RESUMEN
Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in K. phaffii, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.
Asunto(s)
Fitosteroles , Esteroles , Animales , Humanos , Saccharomyces cerevisiae , Ergosterol , Colesterol , MamíferosRESUMEN
The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.
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Carbono/metabolismo , Colesterol/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Técnicas Bacteriológicas , Ciclo del Ácido Cítrico , Glioxilatos/metabolismo , Marcaje Isotópico , Metabolismo de los Lípidos , Redes y Vías Metabólicas , Mycobacterium tuberculosis/metabolismo , FenotipoRESUMEN
The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (-)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-ß-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.
Asunto(s)
Ingeniería Metabólica/métodos , Naftoles/metabolismo , Saccharomyces cerevisiae/metabolismo , Biocatálisis , Furanos , Microorganismos Modificados Genéticamente , Naftalenos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Escualeno/metabolismo , Escualeno-Monooxigenasa/metabolismo , Terbinafina/metabolismo , Triterpenos/metabolismoRESUMEN
Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.
RESUMEN
All intact cells, and their organelles, are surrounded by a â¼30â¯Å hydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.
Asunto(s)
Lípidos de la Membrana/análisis , Lípidos de la Membrana/biosíntesis , Bioingeniería , Glucolípidos/biosíntesis , Proteínas de la Membrana/biosíntesis , Análisis de la Célula IndividualRESUMEN
More than 70,000 different terpenoid structures are known so far; many of them offer highly interesting applications as pharmaceuticals, flavors and fragrances, or biofuels. Extraction of these compounds from their natural sources or chemical synthesis is-in many cases-technically challenging with low or moderate yields while wasting valuable resources. Microbial production of terpenoids offers a sustainable and environment-friendly alternative starting from simple carbon sources and, frequently, safeguards high product specificity. Here, we provide an overview on employing recombinant bacteria and yeasts for heterologous de novo production of terpenoids. Currently, Escherichia coli and Saccharomyces cerevisiae are the two best-established production hosts for terpenoids. An increasing number of studies have been successful in engineering alternative microorganisms for terpenoid biosynthesis, which we intend to highlight in this review. Moreover, we discuss the specific engineering challenges as well as recent advances for microbial production of different classes of terpenoids. Rationalizing the current stages of development for different terpenoid production hosts as well as future prospects shall provide a valuable decision basis for the selection and engineering of the cell factory(ies) for industrial production of terpenoid target molecules.
Asunto(s)
Bacterias/metabolismo , Escherichia coli/metabolismo , Ingeniería Metabólica , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Bacterias/genética , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene ß-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction-oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of ß-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.
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Alquenos/metabolismo , Betaproteobacteria/metabolismo , Hidroliasas/metabolismo , Monoterpenos/metabolismo , Monoterpenos Acíclicos , Alcoholes/química , Alcoholes/metabolismo , Alquenos/química , Catálisis , Escherichia coli/metabolismo , Hidroliasas/química , Hidrólisis , Isomerasas , Monoterpenos/químicaRESUMEN
The addition of water to non-activated carbon-carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio- and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.
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Ácidos Grasos/metabolismo , Flavobacteriaceae/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Ácido Oléico/química , Ácido Oléico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D2O. The combination of solvent contrast variation method with specific deuteration shows that cholesterol is located closer to the lipid head group-tail interface in this natural PC extract rather than in the center of the core of the bilayer as seen for very thin or polyunsaturated membranes.
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Colesterol/química , Deuterio/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/químicaRESUMEN
Water addition to carbon-carbon double bonds provides access to value-added products from inexpensive organic feedstock. This interesting but relatively little-studied reaction is catalysed by hydratases in a highly regio- and enantiospecific fashion with excellent atom economy. Considering that asymmetric hydration of (non-activated) carbon-carbon double bonds is virtually impossible with current organic chemistry, enzymatic hydration reactions are highly attractive for industrial applications. Hydratases have been known for several decades but their biocatalytic potential has only been explored over the past 15 years. As a result, a considerable amount of information on this enzyme group has become available, enabling their development for practical applications. This review focuses on hydratases catalysing water addition to non-activated carbon-carbon double bonds, and examines hydratases from a biochemical, structural and mechanistic angle. Current challenges and opportunities in hydration biocatalysis are discussed, and, ultimately, their potential for organic synthesis is highlighted.
Asunto(s)
Biocatálisis , Hidroliasas/metabolismo , Agua/química , Carbono/química , CatálisisRESUMEN
In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.
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Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Homeostasis del Telómero/fisiología , Telómero/metabolismo , Factores de Transcripción/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esfingolípidos/genética , Telómero/genética , Factores de Transcripción/genéticaRESUMEN
Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Pichia/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Biotransformación , Medios de Cultivo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Mentha piperita/enzimología , Oxidación-Reducción , Pichia/enzimología , Pichia/crecimiento & desarrollo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Regulación hacia ArribaRESUMEN
Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.
Asunto(s)
Flavobacteriaceae/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Ácido Oléico/química , Dominio Catalítico , Estructura MolecularRESUMEN
The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
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Proteínas de Arabidopsis , Ingeniería Metabólica , Pichia , Sesquiterpenos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Cupressus/enzimología , Cupressus/genética , Hyoscyamus/enzimología , Hyoscyamus/genética , Pichia/enzimología , Pichia/genética , Sesquiterpenos PolicíclicosRESUMEN
BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport. RESULTS: The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations. CONCLUSION: This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation.
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Proteínas Fúngicas/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Reactores Biológicos/microbiología , Carboxilesterasa/metabolismo , Secuencia Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , Hierro/farmacología , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Pichia/efectos de los fármacos , Pichia/genética , Pichia/crecimiento & desarrollo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Regulón/genética , Vías Secretoras/efectos de los fármacos , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.
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Expresión Génica , Pichia/genética , Proteínas Recombinantes/genética , Microbiología Industrial , Pichia/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.
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Eucariontes/metabolismo , Proteínas de la Membrana/metabolismo , Levaduras/metabolismo , Eucariontes/genética , Proteínas de la Membrana/genética , Modelos Biológicos , Unión Proteica , Levaduras/genéticaRESUMEN
The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3ß1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3ß1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.
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Colesterol/metabolismo , Expresión Génica , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Vías Biosintéticas/genética , Membrana Celular/enzimología , Ergosterol/metabolismo , Humanos , Ingeniería Metabólica , Isoformas de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Fatty acids have been supplied for diverse non-food, industrial applications from plant oils and animal fats for many decades. Due to the massively increasing world population demanding a nutritious diet and the thrive to provide feedstocks for industrial production lines in a sustainable way, i.e., independent from food supply chains, alternative fatty acid sources have massively gained in importance. Carbohydrate-rich side-streams of agricultural production, e.g., molasses, lignocellulosic waste, glycerol from biodiesel production, and even CO2, are considered and employed as carbon sources for the fermentative accumulation of fatty acids in selected microbial hosts. While certain fatty acid species are readily accumulated in native microbial metabolic routes, other fatty acid species are scarce, and host strains need to be metabolically engineered for their high-level production. We report the metabolic engineering of Pichia pastoris to produce palmitoleic acid from glucose and discuss the beneficial and detrimental engineering steps in detail. Fatty acid secretion was achieved through the deletion of fatty acyl-CoA synthetases and overexpression of the truncated E. coli thioesterase 'TesA. The best strains secreted >1 g/L free fatty acids into the culture medium. Additionally, the introduction of C16-specific ∆9-desaturases and fatty acid synthases, coupled with improved cultivation conditions, increased the palmitoleic acid content from 5.5% to 22%.
RESUMEN
There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.