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.
•We report limonene production in E. coli using a heterologous mevalonate pathway.•Perillyl alcohol was produced in E. coli from endogenously generated limonene.•Limonene and perillyl alcohol titers were highly improved by metabolic engineering.•An anion exchange resin was used to improve specific recovery of perillyl alcohol.
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).
The phenomenon of protein aggregation capture (PAC) on a wide range of different microparticles is described. Exploiting this mechanism enables generation of clean peptide mixtures from cell lines, ...tissues, and protein pulldowns for proteomics, phosphoproteomics, and secretomics analysis. The findings vastly increase the accessibility of the method that may ultimately lead to low cost and automated proteomics workflows.
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Highlights
•Insoluble protein aggregates preferentially precipitate on microparticles.•The protein aggregation capture (PAC) occurs irrespective of microparticle surface chemistry.•This process can be exploited for multi-purpose proteomics sample preparation.•Facilitates potential for low cost, efficient and high-sensitivity proteomics workflows.
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.
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.
► FadR overexpression increased fatty acid yield by 7.5-fold under optimal condition. ► We produced 5.2g/L fatty acids, reaching 73% of theoretical yield. ► FadR expression leads to many transcriptional changes. ► Expression of single gene examined did not result in a high fatty acid yield. ► FadR enhanced fatty acid production by tuning the expression levels of many genes.
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.
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 ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼10,000 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.
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•Multi-shot proteomics quantifies the protein levels of 12,200+ genes in HeLa cells•This essentially complete HeLa proteome has coverage similar to next-gen RNA-seq•Deep coverage of major PTMs is achieved without specific enrichment•The approach is extendable to other human cell lines and patient samples
Bekker-Jensen et al. show that proteomics can now provide an essentially complete HeLa proteome. They provide measurements acquired simultaneously for more than 12,200 protein-coding genes, 10,000 phosphorylation sites, and 7,000 N-acetylation sites. Their approach is fast, accessible, and requires modest amounts of starting material, making it easily extendable to other human cell lines and patient samples.
Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a ...strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.
Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems ...due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in
Escherichia coli strains engineered to produce the sesquiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from
Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoter led to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer (>500
mg/L).
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.
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•We report quantitative analysis method for proteomics data using PCA.•Principal component analysis of proteomics (PCAP) can direct metabolic engineering.•PCAP provides information not apparent by the simple observation of proteomics data.•PCAP was successfully applied to improve the mevalonate pathway (MEV) in E. coli.•Limonene and bisabolene production increased about 40% using PCAP-based strategy.
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