In vivo functional assay of a recombinant aquaporin in Pichia pastoris

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.     

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man₅GlcNAc₂N-glycans, glycan profiling revealed two major species: Man₅GlcNAc₂ and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man₅GlcNAc₂ modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan₅GlcNAc₂ structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.     

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.     

Control of TNF-induced dendritic cell maturation by hybrid-type N-glycans

The activity of α-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that α-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c(+) dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-L(low) and PHA-L(high) subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-L(high). Interestingly, all PHA-L(high) DCs displayed a strongly reduced responsiveness to TNF-α-induced p38-MAPK activation compared with PHA-L(low) DCs, indicating differences in PHA-L-binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing α-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-α-induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). α-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-γ-producing T cells in vivo. In contrast, the activity of α-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs' general capability for phenotypic and functional maturation. Systemic application of an α-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by α-1,2-mannosidase I for alloantigen presentation and T cell priming.     

Expression of l-phosphinothricin synthesis enzymes in Pichia pastoris for synthesis of l-phosphinothricin

Biotechnology Letters - With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide....     

In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.     

毕赤酵母不对称合成阿托伐他汀中间体

阿托伐他汀可通过抑制HMG-CoA还原酶与底物的结合来抑制胆固醇的合成,而 (R)-3-羟基-5-邻苯二甲酰亚胺基戊酸乙酯是阿托伐他汀合成的重要中间体。通过对实验室保藏菌种进行筛选,得到一株巴氏毕赤酵母X-33可将5-邻苯二甲酰亚胺-3-氧代戊酸乙酯还原为 (R)-3-羟基-5-邻苯二甲酰亚胺基戊酸乙酯。在磷酸盐缓冲液体系中考察了初始底物浓度、反应时间、辅助底物、葡萄糖添加量、pH、温度等因素对产物收率和对映体选择性的影响,获得了较佳的反应条件。选择底物投料量为7 g/L时,当菌体浓度120 g/L,葡萄     

In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest. Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2. Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P. pastoris.     

Internal ribosome entry site mediates protein synthesis in yeast Pichia pastoris

The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.     

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml ⁻¹. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.     

Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris

ABSTRACT: BACKGROUND: Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. RESULTS: An efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 x 105 /mug DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60degreesC and 65degreesC were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein. CONCLUSIONS: P. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures.     

Bax-induced cell death in Pichia pastoris

Murine Bax was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase 1 (AOX1) or alcohol oxidase 2 (AOX2) promoter and the AOX1 terminator. Upon induction in methanol medium, transformants containing BAX cDNA under control of the strong AOX1 promoter showed complete growth inhibition and extensive cell death. Except for chromatin condensation, morphological changes typical of apoptosis in mammalian cells could not be observed, indicating that the cell death machinery in P. pastoris is marked different from the endogenous cell death program of higher eukaryotes. Staining of Bax-induced cells with propidium iodide indicated that cell death was not correlated with necrosis. Electron microscopic examination revealed no striking differences in cell morphology, but showed few cells with an enlarged vacuole containing spherical bodies, which suggests autophagic cell death.     

Recombinant protein expression in Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.     

巴斯德毕赤酵母表达系统研究进展

巴斯德毕赤酵母表达系统现在已经发展成为一种高效的外源蛋白基因优秀表达系统,该系统具有高表达、高稳定、高分泌、容易放大和成本低等优点,目前已有多种外源蛋白基因在该系统中实现高效表达,对巴斯德毕赤酵母表达系统的进一步研究将会促进其大规模的工业化应用。     

蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造.将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu.用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA.该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg/ml.     

Expression, purification, and characterization of equistatin in Pichia pastoris

Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina. It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins. Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields. A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P. pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 transformants. Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor. EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI.     

毕赤酵母的密码子用法分析

通过分析Pichia pastoris的28个蛋白编码基因的同义密码子使用情况并计算该酵母的密码子用法,首次确定出P.pastoris的19个高表达优越密码子。这些结果经与已知的Saccharomyces cerevisiae及Kluyveromyces lactis的密码子用法基本相似,但在氨基酸谷氨酸的密码子选择上截然相反,提示这可能属于P.pastoris所偏爱的密码子用法。     

Production of protein-based polymers in Pichia pastoris

Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.     

毕赤酵母基因编辑技术研究进展

巴斯德毕赤酵母是一种重要的蛋白表达系统,基因编辑技术作为代谢工程的基本工具,对于毕赤酵母的代谢改造十分重要。近十年基因编辑技术发展迅速,除传统的同源重组和Cre/loxP重组外,相继出现了许多新的基因编辑技术,例如ZFN、TALEN和CRISPR/Cas9等,这些技术的出现使基因编辑更加简便高效。本文对毕赤酵母中传统和新型基因编辑技术的原理应用和研究进展进行了简要综述,并结合相关领域的发展对毕赤酵母基因编辑技术的发展进行了展望。     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.