RESUMO
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.
Assuntos
Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas p21(ras)/genética , Feminino , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Guanosina Trifosfato/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , MasculinoRESUMO
Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proliferação de Células , Serina-Treonina Quinases TOR , Alvo Mecanístico do Complexo 2 de Rapamicina , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Linhagem Celular TumoralRESUMO
Polyketide natural products are an important class of biologically active compounds. Although substantial progress has been made on the synthesis of repetitive polyketide motifs through the iterative application of a single reaction type, synthetic access to more diverse motifs that require more than one type of carbon-carbon bond connection remains a challenge. Here we describe a catalytic, multicomponent method for the synthesis of the privileged polyketide 1,3-dienyl-6-oxy motif. The method allows for the formation of two new carbon-carbon bonds and two stereodefined olefins. It generates products that contain up to three contiguous sp3 stereocentres with a high stereoselectivity in a single operation and can be used to generate chiral products. The successful development of this methodology relies on the remarkable efficiency of the ruthenium-catalysed alkene-alkyne coupling reaction between readily available vinyl boronic acids and alkynes to provide unsymmetrical 3-boryl-1,4-diene reagents. In the presence of carbonyl compounds, these reagents undergo highly diastereoselective allylations to afford the desired 1,3-dienyl-6-oxy motif and enable complex polyketide synthesis in a rapid and asymmetric fashion.
RESUMO
Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened â¼100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally related chemical groups, suggesting that they may act on common targets. Two small molecules increased C. elegans' stress resistance, and at least 9 extended their lifespan by â¼10-50%. We further evaluated a chalcone that produced relatively large effects on lifespan and were able to implicate the activity of two, stress-response regulators, NRF2/skn-1 and SESN/sesn-1, in its mechanism of action. Our findings suggest that screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds may reveal new ways to extend healthy human lifespan.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Longevidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Envelhecimento/genética , Animais , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Humanos , Imagem Molecular , Estresse Oxidativo/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Estresse Fisiológico/genética , TranscriptomaRESUMO
BACKGROUND: Blood-based methods using cell-free DNA (cfDNA) are under development as an alternative to existing screening tests. However, early-stage detection of cancer using tumor-derived cfDNA has proven challenging because of the small proportion of cfDNA derived from tumor tissue in early-stage disease. A machine learning approach to discover signatures in cfDNA, potentially reflective of both tumor and non-tumor contributions, may represent a promising direction for the early detection of cancer. METHODS: Whole-genome sequencing was performed on cfDNA extracted from plasma samples (N = 546 colorectal cancer and 271 non-cancer controls). Reads aligning to protein-coding gene bodies were extracted, and read counts were normalized. cfDNA tumor fraction was estimated using IchorCNA. Machine learning models were trained using k-fold cross-validation and confounder-based cross-validations to assess generalization performance. RESULTS: In a colorectal cancer cohort heavily weighted towards early-stage cancer (80% stage I/II), we achieved a mean AUC of 0.92 (95% CI 0.91-0.93) with a mean sensitivity of 85% (95% CI 83-86%) at 85% specificity. Sensitivity generally increased with tumor stage and increasing tumor fraction. Stratification by age, sequencing batch, and institution demonstrated the impact of these confounders and provided a more accurate assessment of generalization performance. CONCLUSIONS: A machine learning approach using cfDNA achieved high sensitivity and specificity in a large, predominantly early-stage, colorectal cancer cohort. The possibility of systematic technical and institution-specific biases warrants similar confounder analyses in other studies. Prospective validation of this machine learning method and evaluation of a multi-analyte approach are underway.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genoma Humano , Genômica , Aprendizado de Máquina , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/sangue , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Genômica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Curva ROC , Reprodutibilidade dos Testes , TranscriptomaRESUMO
Vinylcyclopropanes (VCPs) are known to generate 1,3-dipoles with a palladium catalyst that initially serve as nucleophiles to undergo [3 + 2] cycloadditions with electron-deficient olefins. In this report, we reverse this reactivity and drive the 1,3-dipoles to serve as electrophiles by employing 3-alkylated indoles as nucleophiles. This represents the first use of VCPs for the completely atom-economic functionalization of 3-substituted 1 H-indoles and tryptophan derivatives via a Pd-catalyzed asymmetric allylic alkylation (Pd-AAA). Excellent yields and high chemo-, regio-, and enantioselectivities have been realized, providing various indolenine and indoline products. The method is amenable to gram scale and works efficiently with tryptophan derivatives that contain a diketopiperazine or diketomorpholine ring, allowing us to synthesize mollenine A in a rapid and ligand-controlled fashion. The obtained indolenine products bear an imine, an internal olefin, and a malonate motif, giving multiple sites with diverse reactivities for product diversification. Complicated polycyclic skeletons can be conveniently constructed by leveraging this unique juxtaposition of functional groups.
Assuntos
Compostos Alílicos/síntese química , Ciclopropanos/química , Indóis/química , Paládio/química , Triptofano/química , Alquilação , Compostos Alílicos/química , Catálise , Reação de Cicloadição , Estrutura MolecularRESUMO
Peroxisomal membrane proteins (PMPs) traffic to peroxisomes by two mechanisms: direct insertion from the cytosol into the peroxisomal membrane and indirect trafficking to peroxisomes via the endoplasmic reticulum (ER). In mammals and yeast, several PMPs traffic via the ER in a Pex3- and Pex19-dependent manner. In Komagataella phaffii (formerly called Pichia pastoris) specifically, the indirect traffic of Pex2, but not of Pex11 or Pex17, depends on Pex3, but all PMPs tested for indirect trafficking require Pex19. In mammals, the indirect traffic of PMPs also requires PEX16, a protein that is absent in most yeast species. In this study, we isolated PEX36, a new gene in K. phaffii, which encodes a PMP. Pex36 is required for cell growth in conditions that require peroxisomes for the metabolism of certain carbon sources. This growth defect in cells lacking Pex36 can be rescued by the expression of human PEX16, Saccharomyces cerevisiae Pex34, or by overexpression of the endogenous K. phaffii Pex25. Pex36 is not an essential protein for peroxisome proliferation, but in the absence of the functionally redundant protein, Pex25, it becomes essential and less than 20% of these cells show import-incompetent, peroxisome-like structures (peroxisome remnants). In the absence of both proteins, peroxisome biogenesis and the intra-ER sorting of Pex2 and Pex11C are seriously impaired, likely by affecting Pex3 and Pex19 function.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Peroxinas/metabolismo , Peroxissomos/metabolismo , Pichia/metabolismo , Proteínas Fúngicas/genética , Humanos , Proteínas de Membrana/genética , Peroxinas/genética , Pichia/crescimento & desenvolvimento , Transporte ProteicoRESUMO
Efficient synthesis of versatile building blocks for enabling medicinal chemistry research has always challenged synthetic chemists to develop innovative methods. Of particular interest are the methods that are amenable to the synthesis of chemically distinct and diverse classes of pharmaceutically relevant motifs. Herein we report a general method for the one-pot synthesis of cyclic α-amido-ethers containing different amide functionalities including lactams, tetramic acids and amino acids. For the incorporation of the nucleotide bases, a chemo and regioselective palladium-catalyzed transformation has been developed, providing rapid access to nucleoside analogs.
RESUMO
Enamides represent bioactive pharmacophores in various natural products, and have become increasingly common reagents for asymmetric incorporation of nitrogen functionality. Yet the synthesis of the requisite geometrically defined enamides remains problematic, especially for highly substituted and Z-enamides. Herein we wish to report a general atom economic method for the isomerization of a broad range of N-allyl amides to form Z-di-, tri-, and tetrasubstituted enamides with exceptional geometric selectivity. This report represents the first examples of a catalytic isomerization of N-allyl amides to form nonpropenyl disubstituted, tri- and tetrasubstituted enamides with excellent geometric control. Applications of these geometrically defined enamides toward the synthesis of cis vicinal amino alcohols and tetrasubstituted α-borylamido complexes are discussed.
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Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.
Assuntos
DNA Fúngico/genética , Genoma Fúngico/genética , Pichia/genética , Processamento Alternativo , Centrômero/genética , Engenharia Genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
The Ru-catalyzed alkene-alkyne coupling reaction has been demonstrated to be an enabling methodology for the synthesis of complex molecules. However, to date, it has been limited to monosubstituted olefins. Herein we report the first general utilization of disubstituted olefins in the Ru-catalyzed alkene-alkyne coupling reaction by employing carbamate directing groups. The products are stereodefined trisusbstituted enecarbamates. The elaboration of these structures toward the asymmetric synthesis of complex aminocyclopentitols and 1,2-amino alcohols is discussed.
RESUMO
Membrane proteins are the largest group of human drug targets and are also used as biocatalysts. However, due to their complexity, efficient expression remains a bottleneck for high level production. In recent years, the methylotrophic yeast Pichia pastoris has emerged as one of the most commonly used expression systems for membrane protein production. Here, we have analysed the transcriptomes of P. pastoris strains producing different classes of membrane proteins (mitochondrial, ER/Golgi and plasma membrane localized) to understand the cellular response and to identify targets to engineer P. pastoris towards an improved chassis for membrane protein production. Microarray experiments revealed varying transcriptional responses depending on the enzymatic activity, subcellular localization and physiological role of the membrane proteins. While an alternative oxidase evoked primarily a response within the mitochondria, the overexpression of transporters entering the secretory pathway had a wide effect on lipid metabolism and induced the upregulation of the UPR (unfolded protein response) transcription factor Hac1p. Coexpression of P. pastoris endogenous HAC1 increased the levels of ER-resident membrane proteins 1.5- to 2.1-fold. Subsequent transcriptome analysis of HAC1 coexpression revealed an upregulation of the folding machinery correlating with an expansion of the ER membrane capacity, thus boosting membrane protein production. Hence, our study has helped to elucidate the cellular response of P. pastoris to the expression of different classes of membrane proteins and led specifically to new insights into the effect of PpHac1p on membrane proteins entering the secretory pathway.
Assuntos
Proteínas de Membrana/biossíntese , Pichia/metabolismo , Transcrição Gênica , Proteínas de Fluorescência Verde , Proteínas de Membrana/genéticaRESUMO
Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.
Assuntos
Proteínas Fúngicas/metabolismo , Genes Fúngicos/fisiologia , Oxirredutases/metabolismo , Pichia/metabolismo , Esfingolipídeos/biossíntese , Proteínas Fúngicas/genética , Oxirredutases/genética , Pichia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Esfingolipídeos/genéticaRESUMO
Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35∆ mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/fisiologia , Proteínas Fúngicas/fisiologia , Pichia/imunologia , Proteínas de Transporte/genética , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Glucose/química , Metanol/química , Modelos Genéticos , Mutação , Peroxissomos/metabolismo , Pichia/metabolismo , Transporte Proteico , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismoRESUMO
This study evaluates the efficacy of green design principles such as the "12 Principles of Green Chemistry," and the "12 Principles of Green Engineering" with respect to environmental impacts found using life cycle assessment (LCA) methodology. A case study of 12 polymers is presented, seven derived from petroleum, four derived from biological sources, and one derived from both. The environmental impacts of each polymer's production are assessed using LCA methodology standardized by the International Organization for Standardization (ISO). Each polymer is also assessed for its adherence to green design principles using metrics generated specifically for this paper. Metrics include atom economy, mass from renewable sources, biodegradability, percent recycled, distance of furthest feedstock, price, life cycle health hazards and life cycle energy use. A decision matrix is used to generate single value metrics for each polymer evaluating either adherence to green design principles or life-cycle environmental impacts. Results from this study show a qualified positive correlation between adherence to green design principles and a reduction of the environmental impacts of production. The qualification results from a disparity between biopolymers and petroleum polymers. While biopolymers rank highly in terms of green design, they exhibit relatively large environmental impacts from production. Biopolymers rank 1, 2, 3, and 4 based on green design metrics; however they rank in the middle of the LCA rankings. Polyolefins rank 1, 2, and 3 in the LCA rankings, whereas complex polymers, such as PET, PVC, and PC place at the bottom of both ranking systems.
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Química Verde/métodos , Polímeros/síntese química , Biodegradação Ambiental , Conservação dos Recursos Naturais , Poluição Ambiental/prevenção & controle , Petróleo , Polienos/síntese química , Polienos/química , Polímeros/químicaRESUMO
In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an Deltahxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.
Assuntos
Oxirredutases do Álcool/metabolismo , Repressão Catabólica/fisiologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/metabolismo , Pichia/metabolismo , Autofagia/fisiologia , Sequência de Bases , Western Blotting , Primers do DNA/genética , Eletroporação , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Peroxissomos/metabolismo , Pichia/fisiologia , Plasmídeos/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae , Análise de Sequência de DNARESUMO
The yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth. This chapter assumes the reader is proficient in molecular biology and details the more yeast specific procedures involved in utilizing the P. pastoris system for gene expression. Procedures to be found here include: strain construction by classical yeast genetics, the logic in selection of a vector and strain, preparation of electrocompetent yeast cells and transformation by electroporation, and the yeast colony western blot or Yeastern blot method for visualizing secreted proteins around yeast colonies.
Assuntos
Técnicas Microbiológicas/métodos , Pichia/genética , Proteínas Recombinantes/genética , Animais , Clonagem Molecular/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Modelos Genéticos , Pichia/química , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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 psd1Delta and psd2Delta 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.
Assuntos
Carboxiliases/genética , Carboxiliases/metabolismo , Fosfatidiletanolaminas/metabolismo , Pichia/enzimologia , Sequência de Aminoácidos , Membrana Celular/química , Ácidos Graxos/análise , Deleção de Genes , Redes e Vias Metabólicas/genética , Modelos Biológicos , Dados de Sequência Molecular , Filogenia , Pichia/química , Pichia/genética , Pichia/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5-6% of selected clones and had a proportional increase in recombinant protein.
Assuntos
Farmacorresistência Fúngica/genética , Dosagem de Genes , Engenharia Genética/métodos , Marcadores Genéticos/genética , Vetores Genéticos/genética , Pichia/genética , Transformação Genética , Bleomicina/farmacologia , Eletroporação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Pichia/efeitos dos fármacos , Pichia/enzimologia , Pichia/crescimento & desenvolvimento , Seleção Genética , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.