Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy

Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.

Related papers:

title: Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression
abstract: Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris from S. frugiperda, following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.

title: Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor
abstract: BackgroundRecombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker’s yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.ResultsCodon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.ConclusionOur data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

title: Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography
abstract: The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400 SEC, we obtained highly pure, i.e., > 90%, rHBsAg virus-like particles (VLP).

title: High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.
abstract: A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.

title: Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation
abstract: BackgroundPhytase supplied in feeds for monogastric animals is important for improving nutrient uptake and reducing phosphorous pollution. High-thermostability phytases are particularly desirable due to their ability to withstand transient high temperatures during feed pelleting procedures. A comparison of crystal structures of the widely used industrial Aspergillus niger PhyA phytase (AnP) with its close homolog, the thermostable Aspergillus fumigatus phytase (AfP), suggests 18 residues in three segments associated with thermostability. In this work, we aim to improve the thermostability of AnP through site-directed mutagenesis. We identified favorable mutations based on structural comparison of homologous phytases and molecular dynamics simulations.ResultsA recombinant phytase (AnP-M1) was created by substituting 18 residues in AnP with their AfP analogs. AnP-M1 exhibited greater thermostability than AnP at 70 °C. Molecular dynamics simulations suggested newly formed hydrogen bonding interactions with nine substituted residues give rise to the improved themostability. Thus, another recombinant phytase (AnP-M2) with just these nine point substitutions was created. AnP-M2 demonstrated superior thermostability among all AnPs at ≥70 °C: AnP-M2 maintained 56% of the maximal activity after incubation at 80 °C for 1 h; AnP-M2 retained 30-percentage points greater residual activity than that of AnP and AnP-M1 after 1 h incubation at 90 °C.ConclusionsThe resulting AnP-M2 is an attractive candidate in industrial applications, and the nine substitutions in AnP-M2 are advantageous for phytase thermostability. This work demonstrates that a strategy combining structural comparison of homologous enzymes and computational simulation to focus on important interactions is an effective method for obtaining a thermostable enzyme.

title: Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter Engineering by High Resolution Systematic Mutagenesis.
abstract: Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible "tuning knob" for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution is performed. Adjacent triplets of the 200 bp long core promoter are mutated at a time by changing the wild-type sequence into cytosine or adenine triplets, resulting in 130 variants that are cloned upstream of an eGFP reporter gene providing a library for expression fine-tuning. Mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5'UTR had a significant effect on the eGFP fluorescence. Surprisingly, mutations in most other regions are tolerated, indicating that yeast core promoters can show a high tolerance toward small mutations, supporting regulatory models of degenerate motifs, or redundant design. The authors exploited these neutral core promoter positions, not affecting expression, to introduce extrinsic sequence elements such as cloning sites (allowing targeted core promoter/5'UTR modifications) and bacterial promoters (applicable in multi host vectors).

title: Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol
abstract: The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel‐engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (PADH2) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single‐handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA‐box integration; and, (b) nucleosome optimization. PADH2‐Cat8‐L2 and PADH2‐Cat8‐L1 designed by the integration of synthetic Cat8 binding sites were superior, and then PADH2‐NucOpt. Compared to that with PADH2 at t = 20 hr of the fermentations, PADH2‐Cat8‐L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8‐fold on ethanol and 3.8‐fold on methanol; and, PADH2‐NucOpt upregulated the expression 1.5‐fold on ethanol and enhanced 3.2‐fold on methanol. Using the superior two tools, Cat8‐L2 and NucOpt, we designed PADH2‐NucOpt‐Cat8‐L2. With PADH2‐NucOpt‐Cat8‐L2, the expression in the fermentation of ethanol was upregulated 3.7‐fold that is distinctly higher than that with PADH2‐NucOpt but lower than that with PADH2‐Cat8‐L2; while on methanol compared to that with PADH2, the expression was enhanced 8.8‐fold. Extracellular recombinant human serum albumin production was also studied with PADH2‐Cat8‐L2 and PADH2‐NucOpt, and average recombinant human serum albumin yield (YP/X) on ethanol was 1.13 and 0.38 mg/gWCW, respectively; whereas with PADH2, YP/X was 0.26 mg/gWCW. We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid‐promoter architectures of NEPVs in coordination with trans‐acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris.

title: An improved secretion signal enhances the secretion of model proteins from Pichia pastoris
abstract: BackgroundProteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal.ResultsWe engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal.ConclusionsThe improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.

title: High-level expression, purification and properties of an Endochitinase gene without signal peptide from Lecanicillium lecanii 43H in Pichia pastoris
abstract: Chitinases play the key role in hydrolysis of chitin, a huge organic carbon reservoir on earth, into monomeric sugars and their eventual conversion into valuable chemicals and energy sources. The Lecanicillium lecanii strain 43H was used as the source for the Endochitinase gene without signal peptide (mchit1). This mchit1 gene was cloned and sequenced. The recombinant Endochitinase non signal peptide was overexpressed in Pichia pastoris X33 with a level of 2.048 U mL−1 culture supernatant. The molecular mass of the purified recombinant Endochitinase (rmchit1) without signal peptide was 43 kDa. Metal ions, detergents, and organic solvents tested indicated a significantly influence on rmchit1 activity. The obtained results demonstrated that signal peptides affect the yield expression, purification methods, recovery as well as the physicochemical properties of the enzyme.

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

title: Enhancing the efficiency of the Pichia pastoris AOX1 promoter via the synthetic positive feedback circuit of transcription factor Mxr1
abstract: BackgroundThe methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal.ResultsThe characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction.ConclusionsWe successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
abstract: Rtg1p and Rtg3p are two basic helix–loop–helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae. Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris. We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris. We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

title: Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach
abstract: Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV‐G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV‐G in P. pastoris.

title: Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing
abstract: BackgroundThe methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The study of the P. pastoris transcriptome helps to annotate the P. pastoris transcript structures and provide useful information for further improvement of the production of recombinant proteins.ResultsWe used a massively parallel mRNA sequencing platform (RNA-Seq), based on next-generation sequencing technology, to map and quantify the dynamic transcriptome of P. pastoris at the genome scale under growth conditions with glycerol and methanol as substrates. The results describe the transcription landscape at the whole-genome level and provide annotated transcript structures, including untranslated regions (UTRs), alternative splicing (AS) events, novel transcripts, new exons, alternative upstream initiation codons (uATGs), and upstream open reading frames (uORFs). Internal ribosome entry sites (IRESes) were first identified within the UTRs of genes from P. pastoris, encoding kinases and the proteins involved in the control of growth. We also provide a transcriptional regulation model for P. pastoris grown on different carbon sources.ConclusionsWe suggest that the IRES-dependent translation initiation mechanism also exists in P. pastoris. Retained introns (RIs) are determined as the main AS event and are produced predominantly by an intron definition (ID) mechanism. Our results describe the metabolic characteristics of P. pastoris with heterologous protein production under methanol induction and provide rich information for further in-depth studies of P. pastoris protein expression and secretion mechanisms.

title: Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes
abstract: BackgroundPichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.ResultsThe shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation.ConclusionsIn the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.

title: Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures
abstract: Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P. pastoris. Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction.

title: Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level
abstract: BackgroundThe methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale.ResultsWe performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol.ConclusionsP. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

title: Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris
abstract: The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.

title: Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger
abstract: The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h−1 and was constant in the range 0.1–0.2 h−1. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h−1 irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l−1 were obtained even though the strain contained only one copy of the glaA gene.

title: Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review
abstract: The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

title: Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review
abstract: Pichia pastoris is currently one of the most effective and versatile systems for the expression of heterologous proteins. Its success is due to its powerful methanol-inducible alcohol oxidase 1 (AOX1) promoter, capacity to perform post-translational modifications and pathways leading to recombinant products secretion. Despite its advantages, P. pastoris cultivation is plagued by high protease expression levels, high sensitivity to methanol levels, nutrient-deficiency when grown on defined media, difficulties in systematic study due to product-specific effects, and health and safety concerns associated with the storage of large quantities of methanol. Difficulties also arise that are specific to the cultivation methods and control strategies used. The present review discusses several bioprocess engineering aspects related to P. pastoris cultivation, including the different promoters available, both constitutive and inductive, on- and off-line process parameter monitoring methods, fed-batch and continuous cultivation control strategies, proteolytic degradation of products and methods to minimize associated yield reductions, and the different models devised to describe cell growth and protein production. The economics of P. pastoris cultivation, through a case-study involving recombinant phytase production, are also discussed. The study of yeast fermentation is currently very dynamic, and P. pastoris is on its way towards becoming a standard expression system.

title: Enhancing the efficiency of recombinant hepatitis B surface antigen production in Pichia pastoris by employing continuous fermentation
abstract: The recombinant hepatitis B surface antigen (rHBsAg) is a protein-based vaccine which is mainly produced by Pichia pastoris (P. pastoris) in a high-cell-density fed-batch fermentation for large-scale production purposes. In this study, we compared the efficiency and productivity of a chemostat fermentation of P. pastoris for rHBsAg production with the conventional fed-batch fermentation process. For this purpose, we established chemostat fermentation of P. pastoris for rHBsAg production in bench-scale for two weeks. The specific and volumetric productivity for chemostat fermentation were 0.00468 mg HBsAg/g cell/h and 1.699 mg HBsAg/L/h, respectively. These parameters for fed-batch fermentation were 0.00456 mg HBsAg/g cell/h and 1.38 mg HBsAg/L/h, and by considering the downtime for harvesting and initiating the next run, these values dropped to 0.00375 mg HBsAg/g cell/h and 1.13 mg HBsAg/L/h, respectively. According to the PCR analysis, no genetic mutation and contamination were detected after approximately three weeks of fermentation process- including batch, fed-batch and chemostat fermentation. These results indicate large-scale production of rHBsAg in recombinant P. pastoris, using the chemostat operation mode is more cost-effective and time-sparing than the conventional fed-batch production system. Besides, common challenges such as contamination and mutation were absent in the continuous production of rHBsAg in P. pastoris.

title: High cell-density fermentation.
abstract: The purpose of this chapter is to educate the reader about the basic equipment and strategies used in fermentations of P. pastoris in both bench-top and pilot-scale operations. A key element in expression of foreign proteins in this yeast is the need for sufficient aeration, which is achieved by proper mixing of the media and by blending gases to control dissolved oxygen content. Automatic pH control is essential for growth and expression in P. pastoris. Finally, fed-batch fermentations require the use of peristaltic pumps and tubing capable of low rates of delivery for the feeding of nutrients and base. Teflon tubing and peristaltic pump adapters are recommended for fed-batch operations. The information in this chapter should enable a reader with little or no experience to perform a high-cell density fermentation of a P. pastoris expression strain. Although most procedures described here are specifically for the BioFlo III (NBS), it should be possible to achieve high expression levels with almost any good-quality fermentor, modified to accommodate this organism.

title: De novo transcriptome analysis of Inonotus baumii by RNA-seq.
abstract: Inonotus baumii, a basidiomycete white rot fungus, has been widely used as traditional herbal medicine in China, Korea, Japan and other Asian countries for many years. Its extract is of great medicinal importance and plays a valuable role in the immune response and disease resistance. However, limited genetic resources for I. baumii have hindered exploration of this species. In order to gain a molecular understanding of this fungus, Illumina high-throughput technology was used to sequence and analyze the transcriptome of I. baumii, and 280,691 contigs, 43,890 scaffolds and 30,051 unigenes were obtained. Additionally, based on similarity search with known proteins, unigenes were annotated with gene descriptions, gene ontology (GO), clusters of orthologous group (COG), and database of protein families (Pfam) terms. According to the annotation of unigenes, a total of 12 candidate genes involved in the triterpenoid biosynthesis pathway and 21 putative FOLymes (fungal oxidative lignin enzymes) and 176 CAZymes (carbohydrate-active enzymes) were obtained using homology-based BlastX. Moreover, for better understanding of the transcripts function, the BlastX algorithm was used to search for homologous sequences against the Yeast genome. This is the first study on transcriptome analyses of I. baumii, which provided a dataset for functional gene mining and laid a basis for further functional genomics studies of I. baumii.

title: Identification of differentially expressed genes in flax (Linum usitatissimum L.) under saline-alkaline stress by digital gene expression.
abstract: The salinization and alkalization of soil are widespread environmental problems, and alkaline salt stress is more destructive than neutral salt stress. Therefore, understanding the mechanism of plant tolerance to saline-alkaline stress has become a major challenge. However, little attention has been paid to the mechanism of plant alkaline salt tolerance. In this study, gene expression profiling of flax was analyzed under alkaline-salt stress (AS2), neutral salt stress (NSS) and alkaline stress (AS) by digital gene expression. Three-week-old flax seedlings were placed in 25 mM Na2CO3 (pH11.6) (AS2), 50mM NaCl (NSS) and NaOH (pH11.6) (AS) for 18 h. There were 7736, 1566 and 454 differentially expressed genes in AS2, NSS and AS compared to CK, respectively. The GO category gene enrichment analysis revealed that photosynthesis was particularly affected in AS2, carbohydrate metabolism was particularly affected in NSS, and the response to biotic stimulus was particularly affected in AS. We also analyzed the expression pattern of five categories of genes including transcription factors, signaling transduction proteins, phytohormones, reactive oxygen species proteins and transporters under these three stresses. Some key regulatory gene families involved in abiotic stress, such as WRKY, MAPKKK, ABA, PrxR and ion channels, were differentially expressed. Compared with NSS and AS, AS2 triggered more differentially expressed genes and special pathways, indicating that the mechanism of AS2 was more complex than NSS and AS. To the best of our knowledge, this was the first transcriptome analysis of flax in response to saline-alkaline stress. These data indicate that common and diverse features of saline-alkaline stress provide novel insights into the molecular mechanisms of plant saline-alkaline tolerance and offer a number of candidate genes as potential markers of tolerance to saline-alkaline stress.

title: Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function.
abstract: As manually curated and non-automated BLAST analysis of the published Pichia pastoris genome sequences revealed many differences between the gene annotations of the strains GS115 and CBS7435, RNA-Seq analysis, supported by proteomics, was performed to improve the genome annotation. Detailed analysis of sequence alignment and protein domain predictions were made to extend the functional genome annotation to all P. pastoris sequences. This allowed the identification of 492 new ORFs, 4916 hypothetical UTRs and the correction of 341 incorrect ORF predictions, which were mainly due to the presence of upstream ATG or erroneous intron predictions. Moreover, 175 previously erroneously annotated ORFs need to be removed from the annotation. In total, we have annotated 5325 ORFs. Regarding the functionality of those genes, we improved all gene and protein descriptions. Thereby, the percentage of ORFs with functional annotation was increased from 48% to 73%. Furthermore, we defined functional groups, covering 25 biological cellular processes of interest, by grouping all genes that are part of the defined process. All data are presented in the newly launched genome browser and database available at www.pichiagenome.org In summary, we present a wide spectrum of curation of the P. pastoris genome annotation from gene level to protein function.

title: Activity changes of inorganic pyrophosphatase of Streptococcus faecalis during batch culture.
abstract: The state of inorganic pyrophosphatase (EC 3.6.1.1) from Streptococcus faecalis ATCC 8043 was studied in different phases of batch culture. The degree of inactivation (i.e. the ratio of activities observed before and after incubation at 37 degrees C without cysteine) was highest, and the degree of activation (i.e. the ratio of activities after and before incubation in the presence of cysteine) was lowest, in samples taken during the early-exponential growth phase. During the various phases of batch culture, the specific activity before incubation and the degree of inactivation changed in parallel, whereas the specific activity observed after incubation remained nearly constant. During the early-exponential phase of growth almost all the enzyme was in the high-activity form, whereas during the stationary phase the highly active and the less active forms existed in equal amounts. These findings suggest that inorganic pyrophosphatase in S. faecalis is synthesized constitutively and is primarily regulated at the level of activity.

title: Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3
abstract: Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.

title: Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris
abstract: Mitogen-activated protein kinases (MAPKs) are key enzymes in the signal transduction pathways of eukaryotes. We report the isolation of a Pichia pastoris gene, PIM1, which encodes the first MAPK to be identified in this yeast. Pim1 shows the greatest similarity to fungal MAPKs involved in the maintenance of cell integrity. Disruption of the PIM1 gene results in an osmoremediable thermosensitive phenotype reminiscent of that observed in mutants affected in the MAPK Slt2/Mpk1 of Saccharomyces cerevisiae, which is involved in ensuring cell wall integrity. Furthermore, pim1 mutants are hypersensitive to caffeine and cell wall-destabilising compounds. Pim1 is phosphorylated at two sites, and thereby activated, in response to heat stress, caffeine and agents that alter the fungal cell wall, which is consistent with a role in adaptation to these conditions. These results support the idea that the MAPK-based mechanisms which regulate cell wall integrity are conserved in yeast species. Pim1 is alsodoubly phosphorylated in S. cerevisiae in response to stimuli that activate the cell integrity pathway in this yeast. In addition, Pim1 is able to activate the transcription of a reporter gene in one-hybrid experiments, as does its S. cerevisiae counterpart, Slt2. Interestingly, however, Pim1 does not rescue the mutant phenotype of an slt2Δ strain. This indicates some functional divergence in MAPK modulation and signal transmission by cell integrity pathways and provides a tool that may contribute to a better understanding of MAPK signalling.

title: Macrokinetic model for methylotrophic Pichia pastoris based on stoichiometric balance.
abstract: A macrokinetic model for Pichia pastoris expressing recombinant human serum albumin is proposed. The model describes the balances of some key metabolites, ATP and NADH, during glycerol and methanol metabolism. In the glycerol growth phase, the metabolic pathways mainly include phosphorylation, glycolysis, tricarboxylic acid cycle, and respiratory chain. In the methanol growth phase, methanol is oxidized to formaldehyde at first. Then, while a part of formaldehyde is oxidized to formate, the rest is condensed with xylulose-5-monophosphate to form glyceraldehyde-3-phosphate, and further assimilated to form cell constituents. The metabolic pathways following glyceraldehyde-3-phosphate were assumed to be similar to those in the glycerol growth phase. Based on the model, the macrokinetic bioreaction rates such as the specific substrate consumption rate, the specific growth rate, the specific acetyl-CoA formation rate as well as the specific oxygen uptake rate are obtained. The specific substrate consumption rate and the specific growth rate are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, i.e., the medium volume, the concentrations of the biomass, the substrate, and the product, is set up. Experimental results demonstrate that the model can describe the cell growth and the protein production with reasonable accuracy.

title: The significance of peroxisomes in methanol metabolism in methylotrophic yeast.
abstract: The capacity to use methanol as sole source of carbon and energy is restricted to relatively few yeast species. This may be related to the low efficiency of methanol metabolism in yeast, relative to that of prokaryotes. This contribution describes the details of methanol metabolism in yeast and focuses on the significance of compartmentalization of this metabolic pathway in peroxisomes.

title: A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.
abstract: The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform PAOX1 and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.

title: Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence.
abstract: Peroxisomal matrix proteins have to be imported into their target organelle post-translationally. The major translocation pathway depends on a C-terminal targeting signal, termed PTS1. Our previous analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for signal recognition in the PTS1-Pex5 receptor complex. The refined PTS1 motif description was implemented in a prediction tool composed of taxon-specific functions (metazoa, fungi, remaining taxa), capable of recognising potential PTS1s in query sequences. The composite score function consists of classical profile terms and additional terms penalising deviations from the derived physical property pattern over sequence segments. The prediction algorithm has been validated with a self-consistency and three different cross-validation tests. Additionally, we tested the tool on a large set of non-peroxisomal negatives, on mutation data, and compared the prediction rate to the PTS1 component of the PSORT2 program. The sensitivity of our predictor in recognising documented PTS1 signal containing proteins is close to 90% for reliable prediction. The predictor distinguishes even SKL-appended non-peroxisomally targeted proteins such as a mouse dihydrofolate reductase-SKL construct. The corresponding rate of false positives is not worse than 0.8%; thus, the tool can be applied for large-scale unsupervised sequence database annotation. A scan of public protein databases uncovered a number of yet uncharacterised proteins for which the PTS1 signal might be critical for biological function. The predicted presence of a PTS1 signal implies peroxisomal localisation in the absence of N-terminal targeting sequences such as the mitochondrial import signal.

title: Peroxisomal Catalase in the Methylotrophic Yeast Candida boidinii: Transport Efficiency and Metabolic Significance
abstract: In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Δ strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and d-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and d-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 andd-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pΔnkf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

title: Antioxidant System within Yeast Peroxisome
abstract: Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using thePMP20-deleted strain of C. boidinii(pmp20Δ strain). The His6-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activityin vitro toward alkyl hydroperoxides and H2O2. Catalytic activity and dimerization of His6-CbPmp20 depended on the only cysteine residue corresponding to Cys53. The pmp20Δ strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Δ growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Δ strain had a more severe growth defect than the cta1Δ strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, thecta1Δ strain accumulated H2O2, whereas the pmp20Δ strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface,e.g. lipid hydroperoxides, rather than to decompose H2O2. In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

title: Absence of the peroxiredoxin Pmp20 causes peroxisomal protein leakage and necrotic cell death.
abstract: We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death.

title: Fatty acid metabolism in Saccharomyces cerevisiae
abstract: Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid β-oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the β-oxidation products out of the peroxisome to mitochondria for full oxidation to CO2 and H2O.

title: The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae.
abstract: Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of beta-oxidation enzyme activities as well as a set of auxiliary activities to provide the beta-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of beta-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p-Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in beta-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in beta-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

title: Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.
abstract: Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

title: Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures
abstract: BackgroundThe methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production.ResultsThe metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates.ConclusionsOverall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.

title: Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2
abstract: The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.

title: Novel function of Wsc proteins as a methanol‐sensing machinery in the yeast Pichia pastoris
abstract: Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol‐sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high‐temperature stress. We also found that PpRom2, which is known to function downstream of the Wsc family proteins in the cell wall integrity pathway, was also involved in sensing methanol. Based on these results, these PpWsc family proteins were demonstrated to be involved in sensing methanol and transmitting the signal via their cytoplasmic tail to the nucleus via PpRom2, which plays a critical role in regulating expression of a subset of methanol‐inducible genes to coordinate well‐balanced methanol metabolism.

title: Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP2K
abstract: The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11‐Pbs2‐Hog1 MAPK cascade and the Ssk2/Ssk22‐Pbs2‐Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr‐518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser‐514 and Thr‐518 under optimal osmostress conditions. Mono‐phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2‐Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone‐to‐Hog1 crosstalk in the absence of osmostress. We also report that the rapid‐and‐transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

title: Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway.
abstract: Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.

title: Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation
abstract: As one of the most commonly used eukaryotic recombinant protein expression systems, P. pastoris relies heavily on the AOX1 promoter (PAOX1), which is strongly induced by methanol but strictly repressed by glycerol and glucose. However, the complicated signaling pathways involved in PAOX1 regulation when supplemented with different carbon sources are poorly understood. Here we constructed a kinase deletion library in P. pastoris and identified 27 mutants which showed peculiar phenotypes in cell growth or PAOX1 regulation. We analyzed both annotations and possible functions of these 27 targets, and then focused on the MAP kinase Hog1. In order to locate its potential downstream components, we performed the phosphoproteome analysis on glycerol cultured WT and Δhog1 strains and identified 157 differentially phosphorylated proteins. Our results identified important kinases involved in P. pastoris cell growth and PAOX1 regulation, which could serve as valuable targets for further mechanistic studies.

title: Pichia pastoris 14‐3‐3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction
abstract: The zinc‐finger transcription factor, Mxr1 activates methanol utilization and peroxisome biogenesis genes in the methylotrophic yeast, Pichia pastoris. Expression of Mxr1‐dependent genes is regulated in response to various carbon sources by an unknown mechanism. We show here that this mechanism involves the highly conserved 14‐3‐3 proteins. 14‐3‐3 proteins participate in many biological processes in different eukaryotes. We have characterized a putative 14‐3‐3 binding region at Mxr1 residues 212–225 and mapped the major activation domain of Mxr1 to residues 246–280, and showed that phenylalanine residues in this region are critical for its function. Furthermore, we report that a unique and previously uncharacterized 14‐3‐3 family protein in P. pastoris complements Saccharomyces cerevisiae 14‐3‐3 functions and interacts with Mxr1 through its 14‐3‐3 binding region via phosphorylation of Ser215 in a carbon source‐dependent manner. Indeed, our in vivo results suggest a carbon source‐dependent regulation of expression of Mxr1‐activated genes by 14‐3‐3 in P. pastoris. Interestingly, we observed 14‐3‐3‐independent binding of Mxr1 to the promoters, suggesting a post‐DNA binding function of 14‐3‐3 in regulating transcription. We provide the first molecular explanation of carbon source‐mediated regulation of Mxr1 activity, whose mechanism involves a post‐DNA binding role of 14‐3‐3.

title: Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris
abstract: Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.

title: Trm1p, a Zn(II)₂Cys₆-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.
abstract: The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris ΔTrm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii.

title: Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris*
abstract: The alcohol oxidase 1 (AOX1) promoter (PAOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of PAOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated PAOX1 in response to methanol, were bound to PAOX1 at different sites and did not interact with each other. However, these factors cooperatively activated PAOX1 through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (PMIT1), thus increasingly expressing Mit1 and subsequently activating PAOX1.

title: Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation
abstract: The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). We determined the transcriptional scope of the UPR using DNA microarrays. Rather than regulating only ER-resident chaperones and phospholipid biosynthesis, as anticipated from earlier work, the UPR affects multiple ER and secretory pathway functions. Studies of UPR targets engaged in ER-associated protein degradation (ERAD) reveal an intimate coordination between these responses: efficient ERAD requires an intact UPR, and UPR induction increases ERAD capacity. Conversely, loss of ERAD leads to constitutive UPR induction. Finally, simultaneous loss of ERAD and the UPR greatly decreases cell viability. Thus, the UPR and ERAD are dynamic responses required for the coordinated disposal of misfolded proteins even in the absence of acute stress.

title: Decay of Endoplasmic Reticulum-Localized mRNAs During the Unfolded Protein Response
abstract: The unfolded protein response (UPR) allows the endoplasmic reticulum (ER) to recover from the accumulation of misfolded proteins, in part by increasing its folding capacity. Inositol-requiring enzyme–1 (IRE1) promotes this remodeling by detecting misfolded ER proteins and activating a transcription factor, X-box–binding protein 1, through endonucleolytic cleavage of its messenger RNA (mRNA). Here, we report that IRE1 independently mediates the rapid degradation of a specific subset of mRNAs, based both on their localization to the ER membrane and on the amino acid sequence they encode. This response is well suited to complement other UPR mechanisms because it could selectively halt production of proteins that challenge the ER and clear the translocation and folding machinery for the subsequent remodeling process.

title: Regulated Ire1-dependent decay of messenger RNAs in mammalian cells
abstract: Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.

title: Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1
abstract: Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

title: IRE1‐ and HAC1‐independent transcriptional regulation in the unfolded protein response of yeast
abstract: The unfolded protein response (UPR) is a signalling pathway leading to transcriptional activation of genes that protect cells from accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). In yeast, the only known ER stress signalling pathway originates at the type I transmembrane protein kinase/endoribonuclease Ire1p. Ire1p regulates synthesis of the basic leucine‐zipper (bZIP)‐containing transcription factor Hac1p by controlling splicing of HAC1 mRNA. Only spliced HAC1 mRNA (HAC1i) is translated, and Hac1ip activates transcription of genes that contain a conserved UPR element (UPRE) in their promoters. Here, we demonstrate that in addition to this well‐understood ER stress signalling pathway, a second, IRE1, HAC1 and UPRE‐independent mechanism for transcriptional activation upon ER stress, exists in yeast. A genetic screen identified recessive SIN4 alleles as suppressors of a defective UPR in ire1Δ strains. Elevation of basal transcription in sin4 strains or by tethering the RNA polymerase II holoenzyme with LexAp‐holoenzyme component fusion proteins to a promoter allowed for activation of the promoter by ER stress in an IRE1, HAC1 and UPRE‐independent manner. We propose that this novel second ER‐to‐nucleus signal transduction pathway culminates in core promoter activation (CPA) through stimulation of RNA polymerase II holoenzyme activity. Core promoter activation was observed upon diverse cellular stresses, suggesting it represents a primordial stress‐induced gene activation mechanism.

title: Defective ER associated degradation of a model luminal substrate in yeast carrying a mutation in the 4th ER luminal loop of Sec61p.
abstract: The major constituent of the eukaryotic ER protein-translocation channel (Sec61p in yeast, Sec61α in higher eukaryotes) shows a high degree of evolutionary conservation from yeast to humans. The vast majority of eukaryotic species have a conserved di-tyrosine in the 4th ER luminal loop. Previously, we discovered through a screen of ethylnitrosourea- (ENU-) mutagenized mice that substitution of the latter of these tyrosines with histidine (Y344H) of the murine Sec61α protein results in diabetes and hepatic steatosis in mice that is a result of ER stress. To further characterize the mechanism behind ER stress in these mice we made the homologous mutation in yeast Sec61p (Y345H). We found that this mutation increased sensitivity of yeast to ER stressing agents and to reduction of Inositol Requiring Enzyme 1 (IRE1) activity. Furthermore, we found that, while this mutation did not affect translocation, it did delay degradation of the model ER-associated degradation (ERAD) substrate CPY(∗). Replacing both ER luminal tyrosines with alanines resulted in a destabilization of the Sec61 protein that was rescued by over expression of Sss1p. This double mutant still lacked a noticeable translocation defect after stabilization by Sss1p, but exhibited a similar defect in CPY(∗) degradation.

title: Endoplasmic reticulum stress sensing in the unfolded protein response.
abstract: Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1's sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review.

title: Road to Ruin: Targeting Proteins for Degradation in the Endoplasmic Reticulum
abstract: Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.

title: How the Atg1 complex assembles to initiate autophagy
abstract: The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1EAT, is dynamic on its own, but is rigidified in its high-affinity (∼100 nM) complex with Atg13. Atg1EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (∼10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.

title: An overview of macroautophagy in yeast.
abstract: Macroautophagy is an evolutionarily conserved dynamic pathway that functions primarily in a degradative manner. A basal level of macroautophagy occurs constitutively, but this process can be further induced in response to various types of stress including starvation, hypoxia and hormonal stimuli. The general principle behind macroautophagy is that cytoplasmic contents can be sequestered within a transient double-membrane organelle, an autophagosome, which subsequently fuses with a lysosome or vacuole (in mammals, or yeast and plants, respectively), allowing for degradation of the cargo followed by recycling of the resulting macromolecules. Through this basic mechanism, macroautophagy has a critical role in cellular homeostasis; however, either insufficient or excessive macroautophagy can seriously compromise cell physiology, and thus, it needs to be properly regulated. In fact, a wide range of diseases are associated with dysregulation of macroautophagy. There has been substantial progress in understanding the regulation and molecular mechanisms of macroautophagy in different organisms; however, many questions concerning some of the most fundamental aspects of macroautophagy remain unresolved. In this review, we summarize current knowledge about macroautophagy mainly in yeast, including the mechanism of autophagosome biogenesis, the function of the core macroautophagic machinery, the regulation of macroautophagy and the process of cargo recognition in selective macroautophagy, with the goal of providing insights into some of the key unanswered questions in this field.

title: Multilayered Control of Protein Turnover by TORC1 and Atg1.
abstract: The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our in vivo data, combined with targeted in vitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues.

title: Atg1 kinase regulates early and late steps during autophagy
abstract: The notion that phosphorylation constitutes a major mechanism to induce autophagy was established 15 years ago when a conserved Atg1/ULK kinase family was identified as an essential component of the autophagy machinery. The key observation was that starved atg1Δ cells lack autophagosomes in the cytosol and fail to accumulate autophagic bodies in the vacuole. Although many studies have revealed important details of Atg1 activation and function, a cohesive model for how Atg1 regulates the autophagic machinery is lacking. Our recent findings identified conserved steps of temporal and spatial regulation of Atg1/ULK1 kinase at both the PAS and autophagosomal membranes, suggesting that Atg1 not only promotes autophagy induction, but may also facilitate late stages of autophagosome biogenesis.

title: Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy
abstract: Autophagy is a highly conserved degradative process in eukaryotic cells. This process plays an integral role in cellular physiology, and the levels of autophagy must be precisely controlled to prevent cellular dysfunction. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating the induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that Tap42 and protein phosphatase type 2A (PP2A) are involved in the regulation of autophagy in yeast. Temperature-sensitive mutant alleles of TAP42 revealed that autophagy was induced without inactivation of TORC1. Absence of the Tap42-interacting protein Tip41 abolished autophagy induction in the tap42 mutants, whereas overexpression of Tip41 activated autophagy. Furthermore, inactivation of PP2A stimulated autophagy and overexpression of a catalytic subunit of PP2A blocked rapamycin-induced autophagy. Our data support a model in which autophagy is negatively regulated by the Tap42-PP2A pathway.

title: Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt
abstract: Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lőrincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.

title: The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS39.
abstract: The formation of the autophagic vesicles requires the recruitment of ubiquitin-like Atg8 proteins to the membrane of nascent autophagosomes. Seven Atg8 homologs are present in mammals, split into the LC3 and the GABARAP/GATE-16 families, whose respective functions are unknown. Using Caenorhabditis elegans, we investigated the functions of the GABARAP and the LC3 homologs, LGG-1 and LGG-2, in autophagosome biogenesis. Both LGG-1 and LGG-2 localize to the autophagosomes but display partially overlapping patterns. During allophagy, a developmentally stereotyped autophagic flux, LGG-1 acts upstream of LGG-2 to allow its localization to autophagosomes. LGG-2 controls the maturation of LGG-1-positive autophagosomes and facilitates the tethering with the lysosomes through a direct interaction with the VPS-39 HOPS complex subunit. Genetic analyses sustain a sequential implication of LGG-1, LGG-2, RAB-7, and HOPS complex to generate autolysosomes. The duplications of Atg8 in metazoans thus allowed the acquisition of specialized functions for autophagosome maturation.

title: Peroxisomal Pex3 Activates Selective Autophagy of Peroxisomes via Interaction with the Pexophagy Receptor Atg30*
abstract: Pex3 is a docking factor for pexophagy receptors in yeast.  A specific domain on Pex3 is responsible for interaction and activation of the pexophagy receptor Atg30 during pexophagy.  Pex3 regulates the initiation of pexophagy.  The peroxisomal ligand of the pexophagy receptor contributes to pexophagy signaling. Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.

title: A new role for ATM in selective autophagy of peroxisomes (pexophagy)
abstract: abstract Peroxisomes are autonomously replicating and highly metabolic organelles necessary for β-oxidation of fatty acids, a process that generates large amounts of reactive oxygen species (ROS). Maintaining a balance between biogenesis and degradation of peroxisomes is essential to maintain cellular redox balance, but how cells do this has remained somewhat of a mystery. While it is known that peroxisomes can be degraded via selective autophagy (pexophagy), little is known about how mammalian cells regulate pexophagy to maintain peroxisome homeostasis. We have uncovered a mechanism for regulating pexophagy in mammalian cells that defines a new role for ATM (ATM serine/threonine kinase) kinase as a “first responder” to peroxisomal ROS. ATM is delivered to the peroxisome by the PEX5 import receptor, which recognizes an SRL sequence located at the C terminus of ATM to localize this kinase to peroxisomes. In response to ROS, the ATM kinase is activated and performs 2 functions: i) it signals to AMPK, which activates TSC2 to suppresses MTORC1 and phosphorylates ULK1 to induce autophagy, and ii) targets specific peroxisomes for pexophagy by phosphorylating PEX5 at Ser141, which triggers ubiquitnation of PEX5 at Lys209 and binding of the autophagy receptor protein SQSTM1/p62 to induce pexophagy.

title: Intracellular ATP Correlates with Mode of Pexophagy in Pichia pastoris
abstract: The methylotrophic yeast Pichia pastoris can degrade peroxisomes selectively though two distinct pexophagic pathways, viz., micropexophagy and macropexophagy. These micro- and macropexophagy pathways are induced by adaptation of methanol-grown cells to glucose-containing and ethanol-containing media respectively. However, our understanding of the molecular signal(s) that determine which pathway is activated or repressed in response to environmental changes is limited. In this study, the determinant for these pathways was sought using cells undergoing pexophagy under a variety of conditions. Micropexophagy and macropexophagy were distinguished in living cells by fluorescence microscopy. Our results indicate that glucose and ethanol were not specific inducers of micro- and macropexophagy respectively. Micropexophagy was found to be more sensitive to ATP-depletion than macropexophagy, suggesting that the micropexophagic process requires a higher level of ATP than the macropexophagic process. From these and other results, we postulate that intracellular ATP levels play an important role in determining which pexophagic pathway is activated.

title: Damaged peroxisomes are subject to rapid autophagic degradation in the yeast Hansenula polymorpha
abstract: Evidence is accumulating that damaged components of eukaryotic cells are removed by autophagic degradation (e.g., mitophagy). Here we show that peroxisomes that are damaged by the abrupt removal of the membrane protein Pex3 are massively and rapidly degraded even when the cells are placed at peroxisome-inducing conditions and hence need the organelles for growth. Pex3 degradation was induced by a temperature shift using Hansenula polymorpha pex3Δ cells producing a Pex3 fusion protein containing an N-terminal temperature sensitive degron sequence. The massive peroxisome degradation process, associated with Pex3 degradation, showed properties of both micro- and macropexophagy and was dependent on Atg1 and Ypt7. This mode of peroxisome degradation is of physiological significance as it was also observed at conditions that excessive ROS is formed from peroxisome metabolism, i.e., when methanol-grown wild-type cells are exposed to methanol excess conditions.