Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

Sánchez-Pascuala, Alberto; Fernández-Cabezón, Lorena; Lorenzo, Vı́ctor de; Nikel, Pablo I.

doi:10.1016/j.ymben.2019.04.005

Cited by 61 publications

(48 citation statements)

References 94 publications

(89 reference statements)

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“…glycolytic conditions). In a Δ glk mutant of strain KT2440, the bulk of hexoses (80–90%) is oxidized into gluconate, whereas in a Δ gcd mutant, sugars are exclusively phosphorylated to glucose‐6‐ P (Dvořák et al ., ; Sánchez‐Pascuala et al ., , ). Once again, cultures of P. putida KT2440 grown in the aforementioned medium supplemented with CCCP were used to calculate the calibration curve for pH i quantification in all strains (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The oxidation of glucose in the periplasm yields low‐molecular‐weight organic acids, e.g. gluconate and 2‐ketogluconate (Hirshfield et al ., ; Sánchez‐Pascuala et al ., ). Hence, we set to study how the use of this peripheral pathway influences the pH i in strain KT2440 and its glycolytic mutant derivatives.…”

Section: Resultsmentioning

confidence: 99%

“…coli DH5α was used both for the propagation and construction of plasmids and for pH i calculations, along with the well‐characterized K‐12 strain MG1655 (Blattner et al ., ). The P. putida Δ glk and Δ gcd mutants are derivatives of the parental strain KT2440 with specific mutations in the genes encoding the enzymes that execute the first steps of glucose utilization (Sánchez‐Pascuala et al ., , ). P. aeruginosa CH2682 is a meropenem‐resistant clinical strain isolated from a patient with a rectal infection (Hornischer et al ., ).…”

Section: Methodsmentioning

confidence: 99%

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Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

Arce-Rodríguez

Volke

Bense

et al. 2019

Microbial Biotechnology

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Summary The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH (pHi) homoeostasis is a particularly important feature, since the pHi influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy‐to‐implement tools have been designed for quantifying in vivo pHi changes in Gram‐negative bacteria with minimal manipulations. Here we describe a convenient, non‐invasive protocol for the quantification of the pHi in bacteria, which is based on the ratiometric fluorescent indicator protein PHP (pH indicator for Pseudomonas). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP‐based quantification method demonstrated that pHi is tightly regulated over a narrow range of pH values not only in Pseudomonas, but also in other Gram‐negative bacterial species such as Escherichia coli. The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P. putida) and external (e.g. antibiotic exposure in P. aeruginosa) perturbations, and the PHP indicator was also used to follow dynamic changes in the pHi upon external pH shifts. In summary, our work describes a reliable method for measuring pHi in Pseudomonas, allowing for the detailed investigation of bacterial pHi homoeostasis and its regulation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

Arce-Rodríguez

Volke

Bense

et al. 2019

Microbial Biotechnology

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“…For example, a number of genes related to genomic instability (about 4.3 % of the genome) were deleted in KT2440 that resulted in cell-factory strains EM42 and EM383 (a derivative of EM42 deleted for recA gene) with significant increase in ATP and NAD(P)H availability and faster growth of bacteria, reduced sensitivity to oxidative stress and increased expression of heterologous genes [32,123]. Most recently, the glucose catabolism was deeply rewired in P. putida by implanting linear EMP glycolysis route, and the strains with synthetic glycolysis were further engineered for the improved carotenoid synthesis from glucose [124]. The results of this study clearly demonstrated that conserved metabolic features in a platform bacterium can be successfully reshaped for specific biotechnological purposes.…”

Section: P Putida As a Host For Industrial Biocatalysismentioning

confidence: 99%

Narrative of a versatile and adept species Pseudomonas putida

Kivisaar

2020

Journal of Medical Microbiology

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Pseudomonas putida is a fast-growing bacterium found mostly in temperate soil and water habitats. The metabolic versatility of P. putida makes this organism attractive for biotechnological applications such as biodegradation of environmental pollutants and synthesis of added-value chemicals (biocatalysis). This organism has been extensively studied in respect to various stress responses, mechanisms of genetic plasticity and transcriptional regulation of catabolic genes. P. putida is able to colonize the surface of living organisms, but is generally considered to be of low virulence. A number of P. putida strains are able to promote plant growth. The aim of this review is to give historical overview of the discovery of the species P. putida and isolation and characterization of P. putida strains displaying potential for biotechnological applications. This review also discusses some major findings in P. putida research encompassing regulation of catabolic operons, stress-tolerance mechanisms and mechanisms affecting evolvability of bacteria under conditions of environmental stress.

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“…Thereafter, pathway modules have been edited for increasing bioproduction of several molecules, including isoprenoids (48,167,212,(253)(254)(255). Moreover, exclusive exploitation of non-native modules allowed to reduce endogenous control over biochemical routes (256,257). As proof of principle, we assessed if this approach could be applied also to isoprenoid metabolism.…”

Section: Modular Pathway Engineering Allows To Replace Isoprenoid Biomentioning

confidence: 99%

Racing-red chassis : Advancing Rhodobacter sphaeroides as platform for isoprenoid production

Orsi¹

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Diminishing the turnaround time of DBTL cycles is a crucial aspect for accelerating the development of microbial chassis (31, 32). Cutting edge advances in fields like systems biology (33), machine learning (34), metabolomics (35), automation (31), or genome engineering (36-38) can contribute to accelerate one or more parts of the DBTL cycle. Biofoundries have been realized, in which automated and high-throughput DBTL cycles are integrated for rapidly and efficiently reprogramming cell factories (31, 39). While rapid and standardized DBTL cycles can be performed for model organisms like E. coli or S. cerevisiae, their use for non-model microorganisms is not trivial (15). For example, potentially interesting chassis might lack genetic accessibility, which would render the 'build' phase cumbersome. Also, the metabolic pathways supporting product formation could be unknown. Therefore, implementation of DBTL cycles in non-traditional microorganisms is an intriguing challenge for biotechnology. Succeeding in such a task would allow to investigate and assess non-traditional microorganisms as potential cell factories. Isoprenoid compounds: a successful example for the bioeconomy obtained via DBTL cycles The case of semi-synthetic artemisinin synthesis in yeast Probably the most scientifically relevant achievement obtained via DBTL cycles is the microbial synthesis of the antimalarial drug artemisinin (40). Such a compound is natively produced by the plant Artemisia annua, but dependence on yearly harvesting makes its production challenging (41). Therefore, alternatives like microbial synthesis were explored for feasible and costcompetitive artemisinin production (40, 41). This endeavour started with the implementation of a heterologous production pathway in E. coli (42), and was concluded a decade later by producing up to 25 g/L of artemisinic acid in Fld/Fd red Fld/Fd ox

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Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

Cited by 61 publications

References 94 publications

Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

Narrative of a versatile and adept species Pseudomonas putida

Racing-red chassis : Advancing Rhodobacter sphaeroides as platform for isoprenoid production

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