Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.     

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth.     

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.     

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.     

Molecular characterization of the PEX14 gene from the methylotrophic yeast Pichia methanolica

In this study, we describe the molecular characterization of the PmPEX14 gene encoding the peroxisomal membrane protein from the methylotrophic yeast Pichia methanolica. The pex14Δ strain of P. methanolica lost its ability to grow on methanol and oleate but grew normally on glucose. Disruption of the PmPEX14 caused a decrease in the activities of peroxisomal methanol-metabolizing enzymes and mislocalization of those proteins into the cytosol and vacuole. Taken together, these findings show that PmPex14p has an essential physiological role in methanol metabolism in P. methanolica.     

Heterologous protein production using the Pichia pastoris expression system

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.     

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.     

Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus

The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcr1 and ecr1 mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L -sorbose and xylose) was impaired only in the gcr1 mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecr1 mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L -arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcr1 and ecr1 strains. Mutation gcr1 causes a significant decrease in phosphofructokinase activity. It also led to a six- to seven-fold increase in intracellular pools of glucose-6-phosphate and fructose-6-phosphate and to a two-fold decrase in the intracellular pool of fructose-1,6-bisphosphate. In ecr1 strains, a decrese in 2-oxoglutarate dehydrogenase activity accompanied by an increae in activities of NAD- and NADP-dependent isocitrate dehydrogenases and NAD- and NADP-dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2-oxoglutarate was increased 2·5-fold in ecr1 strains. Genes GCR1 and ECR1 are not linked. The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed.     

Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly.

We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.     

The human c‐fos and TNFα AU‐rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris

AU‐rich elements (AREs) are located in the 3′ untranslated region (3′ UTR) of their host genes and tightly regulate mRNA degradation and expression. Examples for this kind of regulation are the human proto‐oncogene c‐fos and the cytokine TNFα. Despite large effort in this field, the exact mechanism of ARE‐mediated mRNA turnover remains unclear. In this work we analysed the effects of c‐fos‐ and TNFα AREs on mRNA abundance and protein expression of selected human cDNAs in the yeast Pichia pastoris. This yeast is exceedingly well known for its excellent protein production capacity; however, ARE‐like mechanisms have not been studied in this yeast to date. Interestingly, we observed both stabilizing and destabilizing effects of the c‐fos ARE, whereas the TNFα ARE has a destabilizing or expression‐reducing function in all tested cDNAs. Based on this observation, we introduced a number of single‐point mutations upstream of the introduced c‐fos ARE into the 3′ UTR of a single cDNA in order to demonstrate the importance of ARE‐flanking sequences for their own regulation. In conclusion, we illustrate that the analysis of ARE‐mediated effects on mRNA abundance and protein expression of a reporter depends on the sequence of the reporter itself as well as the ARE‐surrounding sequences within the 3′ UTR. For this reason, we question whether already established reporter constructs in other cellular systems display the true type of regulation of the tested AREs for its original host gene. Finally, we propose that AREs should be analysed in their native sequence context. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri

This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322-based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per μg of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild-type URA3 gene by an in vitro generated deletion. Sequences conferring extrachromosomal replication were isolated from Y. ohmeri DNA. Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 10⁴/μg and were shown to replicate freely in Y. ohmeri at an approximate copy number of 40. Unexpectedly, we observed that BS-SK^R derivatives carrying either YoLEU2 or YoURA3 but no Y. ohmeri ARS also replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four-fold. The sequences are available for consultation under EMBL accession number Z35101 for YoLEU2 and Z35100 for YOURA3.