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Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

ABSTRACT: Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

SUBMITTER: Ruiz JA

PROVIDER: S-EPMC3962086 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

Ruiz Jimena A JA de Almeida Alejandra A Godoy Manuel S MS Mezzina Mariela P MP Bidart Gonzalo N GN Méndez Beatriz S BS Pettinari M Julia MJ Nikel Pablo I PI

Computational and structural biotechnology journal 20120101

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction s ...[more]

PMID: 24688679

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Project description:BackgroundCrocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis.ResultsIn this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS.ConclusionsA heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Project description:BackgroundThe modification of glucose import capacity is an engineering strategy that has been shown to improve the characteristics of Escherichia coli as a microbial factory. A reduction in glucose import capacity can have a positive effect on production strain performance, however, this is not always the case. In this study, E. coli W3110 and a group of four isogenic derivative strains, harboring single or multiple deletions of genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent transporters as well as non-PTS transporters were characterized by determining their transcriptomic response to reduced glucose import capacity.ResultsThese strains were grown in bioreactors with M9 mineral salts medium containing 20 g/L of glucose, where they displayed specific growth rates ranging from 0.67 to 0.27 h-1, and specific glucose consumption rates (qs) ranging from 1.78 to 0.37 g/g h. RNA-seq analysis revealed a transcriptional response consistent with carbon source limitation among all the mutant strains, involving functions related to transport and metabolism of alternate carbon sources and characterized by a decrease in genes encoding glycolytic enzymes and an increase in gluconeogenic functions. A total of 107 and 185 genes displayed positive and negative correlations with qs, respectively. Functions displaying positive correlation included energy generation, amino acid biosynthesis, and sugar import.ConclusionChanges in gene expression of E. coli strains with impaired glucose import capacity could be correlated with qs values and this allowed an inference of the physiological state of each mutant. In strains with lower qs values, a gene expression pattern is consistent with energy limitation and entry into the stationary phase. This physiological state could explain why these strains display a lower capacity to produce recombinant protein, even when they show very low rates of acetate production. The comparison of the transcriptomes of the engineered strains employed as microbial factories is an effective approach for identifying favorable phenotypes with the potential to improve the synthesis of biotechnological products.

Project description:Manipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary under different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra, and Rob using single deletion mutants of the well-characterized and completely sequenced Escherichia coli strain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds, and synthesis of native and nonnative bioproducts under different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3-propanediol (1,3-PDO). The Δcra mutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO2 availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the ΔarcA mutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semidefined medium using glycerol, resulting in 24 g · liter-1 of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g · liter-1 h-1 IMPORTANCE Although the effects of many global regulators, especially ArcA and Cra, have been studied in Escherichia coli, the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, which is essential for the design of metabolic engineering strategies. The genome of Escherichia coli BW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion, eliminating possible strain-dependent results. As a proof of concept, Δcra and ΔarcA mutants were subjected to further manipulations to obtain large amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts.

Project description:BACKGROUND: Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. RESULTS: Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far. CONCLUSIONS: This study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield.

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Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

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Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

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