Gss1 protein of the methylotrophic yeast Pichia pastoris is involved in glucose sensing, pexophagy and catabolite repression

In the yeast Saccharomyces cerevisiae, the one-at-a-time deletions of either the high-affinity glucose sensor gene SNF3 or the low-affinity glucose sensor gene RGT2 only slightly reduced pexophagy; however, deleting both genes greatly reduced pexophagy, evincing interaction beyond the sum of the additive effects, as recently shown. The present study identifies the only ScSNF3/RGT2 ortholog in the methylotrophic yeast Pichia pastoris (designated as PpGSS1, from GlucoSe Sensor) and describes its roles in autophagic pathways (non-selective and selective). GSS1 knock-out strain has been constructed. The experiments support the hypothesis that Gss1 plays an important role in autophagic degradation of peroxisomes and glucose catabolite repression in P. pastoris.     

敲除MIG1和SNF1基因对酿酒酵母共利用葡萄糖和木糖的影响

Mig1和Snf1是酿酒酵母葡萄糖阻遏效应的两个关键调控因子。为了提高酿酒酵母工程菌同时利用葡萄糖和木糖的能力,分别对MIG1和SNF1基因进行了单敲除和双敲除,并通过摇瓶发酵实验和RNA-Seq转录组分析,初步揭示了Mig1和Snf1可能影响葡萄糖和木糖共利用表达差异基因的层级调控机制。研究结果表明,MIG1单敲除对混合糖的共利用影响不大;SNF1单敲除会加快混合糖中木糖的利用而且葡萄糖和木糖可以被同时利用,这可能归因于SNF1单敲除会解除对一些氮分解代谢阻遏基因表达的抑制,从而促进了细胞对氮源营养的利用;进一步敲除MIG1,会解除更多氮分解代谢阻遏基因表达的抑制,以及一些碳中心代谢途径基因表达上调。虽然MIG1和SNF1双敲除菌株利用葡萄糖加快而利用木糖变慢,但是葡萄糖和木糖可以被同时利用,进而加快乙醇的积累。综上所述,MIG1和SNF1的敲除导致氮分解阻遏基因表达上调,有助于促进葡萄糖和木糖的共利用;解析Mig1和Snf1对氮分解阻遏基因的层级调控作用,为进一步提高葡萄糖和木糖的共利用提供新的靶点。     

Conditions with high intracellular glucose inhibit sensing through glucose sensor Snf3 in Saccharomyces cerevisiae

Gene expression in micro‐organisms is regulated according to extracellular conditions and nutrient concentrations. In Saccharomyces cerevisiae, non‐transporting sensors with high sequence similarity to transporters, that is, transporter‐like sensors, have been identified for sugars as well as for amino acids. An alternating‐access model of the function of transporter‐like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose through the transporter‐like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity of extracellular glucose to Snf3 was measured for cells grown in non‐fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating‐access model for transporter‐like sensors. J. Cell. Biochem. 110: 920–925, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.     

Activity of Pichia pastoris alternative cis-prenyltransferase is correlated with proliferation of peroxisomes

We have investigated dolichol synthesis in yeast Pichia pastoris. Growth of these cells on methanol causes peroxisome proliferation and induction of peroxisomal enzymes. Twenty-four hours methanol treatment was sufficient for the appearance of longer-chain dolichols. Less specific oleic acid induction needed 48 h for the synthesis of longer dolichol family with typical one still present. Cells cultured in non-inducing conditions for 48 h did not reveal the presence of additional dolichol family. Peroxisomes purified from oleic acid treated cells synthesize in vitro polyprenols longer by two isoprene residues than those synthesized by microsomal fraction from glucose culture. These observations lead us to suggest that chain length of dolichols synthesized in yeast cell may depend on the carbon and energy source supply which mobilizes metabolic pathways localized to different cellular compartments.     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.     

Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst

The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-d-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-d-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester.     

The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Development of a responsive methanol sensor and its application in Pichia pastoris fermentation

A methanol sensor was developed with response time less than 2 min. It was unaffected by the dissolved O₂ concentration, agitation speed or pH value. When the sensor was used to monitor the methanol concentration on-line during hirudin production by recombinant Pichia pastoris, the cell dry weight was up to 155 g l^–1, and hirudin was 1.4 g l^–1.     

Derepression of a baker's yeast strain for maltose utilization is associated with severe deregulation of HXT gene expression

Aims: We undertook to improve an industrial Saccharomyces cerevisiae strain by derepressing it for maltose utilization in the presence of high glucose concentrations. Methods and Results: A mutant was obtained from an industrial S. cerevisiae strain following random UV mutagenesis and selection on maltose/5‐thioglucose medium. The mutant acquired the ability to utilize glucose simultaneously with maltose and possibly also sucrose and galactose. Aerobic sugar metabolism was still largely fermentative, but an enhanced respirative metabolism resulted in a 31% higher biomass yield on glucose. Kinetic characterization of glucose transport in the mutant revealed the predominance of the high‐affinity component. Northern blot analysis showed that the mutant strain expresses only the HXT6/7 gene irrespective of the glucose concentration in the medium, indicating a severe deregulation in the induction/repression pathways modulating HXT gene expression. Interestingly, maltose‐grown cells of the mutant display inverse diauxy in a glucose/maltose mixture, preferring maltose to glucose. Conclusion: In the mutant here reported, the glucose transport step seems to be uncoupled from downstream regulation, because it seems to be unable to sense abundant glucose, via both repression and induction pathways. Significance and Impact of the Study: We report here the isolation of a S. cerevisiae mutant with a novel derepressed phenotype, potentially interesting for the industrial fermentation of mixed sugar substrates.     

人组织型基质金属蛋白酶抑制剂-1在Pichia pastoris酵母中重组表达研究

 为研究组织型基质金属蛋白酶抑制剂 (TIMPs)的分子作用机制 ,探讨了在 Pichia pastoris酵母中高效表达分泌型人组织型基质金属蛋白酶抑制剂 - 1 (TIMP- 1 )的技术路线 ,并对产物性质进行初步研究 .通过 PCR从含有 TIMP- 1基因的 p BS质粒获得了该基因的全长序列 ,构建了 p PIC9/T1表达载体 ,电击法转化酵母 ,通过表型筛选和 PCR鉴定证实了目的基因已稳定整合入 Pichiapastoris酵母基因组中 .SDS- PAGE表明表达量高达 40 mg/L培养上清 .用免疫印迹法确定了产物的正确性 ;同时 ,反向明胶酶谱法证明了重组蛋白具有抑制基质金属蛋白酶的活性 .     

Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.