Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida

Kukurugya, Matthew A.; Mendonca, Caroll M.; Solhtalab, Mina; Wilkes, Rebecca A.; Thannhauser, Theodore W.; Aristilde, Ludmilla

doi:10.1074/jbc.ra119.007885

Cited by 60 publications

(93 citation statements)

References 54 publications

(96 reference statements)

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“…Several earlier studies described the systems-level cellular adjustments that accompany the growth of industrially relevant model organisms such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, Corynebacterium glutamicum, Basfia succiniciproducens, and P. putida in single carbon sources (59)(60)(61)(62)(63)(64). However, and although the accrued metabolic models have made a valuable contribution toward our "textbook understanding" of microbial metabolism, it is clear that they can be extrapolated to other organisms, such as P. aeruginosa, only loosely (65).…”

Section: Discussionmentioning

confidence: 99%

Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

Dolan

Kohlstedt

Trigg

et al. 2020

mBio

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Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized “EDEMP cycle” (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

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Section: Discussionmentioning

confidence: 99%

Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

Dolan

Kohlstedt

Trigg

et al. 2020

mBio

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“…Pseudomonas putida has attracted great attention as a potential chassis organism for metabolic engineering due in large part to its ability to metabolize a wide variety of carbon sources, particularly aromatic compounds that can be derived from lignin [2,3]. To more fully realize this vision, much effort has been put forth recently to better characterize the central metabolism of P. putida with updated genome-scale models [4], C 13 flux experiments [5,6], and high-throughput fitness assays, which have all contributed to a more complete understanding of the bacterium [7,8]. Despite these advances, the metabolic capabilities of P. putida are not yet fully understood.…”

Section: Introductionmentioning

confidence: 99%

Host engineering for improved valerolactam production inPseudomonas putida

Thompson

et al. 2019

Preprint

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25Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its 26 ability to metabolize a wide array of carbon sources, especially aromatic compounds derived 27 from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally 28 metabolize products produced from engineered pathways. Herein we show that P. putida is able 29 to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent 30 important nylon precursors, and are produced in quantities exceeding one million tons per 31 year[1]. To better understand this metabolism we use a combination of Random Barcode 32 Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the 33 likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA 34 genes prevented P. putida from growing on valerolactam, prevented the degradation of 35 valerolactam in rich media, and dramatically reduced caprolactam degradation under the same 36 conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-37 aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production 38 to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in 39 non-model hosts in future metabolic engineering efforts. 40 1 INTRODUCTION 41 Pseudomonas putida has attracted great attention as a potential chassis organism for 42 metabolic engineering due in large part to its ability to metabolize a wide variety of carbon 43 sources, particularly aromatic compounds that can be derived from lignin [2,3]. To more fully 44 realize this vision, much effort has been put forth recently to better characterize the central 45 metabolism of P. putida with updated genome-scale models [4], C 13 flux experiments [5,6], and 46high-throughput fitness assays, which have all contributed to a more complete understanding of 47 48 fully understood. 49 One consequence of this omnivorous metabolism is that P. putida possesses the ability to 50 degrade or fully catabolize chemicals metabolic engineers seek to produce in the host. An 51 ongoing challenge for P. putida host engineering will be to rapidly identify catabolic pathways 52 of important target molecules and eliminate them from the genome. The recent report of a novel 53 pathway for levulinic acid catabolism in P. putida KT2440 underscores the catabolic flexibility 54 of the host, and an additional obstacle towards producing high product titer [8]. While 55 challenging, this is not surprising; as a genus, Pseudomonads are well known for their ability to 56 degrade a wide range of naturally occurring or xenobiotic chemicals [9,10]. 57 Caprolactam and valerolactam are both important commodity chemicals used in the 58 synthesis of nylon polymers, with global production of caprolactam reaching over four million 59 metric tons [1]. Multiple efforts have been made to produce these chemicals biologically, with 60the titers of valerolactam...

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“…Several earlier studies have described the systems-level cellular adjustments that accompany the growth of industrially-relevant model organisms such as E. coli, Saccharomyces cerevisiae, B. subtilis, C. glutamicum, B. succiniciproducens , and P. putida in single carbon sources [61–66]. However, and although the accrued metabolic models have made a valuable contribution towards our “textbook understanding” of microbial metabolism, it is clear that they can be only loosely extrapolated to other organisms, such as P. aeruginosa [67].…”

Section: Discussionmentioning

confidence: 99%

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

Dolan

Kohlstedt

Trigg

et al. 2019

Preprint

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Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge towards a common metabolic program as the organism adapts to the CF airway environment. However, at a systems level, we still have only a limited understanding of how these transcriptional changes impact on metabolic flux. To address this, we analysed the transcriptome, proteome and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in the CF airways. We show that the flux of carbon through central metabolism is radically different on each carbon source. For example, the newly-recognised EDEMP cycle plays an important role in supplying NADPH during growth on glycerol. By contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the isocitrate dehydrogenase(s)-catalyzed reaction. Perhaps more importantly, our proteomic and transcriptomic analyses reveal a global remodelling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; a plasticity which allows the organism to seamlessly segue between different carbon sources, maximising the energetic yield from each. Importance Pseudomonas aeruginosa is an opportunistic human pathogen, well-known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well-adapted to life in the CF lung, little is currently known about how the organism metabolises the nutrients available in the airways. In this work, we use a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We find that carbon is routed (“fluxed”) through very different pathways during growth on these substrates, and that this is accompanied by an unexpected remodelling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognised as a target for the development of much-needed antimicrobial agents.

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Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida

Cited by 60 publications

References 54 publications

Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

Host engineering for improved valerolactam production inPseudomonas putida

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

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