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1.
Mol Cell Proteomics ; 18(5): 1027-1035, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833379

RESUMO

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Agregados Proteicos , Proteômica/métodos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Células RAW 264.7
2.
J Biol Chem ; 294(45): 17117-17130, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31471321

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.


Assuntos
Histidina , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Ligantes , Oxigenases de Função Mista/genética , Modelos Moleculares , Fosforilação , Filogenia
3.
J Proteome Res ; 17(11): 4008-4016, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30220210

RESUMO

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT), which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here, we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments, relying on isobaric TMT reporter ion quantification.


Assuntos
Peptídeos/análise , Proteoma/isolamento & purificação , Proteômica/métodos , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/química , Células Epiteliais/citologia , Células HeLa , Humanos , Íons , Células Jurkat , Neurônios/química , Neurônios/patologia , Osteoblastos/química , Osteoblastos/patologia , Proteólise , Proteoma/genética , Proteoma/metabolismo , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/citologia
4.
Metab Eng ; 28: 123-133, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25554074

RESUMO

Targeted proteomics is a convenient method determining enzyme expression levels, but a quantitative analysis of these proteomic data has not been fully explored yet. Here, we present and demonstrate a computational tool (principal component analysis of proteomics, PCAP) that uses quantitative targeted proteomics data to guide metabolic engineering and achieve higher production of target molecules from heterologous pathways. The method is based on the application of principal component analysis to a collection of proteomics and target molecule production data to pinpoint specific enzymes that need to have their expression level adjusted to maximize production. We illustrated the method on the heterologous mevalonate pathway in Escherichia coli that produces a wide range of isoprenoids and requires balanced pathway gene expression for high yields and titers. PCAP-guided engineering resulted in over a 40% improvement in the production of two valuable terpenes. PCAP could potentially be productively applied to other heterologous pathways as well.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica/métodos , Proteômica , Terpenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética
5.
J Proteome Res ; 13(12): 6176-86, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25338131

RESUMO

Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC-MS/MS gradients. From 14 HpH fractions, we routinely identified over 30,000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404).


Assuntos
Cromatografia de Fase Reversa/métodos , Fosfopeptídeos/análise , Proteoma/análise , Proteômica/métodos , Métodos Analíticos de Preparação de Amostras , Animais , Resinas de Troca de Cátion , Fracionamento Químico , Cromatografia por Troca Iônica/métodos , Cromatografia Líquida/métodos , Concentração de Íons de Hidrogênio , Camundongos , Células NIH 3T3 , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Titânio
6.
J Proteome Res ; 13(12): 6187-95, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25349961

RESUMO

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 µg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Benchmarking/métodos , Fracionamento Químico , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Peptídeos/metabolismo , Proteoma/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de Proteína/métodos
7.
Metab Eng ; 26: 48-56, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25205128

RESUMO

Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Peptídeos/genética , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos
8.
Biotechnol Bioeng ; 111(8): 1648-58, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24615242

RESUMO

The ability to rapidly assess and optimize heterologous pathway function is critical for effective metabolic engineering. Here, we develop a systematic approach to pathway analysis based on correlations between targeted proteins and metabolites and apply it to the microbial production of isopentenol, a promising biofuel. Starting with a seven-gene pathway, we performed a correlation analysis to reduce pathway complexity and identified two pathway proteins as the primary determinants of efficient isopentenol production. Aided by the targeted quantification of relevant pathway intermediates, we constructed and subsequently validated a conceptual model of isopentenol pathway function. Informed by our analysis, we assembled a strain which produced isopentenol at a titer 1.5 g/L, or 46% of theoretical yield. Our engineering approach allowed us to accurately identify bottlenecks and determine appropriate pathway balance. Paired with high-throughput cloning techniques and analytics, this strategy should prove useful for the analysis and optimization of increasingly complex heterologous pathways.


Assuntos
Biocombustíveis/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Pentanóis/metabolismo , Acetatos/metabolismo , Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Glucose/metabolismo , Modelos Biológicos , Proteômica/métodos
9.
Appl Microbiol Biotechnol ; 98(4): 1567-81, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24257840

RESUMO

The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5' of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.


Assuntos
Ribossomos/metabolismo , Sítios de Ligação , Carboxiliases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo
10.
Metab Eng ; 19: 33-41, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23727191

RESUMO

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.


Assuntos
Antineoplásicos/metabolismo , Cicloexenos/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica , Monoterpenos/metabolismo , Terpenos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidroxilação/genética , Limoneno , Ácido Mevalônico/metabolismo
11.
Plant Physiol ; 159(1): 12-26, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22430844

RESUMO

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.


Assuntos
Arabidopsis/metabolismo , Parede Celular/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteoma/isolamento & purificação , Apirase/genética , Apirase/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , Ensaios Enzimáticos , Genes de Plantas , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/ultraestrutura , Immunoblotting , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Células Vegetais/enzimologia , Células Vegetais/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Pirofosfatases/genética , Pirofosfatases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Proc Natl Acad Sci U S A ; 107(11): 4967-72, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20194787

RESUMO

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.


Assuntos
Chaperonina com TCP-1/química , Microscopia Crioeletrônica , Subunidades Proteicas/química , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes , Eletricidade Estática , Propriedades de Superfície
13.
Nat Commun ; 14(1): 4202, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37452022

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.


Assuntos
Histidina , Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Histidina/genética , Histidina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional
14.
Proteomics ; 12(8): 1289-99, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22577029

RESUMO

As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas Fúngicas/análise , Engenharia Metabólica/métodos , Plasmídeos/genética , Proteômica/métodos , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Variação Genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Ácido Mevalônico/metabolismo , Óperon/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/química , Transformação Bacteriana , Tirosina/biossíntese , Tirosina/genética , Proteína Vermelha Fluorescente
15.
Metab Eng ; 14(6): 653-60, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23026122

RESUMO

Fatty acids are important precursors to biofuels. The Escherichia coli FadR is a transcription factor that regulates several processes in fatty acid biosynthesis, degradation, and membrane transport. By tuning the expression of FadR in an engineered E. coli host, we were able to increase fatty acid titer by 7.5-fold over our previously engineered fatty acid-producing strain, reaching 5.2±0.5g/L and 73% of the theoretical yield. The mechanism by which FadR enhanced fatty acid yield was studied by whole-genome transcriptional analysis (microarray) and targeted proteomics. Overexpression of FadR led to transcriptional changes for many genes, including genes involved in fatty acid pathways. The biggest transcriptional changes in fatty acid pathway genes included fabB, fabF, and accA. Overexpression of any of these genes alone did not result in a high yield comparable to fadR expression, indicating that FadR enhanced fatty acid production globally by tuning the expression levels of many genes to optimal levels.


Assuntos
Proteínas de Bactérias/fisiologia , Escherichia coli/fisiologia , Ácido Graxo Sintase Tipo II/genética , Ácidos Graxos/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Melhoramento Genético/métodos , Elementos Reguladores de Transcrição/genética , Proteínas Repressoras/fisiologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase , Acetil-CoA Carboxilase , Acetiltransferases , Proteínas de Escherichia coli , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/isolamento & purificação
16.
Appl Environ Microbiol ; 78(7): 2221-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22286982

RESUMO

Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy of xylR was inserted into the araBAD operon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of an E. coli biocatalyst that can simultaneously convert all biomass sugars.


Assuntos
Arabinose/metabolismo , Biotecnologia/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Polissacarídeos/metabolismo , Fatores de Transcrição/metabolismo , Xilose/metabolismo , Biocombustíveis , Meios de Cultura/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Etanol/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Polissacarídeos/química , Fatores de Transcrição/genética
17.
Appl Environ Microbiol ; 78(1): 89-98, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22020510

RESUMO

Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Tirosina/biossíntese , Oxirredutases do Álcool/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Proteômica , Fosfatos Açúcares/metabolismo
18.
Commun Biol ; 5(1): 150, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35194133

RESUMO

Multiplexing approaches using tandem mass tags with a carrier proteome to boost sensitivity have advanced single cell proteomics by mass spectrometry (SCoPE-MS). Here, we probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided.


Assuntos
Proteoma , Proteômica , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
19.
Cells ; 11(23)2022 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-36497055

RESUMO

Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10-200 mGy).


Assuntos
Quebras de DNA de Cadeia Dupla , Radiação Ionizante , Pontos de Checagem da Fase G2 do Ciclo Celular , Espécies Reativas de Oxigênio , Transdução de Sinais
20.
J Proteome Res ; 10(4): 1571-82, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21166475

RESUMO

The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana was isolated and analyzed using offline strong cation exchange liquid chromatography and LC-MS/MS. This generated a robust set of 1071 cytosolic proteins. Functional annotation of this set revealed major activities in protein synthesis and degradation, RNA metabolism and basic sugar metabolism. This included an array of important cytosol-related functions, specifically the ribosome, the set of tRNA catabolic enzymes, the ubiquitin-proteasome pathway, glycolysis and associated sugar metabolism pathways, phenylpropanoid biosynthesis, vitamin metabolism, nucleotide metabolism, an array of signaling and stress-responsive molecules, and NDP-sugar biosynthesis. This set of cytosolic proteins provides for the first time an extensive analysis of enzymes responsible for the myriad of reactions in the Arabidopsis cytosol and defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol.


Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/química , Citosol/química , Proteoma/análise , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Resinas de Troca de Cátion , Cromatografia Líquida/métodos , Biologia Computacional , Espectrometria de Massas/métodos , Redes e Vias Metabólicas/fisiologia , Dados de Sequência Molecular
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