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Methanol-essential growth of Escherichia coli.

ABSTRACT: Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically coupled to the metabolization of gluconate and the designed strain was subjected to laboratory evolution experiments. Evolved strains incorporate up to 24% methanol into core metabolites under a co-consumption regime and utilize methanol at rates comparable to natural methylotrophs. Genome sequencing reveals mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.

SUBMITTER: Meyer F

PROVIDER: S-EPMC5904121 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Methanol-essential growth of Escherichia coli.

Meyer Fabian F Keller Philipp P Hartl Johannes J Gröninger Olivier G OG Kiefer Patrick P Vorholt Julia A JA

Nature communications 20180417 1

Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically c ...[more]

PMID: 29666370

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Project description:BACKGROUND:Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS:Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (?speB, ?speC, ?speF, ?speB/C and ?speD/E), and in polyamines transport systems (?potE, ?yeeF, ?potABCD and ?potFGHI). Contrary to what was observed for MG1655, the ?potE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ?speB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ?speB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS:Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

Project description:Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCE Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.

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Methanol-essential growth of Escherichia coli.

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Methanol-essential growth of Escherichia coli.

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