The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.     

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.     

Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression

A new method for the isolation of glucose repression-insensitive mutants in the methylotrophic yeast Pichia pinus was developed. The method is based on screening of small suspension samples derived from 2-deoxyglucose-resistant colonies for alcohol oxidase activity. Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells. Mutants with glucose non-repressible alcohol oxidase and catalase synthesis were obtained. All mutants grew poorly on D -xylose compared to the wild type, whereas growth on L -arabinose was similar to the wild type. Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.     

Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris

The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1 utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1 utilizing enzymes, especially in a shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress beta-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, mannitol or trehalose can be used as a sole carbon and energy source for P. pastoris, although the doubling time on trehalose was very long. Mut- strains growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher amount of beta-gal as compared to the mut+ growing in methanol containing media.     

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

Isolation and bioenergetic characterization of mitochondria from Pichia pastoris

Pichia pastoris is a methylotrophic yeast of high biotechnological interest. The bioenergetic properties of mitochondria from Pichia pastoris have not yet been determined. We report on a protocol for the isolation of the mitochondria in a state that shows good energy coupling. Analysis of Pichia pastoris growth and bioenergetic properties of the isolated mitochondria reveals that glycerol is the carbon source that yields the best results. Under our growth conditions, mitochondria oxidize external NADH but do not possess an alternative oxidase. Finally, Pichia pastoris mitochondria also lack the nucleotide-stimulated uncoupling pathway previously identified in Saccharomyces cerevisiae.     

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.     

Chromosomal DNA patterns and gene stability of Pichia pastoris

We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1·7 Mb to 3·5 Mb and total genome size was estimated to be 9·5 Mb to 9·8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation. © 1998 John Wiley & Sons, Ltd.     

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris

Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirDeltakex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirDeltakex1 while it was only 1.1 g/l in GS115Hir.     

Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.     

人对氧磷酶1在巴斯德毕赤酵母中的表达与纯化

人对氧磷酶1（Human Paraoxonase 1,hPON1）是人体血液中的一种非专一性酯酶,可以水解多种有机磷化合物,被认为是一种有机磷农药中毒的解毒剂。为了得到较纯的具有活性的hPON1,本文采用巴斯德毕赤酵母Pichia pastoris表达系统,对hPON1进行胞内表达。hPON1基因经过密码子优化后克隆至pPICZA表达载体上,构建得到pPICZA-PON1重组表达质粒,经线性化后转化至巴斯德毕赤酵母X33菌株中,筛选得到阳性重组菌株。将重组菌株进行摇瓶发酵120 h,酶活达0.15 U/mL。收集发酵液并细胞裂解后,用Ni-NTA螯合亲和层析的方法进行纯化,得到纯化的hPON1,SDS-PAGE结果显示表达产物的大小约37 ku,与预期的蛋白分子量相符。表达的重组hPON1的最适反应温度为45 ℃,最适pH为9.0。以上结果表明,本研究成功地表达并得到较纯的有活性的hPON1蛋白。     

Coprinopsis cinerea 漆酶基因的克隆及其在毕赤酵母中的表达

本文通过RT-PCR从Coprinopsis cinerea okayama7#130中克隆得到laccase1,应用SignalP3.0Server软件分析其氨基酸序列后设计不含信号肽序列的新引物扩增得到不含信号肽的漆酶基因1（lac1）,并构建重组酵母表达载体pPIC9K-lac1,电击转化毕赤酵母GS1115并用甲醇诱导表达,之后研究重组酶的酶学性质。克隆得到的laccase1全长为1593bp,编码530个氨基酸,其中信号肽包含18个氨基酸。SDS-PAGE显示重组蛋白大小约为65KDa。酶学性质研究表明,该酶最适反应温度为45℃,最适pH为4.3。重组漆酶在45℃保存3h后活性基本保持不变,在pH4.0～10.0的范围内稳定性较好。成功分泌表达的漆酶活力达到1.108U/mL。     

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.     

A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial β-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF027959; pIB3, AF027960; pIB4, AF027961. © 1998 John Wiley & Sons, Ltd.     

黑曲霉内切菊粉酶基因在毕赤酵母中的表达及应用研究

利用PCR方法从黑曲霉（Aspergillus niger）基因组DNA中扩增内切菊粉酶（endo I）全长基因（1551 bp）,经序列分析后将其连接到表达载体p PIC9K上,得到重组表达载体p PIC9K-endo I。重组质粒经Sac I线性化并电转化到毕赤酵母GS115,阳性转化子进行发酵产酶后考察其酶学性质。酶学性质研究表明,重组酶的最适p H和最适温度分别为5和60℃,重组酶在55℃下保温9 h后,重组菊粉内切酶仍残余83.2%的活性,表现出极高的热稳定性,Cu²⁺、Ag⁺对重组酶有明显的抑制作用。对内切菊粉酶水解菊粉的过程研究表明,重组内切菊粉酶能在8 h内将4%（w/v）菊粉水解为3⁶个聚合度的低聚果糖,水解11 h后,低聚果糖得率达到63.5%,本研究为进一步开发以菊粉为原料生产低聚果糖的应用奠定了基础。      

Identification and characterization of GRIP domain Golgin PpImh1 from Pichia pastoris

Budding yeast Pichia pastoris has highly advanced secretory pathways resembling mammalian systems, an advantage that makes it a suitable model system to study vesicular trafficking. Golgins are large Golgi‐resident proteins, primarily reported to play role in cargo vesicle capture, but details of such mechanisms are yet to be deciphered. Golgins that localize to the Golgi via their GRIP domain, a C‐terminal Golgi anchoring domain, are known as GRIP domain Golgins. In this present study, we have identified and functionally characterized a homologue of one such GRIP domain Golgin protein, Imh1, from the budding yeast P. pastoris. We have demonstrated that the GRIP domain present at the C‐terminal of P. pastoris Imh1 (PpImh1) functions as its Golgi‐targeting sequence. Using a combination of yeast two‐hybrid analysis, dynamic light scattering and electron microscopy, we have shown that PpImh1 can self‐associate and form a homodimer. Analysis of purified recombinant PpImh1 by CD spectroscopy indicates the presence of an 85% α‐helical structure, a characteristic of high‐content α‐helical coiled‐coil sequences normally present in other Golgin family proteins. Two‐hybrid analysis indicated self‐interaction between C‐terminal fragments, yet N‐terminal fragments do not mediate any such form of self‐interaction, suggesting that PpImh1 may form a parallel dimer. Electron microscopy data indicates that PpImh1 forms extended rod‐like homo‐dimeric molecules with splayed N‐terminal end which can act as a tether for capturing vesicles. Our study provides the first evidence in support of the dimeric Y‐shaped structure for any Golgin in the budding yeast.     

Isolation and sequence of the MIG1 homologue from the yeast Candida utilis

The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe. This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors. The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain). The MIG1 repressor gene from C. utilis was able to complement functionally the mig1 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ277830.     

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.