ABSTRACT
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.
Subject(s)
Histidine , Mixed Function Oxygenases , Mixed Function Oxygenases/metabolism , Histidine/genetics , Histidine/metabolism , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Polysaccharides/metabolism , Protein Processing, Post-TranslationalABSTRACT
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).
Subject(s)
DNA Breaks, Double-Stranded , Radiation, Ionizing , G2 Phase Cell Cycle Checkpoints , Reactive Oxygen Species , Signal TransductionABSTRACT
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.
Subject(s)
Proteome , Proteomics , Proteome/metabolism , Proteomics/methods , Tandem Mass Spectrometry/methodsABSTRACT
Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a five-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases could also target the factors inducing their upregulation rather than their activity. This study, therefore, presents markers that could help predict AML relapse and direct therapeutic strategies.
ABSTRACT
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.
Subject(s)
Histidine , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Polysaccharides/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Ligands , Mixed Function Oxygenases/genetics , Models, Molecular , Phosphorylation , PhylogenyABSTRACT
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.
Subject(s)
Cell-Derived Microparticles/metabolism , Protein Aggregates , Proteomics/methods , Animals , Cell Line, Tumor , Humans , Mice , Protein Processing, Post-Translational , RAW 264.7 CellsABSTRACT
Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner. Similar to TTP, ZNF598 harbors three proline-rich motifs that bind the GYF domain of GIGYF1. RNA sequencing experiments showed that ZNF598 is required for the regulation of known TTP targets, including IL-8 and CSF2 mRNA. Furthermore, we demonstrate that ZNF598 binds to IL-8 mRNA, but not TNF mRNA. Collectively, our findings highlight that ZNF598 functions as an RBP that buffers the level of a range of mRNAs. We propose that ZNF598 is a TTP-like factor that can contribute to the regulation of the inflammatory potential of cytokine-producing cells.
Subject(s)
Carrier Proteins/metabolism , Inflammation/metabolism , RNA Processing, Post-Transcriptional , Signal Transduction , Tristetraprolin/metabolism , Amino Acid Motifs , Animals , Cell Line, Tumor , Cytokines/metabolism , Humans , Inflammation/genetics , Protein Binding , RNA, Messenger/metabolismABSTRACT
PURPOSE: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood.Experimental Design: We applied mass spectrometry-based proteomics to a unique set of 17 prostate cancer patient-derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation. RESULTS: An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy. CONCLUSIONS: This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.
Subject(s)
Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/metabolism , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Proteogenomics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Animals , Carcinoma, Neuroendocrine/pathology , Cell Cycle/genetics , Cell Line, Tumor , Computational Biology/methods , Disease Models, Animal , Disease Susceptibility , Gene Expression Profiling , Heterografts , Humans , Male , Mice , Proportional Hazards Models , Prostatectomy , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/surgery , Proteogenomics/methodsABSTRACT
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.
Subject(s)
Peptides/analysis , Proteome/isolation & purification , Proteomics/methods , Staining and Labeling/methods , Tandem Mass Spectrometry/methods , Cell Line , Cell Line, Tumor , Epithelial Cells/chemistry , Epithelial Cells/cytology , HeLa Cells , Humans , Ions , Jurkat Cells , Neurons/chemistry , Neurons/pathology , Osteoblasts/chemistry , Osteoblasts/pathology , Proteolysis , Proteome/genetics , Proteome/metabolism , Retinal Pigment Epithelium/chemistry , Retinal Pigment Epithelium/cytologyABSTRACT
Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ßß, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks.
Subject(s)
Phosphoproteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proteomics , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , Animals , Antibodies/metabolism , Becaplermin/pharmacology , Enzyme Activation , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Ligands , Mice , NIH 3T3 Cells , Phosphorylation , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Proteome/metabolismABSTRACT
This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of â¼584,000 unique peptide sequences and â¼14,200 protein isoforms (â¼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including â¼7,000 N-acetylation sites and â¼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.
Subject(s)
Proteome/metabolism , Proteomics/methods , A549 Cells , Acetylation , Cell Line , Cell Line, Tumor , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Mass Spectrometry/methods , Peptides/metabolism , Protein Isoforms/metabolism , Protein Processing, Post-Translational/physiology , Proteins/metabolismABSTRACT
SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2-/- mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.
Subject(s)
Colitis/metabolism , NF-kappa B/metabolism , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/chemistry , Proteomics/methods , Suppressor of Cytokine Signaling Proteins/metabolism , Animals , Cell Line, Tumor , Colitis/genetics , Disease Models, Animal , Enzyme Stability , Gene Expression Regulation , HEK293 Cells , Humans , Male , Mass Spectrometry , Mice , Protein Serine-Threonine Kinases/metabolism , Suppressor of Cytokine Signaling Proteins/deficiency , Transcriptional Activation , Tumor Necrosis Factor-alpha/metabolism , UbiquitinationABSTRACT
Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proof of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks.
Subject(s)
Escherichia coli , Biofuels , Computational Biology , Escherichia coli Proteins , Metabolic Engineering , Models, Biological , Synthetic Biology , WorkflowABSTRACT
Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis.
Subject(s)
Chromatography, Reverse-Phase , Phosphoproteins/analysis , Protein Kinases/metabolism , Proteomics/methods , Animals , Chromatography, Affinity , Chromatography, High Pressure Liquid , Computational Biology , Databases, Protein , Humans , Hydrogen-Ion Concentration , Mass Spectrometry , Peptide Mapping , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Processing, Post-Translational , Substrate Specificity , Titanium/chemistry , WorkflowABSTRACT
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.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Gene Expression Regulation, Bacterial , Metabolic Engineering/methods , Proteomics , Terpenes/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/geneticsABSTRACT
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.
Subject(s)
Mass Spectrometry/methods , Peptides/analysis , Proteome/analysis , Proteomics/methods , Benchmarking/methods , Chemical Fractionation , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , HeLa Cells , Humans , Hydrogen-Ion Concentration , Peptides/metabolism , Proteome/metabolism , Reproducibility of Results , Sequence Analysis, Protein/methodsABSTRACT
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).
Subject(s)
Chromatography, Reverse-Phase/methods , Phosphopeptides/analysis , Proteome/analysis , Proteomics/methods , Analytic Sample Preparation Methods , Animals , Cation Exchange Resins , Chemical Fractionation , Chromatography, Ion Exchange/methods , Chromatography, Liquid/methods , Hydrogen-Ion Concentration , Mice , NIH 3T3 Cells , Phosphopeptides/metabolism , Phosphoproteins/analysis , Phosphoproteins/metabolism , Phosphorylation , Proteome/metabolism , Reproducibility of Results , Tandem Mass Spectrometry/methods , TitaniumABSTRACT
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.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Gene Expression Profiling/methods , High-Throughput Screening Assays/methods , Peptides/metabolism , Protein Engineering/methods , Peptides/genetics , Protein Interaction Mapping/methods , Proteomics/methodsABSTRACT
The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.
Subject(s)
Escherichia coli/drug effects , Ionic Liquids/pharmacology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Promoter Regions, Genetic , RNA, Bacterial/genetics , TranscriptomeABSTRACT
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.