Identification of major ADH genes in ethanol metabolism of Pichia pastoris

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.     

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.     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH₂-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59·44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH₂-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.     

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.     

Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.     

Disruption of the KEX1 gene in Pichia pastoris allows expression of full‐length murine and human endostatin

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright © 1999 John Wiley & Sons, Ltd.     

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch‐wise sorbitol addition as a co‐substrate at the induction phase of methanol fed‐batch fermentation by Pichia pastoris (Mut⁺) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch‐wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag‐phase for the cells and reaching ‘high cell density production’ at t = 24 h of the process (C_X = 70 g CDW/l); (b) achieving 1.8‐fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2‐fold; (d) eliminating the lactic acid build‐up period; (e) lowering the oxygen uptake rate two‐fold; and (f) obtaining 1.4‐fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut⁺ strains of P. pastoris for the production of any bio‐product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast,Pichia pastoris and characterization of the secreted product

Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q³⁰²) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q³⁰² were attempted. Addition of Triton X-100, l-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q³⁰² and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q³⁰² and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q³⁰² was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q³⁰² closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.     

Metabolic regulation in the yeast Hansenula polymorpha. Growth of dihydroxyacetone kinase/glycerol kinase-negative mutants on mixtures of methanol and xylose in continuous cultures

The physiological responses of Hansenula polymorpha wild-type and mutant strains 17B (dihydroxyacetone kinase-negative) and 17BG51 (dihydroxyacetone kinase- and glycerol kinase-negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0–110 mM ) in the feed of the wild-type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose-5-phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash-out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.     

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.     

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1‐L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut^s) and GS115 (Mut⁺), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV‐16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed‐batch cultures of P. pastoris Mut^s were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut^s strains than in the Mut⁺ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L^?1 culture while P. pastoris Mut^s only produced 23.61 mg L^?1. H. polymorpha showed greater potential for the expression of HPV‐16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high‐level producer of heterologous proteins.     

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.