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/metabolismoRESUMO
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 SuperfícieRESUMO
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/metabolismoRESUMO
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 MolecularRESUMO
Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD(+)-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.
Assuntos
Proteínas de Bactérias , Delftia acidovorans , Escherichia coli , Hidroximetilglutaril-CoA Redutases NAD-Dependentes/biossíntese , Ácido Mevalônico/metabolismo , Organismos Geneticamente Modificados , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Candida/enzimologia , Candida/genética , Delftia acidovorans/enzimologia , Delftia acidovorans/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Formiato Desidrogenases/biossíntese , Formiato Desidrogenases/genética , Formiatos/metabolismo , Formiatos/farmacologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Hidroximetilglutaril-CoA Redutases NAD-Dependentes/genética , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Organismos Geneticamente Modificados/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sesquiterpenos Policíclicos , Sesquiterpenos/metabolismoRESUMO
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).
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Redes e Vias Metabólicas/genética , Proteômica/métodos , Sesquiterpenos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Ácido Mevalônico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Sesquiterpenos Policíclicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
To understand how sulphate-reducing bacteria respond to oxidative stresses, the responses of Desulfovibrio vulgaris Hildenborough to H(2)O(2)-induced stresses were investigated with transcriptomic, proteomic and genetic approaches. H(2)O(2) and induced chemical species (e.g. polysulfide, ROS) and redox potential shift increased the expressions of the genes involved in detoxification, thioredoxin-dependent reduction system, protein and DNA repair, and decreased those involved in sulfate reduction, lactate oxidation and protein synthesis. A gene coexpression network analysis revealed complicated network interactions among differentially expressed genes, and suggested possible importance of several hypothetical genes in H(2)O(2) stress. Also, most of the genes in PerR and Fur regulons were highly induced, and the abundance of a Fur regulon protein increased. Mutant analysis suggested that PerR and Fur are functionally overlapped in response to stresses induced by H(2)O(2) and reaction products, and the upregulation of thioredoxin-dependent reduction genes was independent of PerR or Fur. It appears that induction of those stress response genes could contribute to the increased resistance of deletion mutants to H(2)O(2)-induced stresses. In addition, a conceptual cellular model of D. vulgaris responses to H(2)O(2) stress was constructed to illustrate that this bacterium may employ a complicated molecular mechanism to defend against the H(2)O(2)-induced stresses.
Assuntos
Desulfovibrio vulgaris/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo , Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/metabolismo , Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteoma/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Perfilação da Expressão Gênica/métodos , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Estresse Fisiológico/genética , Biologia de Sistemas/métodos , Análise por Conglomerados , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sesquiterpenos Policíclicos , Fosfatos de Poli-Isoprenil , Sesquiterpenos/metabolismoRESUMO
Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism.