space-time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.

We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11.