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Engineering glucose metabolism of Escherichia coli under nitrogen starvation.

ABSTRACT: A major aspect of microbial metabolic engineering is the development of chassis hosts that have favorable global metabolic phenotypes, and can be further engineered to produce a variety of compounds. In this work, we focus on the problem of decoupling growth and production in the model bacterium Escherichia coli, and in particular on the maintenance of active metabolism during nitrogen-limited stationary phase. We find that by overexpressing the enzyme PtsI, a component of the glucose uptake system that is inhibited by α-ketoglutarate during nitrogen limitation, we are able to achieve a fourfold increase in metabolic rates. Alternative systems were also tested: chimeric PtsI proteins hypothesized to be insensitive to α-ketoglutarate did not improve metabolic rates under the conditions tested, whereas systems based on the galactose permease GalP suffered from energy stress and extreme sensitivity to expression level. Overexpression of PtsI is likely to be a useful arrow in the metabolic engineer's quiver as productivity of engineered pathways becomes limited by central metabolic rates during stationary phase production processes.

SUBMITTER: Chubukov V

PROVIDER: S-EPMC5516864 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Engineering glucose metabolism of <i>Escherichia coli</i> under nitrogen starvation.

Chubukov Victor V Desmarais John James JJ Wang George G Chan Leanne Jade G LJG Baidoo Edward Ek EE Petzold Christopher J CJ Keasling Jay D JD Mukhopadhyay Aindrila A

NPJ systems biology and applications 20170105

A major aspect of microbial metabolic engineering is the development of chassis hosts that have favorable global metabolic phenotypes, and can be further engineered to produce a variety of compounds. In this work, we focus on the problem of decoupling growth and production in the model bacterium <i>Escherichia coli</i>, and in particular on the maintenance of active metabolism during nitrogen-limited stationary phase. We find that by overexpressing the enzyme PtsI, a component of the glucose upt ...[more]

PMID: 28725483

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Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

Project description:Microbial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individual species' physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris In this coculture, E. coli anaerobically ferments sugars into excreted organic acids as a carbon source for R. palustris In return, a genetically engineered R. palustris strain constitutively converts N2 into NH4+, providing E. coli with essential nitrogen. Using transcriptome sequencing (RNA-seq) and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture with R. palustris, E. coli gene expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disrupting E. coli NtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at low R. palustris NH4+ excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and, in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.IMPORTANCE Mutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting of Rhodopseudomonas palustris and Escherichia coli growing cooperatively through bidirectional nutrient exchange, we determined that an E. coli nitrogen starvation response is important for maintaining a stable coexistence. The lack of an E. coli nitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

Dataset Information

Engineering glucose metabolism of Escherichia coli under nitrogen starvation.

Publications

Engineering glucose metabolism of <i>Escherichia coli</i> under nitrogen starvation.

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