Showing papers on "Pichia pastoris published in 2016"

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 ...

Comparative genomics and transcriptomics of Pichia pastoris

Abstract: Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species. Here, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts. Our studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.

Lignocellulose degrading extremozymes produced by Pichia pastoris: current status and future prospects

Abstract: In this review article, extremophilic lignocellulosic enzymes with special interest on xylanases, β-mannanases, laccases and finally cellulases, namely, endoglucanases, exoglucanases and β-glucosidases produced by Pichia pastoris are reviewed for the first time. Recombinant lignocellulosic extremozymes are discussed from the perspectives of their potential application areas; characteristics of recombinant and native enzymes; the effects of P. pastoris expression system on recombinant extremozymes; and their expression levels and applied strategies to increase the enzyme expression yield. Further, effects of enzyme domains on activity and stability, protein engineering via molecular dynamics simulation and computational prediction, and site-directed mutagenesis and amino acid modifications done are also focused. Superior enzyme characteristics and improved stability due to the proper post-translational modifications and better protein folding performed by P. pastoris make this host favourable for extremozyme production. Especially, glycosylation contributes to the structure, function and stability of enzymes, as generally glycosylated enzymes produced by P. pastoris exhibit better thermostability than non-glycosylated enzymes. However, there has been limited study on enzyme engineering to improve catalytic efficiency and stability of lignocellulosic enzymes. Thus, in the future, studies should focus on protein engineering to improve stability and catalytic efficiency via computational modelling, mutations, domain replacements and fusion enzyme technology. Also metagenomic data need to be used more extensively to produce novel enzymes with extreme characteristics and stability.

Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization

Abstract: The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity. In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event. Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.

Recent advances in the production of recombinant subunit vaccines in Pichia pastoris

Abstract: Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines.

Scaling-up Fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement

Abstract: Scaling-up of high-cell-density fermentation (HCDF) of Pichia pastoris from the lab or pilot scale to the demonstration scale possesses great significance because the latter is the final technological hurdle in the decision to go commercial. However, related investigations have rarely been reported. In this paper, we study the scaling-up processes of a recombinant P. pastoris from the pilot (10 to 100-L) to the demonstration (1,000-L) scales, which can be used to convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol by the β-xylosidase for semi-synthesis of Taxol. We demonstrated that a pure oxygen supplement can be omitted from the HCDF if the super atmospheric pressure was increased from 0.05 to 0.10 ± 0.05 MPa, and we developed a new methanol feeding biomass-stat strategy (0.035 mL/g/h) with 1% dissolved oxygen and 100 g/L initial induction biomass (dry cell weight). The scaling-up was reproducible, and the best results were obtained from the 1,000-L scale, featuring a shorter induction time and the highest enzyme activities and productions, respectively. The specific growth and specific production rates were also determined. This study lays a solid foundation for the commercial preparation of 10-deacetyltaxol through the recombinant yeast. It also provides a successful paradigm for scaling-up HCDF of P. pastoris to the demonstration scale.

A novel methanol-free Pichia pastoris system for recombinant protein expression

Abstract: As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P AOX1 ) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in P AOX1 activation or repression by different carbon sources may be promising targets. We identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (P AOX1 induced by glycerol) and Δdak-DHA (P AOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50–60 % of the traditional methanol induced system. We successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of P AOX1 , providing a potential alternative to the traditional system.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Abstract: Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris

Abstract: Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of (13)C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.

Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

Abstract: The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker9s yeast.

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

Abstract: The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.

Genome‐scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

Abstract: Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes.

Crystal Structure of Alcohol Oxidase from Pichia pastoris.

Abstract: FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring.

Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates

Abstract: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery. While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay. Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production. In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications

Abstract: Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence (“panARS”) in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells. A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest. An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.

Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains.

Abstract: Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. © 2015 Wiley Periodicals, Inc.

Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.

Abstract: In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study’s aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies. This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.

PpNrg1 is a transcriptional repressor for glucose and glycerol repression of AOX1 promoter in methylotrophic yeast Pichia pastoris

Abstract: The regulator in glycerol repression of Pichia pastoris AOX1 promoter (P AOX1 ) is still unclear. A Cys2His2 zinc finger transcriptional repressor PpNrg1 localized to nucleus and participated in the repression of P AOX1 in P. pastoris in glucose and glycerol. Quantitative real-time PCR revealed that PpNrg1 repressed expression of numerous genes involved in methanol utilization and peroxisome biogenesis in 0.02 % glucose and 1 % (v/v) glycerol. Electrophoretic mobility shift assay and DNase I footprinting assay revealed that PpNrg1 bound to five sites of P AOX1 , including two binding sites of PpMxr1, which is an indispensable activator of P AOX1 in P. pastoris. Transcriptional repressor PpNrg1 suppresses P AOX1 in glucose and glycerol by directly binding to five sites of P AOX1 , including two binding sites of transcriptional activator PpMxr1.

Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy.

Abstract: The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 A. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.