Metabolic Profiling from an Asymptomatic Ferret Model of SARS-CoV-2 Infection

COVID-19 is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of a SARS-CoV-2 infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n=6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic MRM (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using an LC-QToF-MS methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other reported metabolomic-based findings found in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or re-purposed drug therapies.

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Metabolic Profiling from an Asymptomatic Ferret Model of SARS-CoV-2 Infection

Metabolites ◽

10.3390/metabo11050327 ◽

2021 ◽

Vol 11 (5) ◽

pp. 327

Author(s):

David J. Beale ◽

Rohan Shah ◽

Avinash V. Karpe ◽

Katie E. Hillyer ◽

Alexander J. McAuley ◽

...

Keyword(s):

Carbon Metabolism ◽

Impact Analysis ◽

Metabolic Response ◽

Multiple Reaction Monitoring ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Nasal Wash ◽

Viral Shedding ◽

Central Carbon ◽

The Impact

Coronavirus disease (COVID-19) is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n = 6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic Multiple Reaction Monitoring (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other metabolomic-based findings in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or repurposed drug therapies.

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Metabolic Changes Induced by Deletion of Transcriptional Regulator GCR2 in Xylose-Fermenting Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms8101499 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1499

Author(s):

Minhye Shin ◽

Soo Rin Kim

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Pentose Phosphate Pathway ◽

Carbon Metabolism ◽

Transcriptional Regulator ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Metabolic Changes ◽

Regulatory Systems ◽

Central Carbon

Glucose repression has been extensively studied in Saccharomyces cerevisiae, including the regulatory systems responsible for efficient catabolism of glucose, the preferred carbon source. However, how these regulatory systems would alter central metabolism if new foreign pathways are introduced is unknown, and the regulatory networks between glycolysis and the pentose phosphate pathway, the two major pathways in central carbon metabolism, have not been systematically investigated. Here we disrupted gcr2, a key transcriptional regulator, in S. cerevisiae strain SR7 engineered to heterologously express the xylose-assimilating pathway, activating genes involved in glycolysis, and evaluated the global metabolic changes. gcr2 deletion reduced cellular growth in glucose but significantly increased growth when xylose was the sole carbon source. Global metabolite profiling revealed differential regulation of yeast metabolism in SR7-gcr2Δ, especially carbohydrate and nucleotide metabolism, depending on the carbon source. In glucose, the SR7-gcr2Δ mutant showed overall decreased abundance of metabolites, such as pyruvate and sedoheptulose-7-phosphate, associated with central carbon metabolism including glycolysis and the pentose phosphate pathway. However, SR7-gcr2Δ showed an increase in metabolites abundance (ribulose-5-phosphate, sedoheptulose-7-phosphate, and erythrose-4-phosphate) notably from the pentose phosphate pathway, as well as alteration in global metabolism when compared to SR7. These results provide insights into how the regulatory system GCR2 coordinates the transcription of glycolytic genes and associated metabolic pathways.

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The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

mBio ◽

10.1128/mbio.02060-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 2

Author(s):

Laure Dumont ◽

Mark B. Richardson ◽

Phillip van der Peet ◽

Danushka S. Marapana ◽

Tony Triglia ◽

...

Keyword(s):

Pentose Phosphate Pathway ◽

Carbon Metabolism ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Repair Enzyme ◽

Content Type ◽

Phosphoglycolate Phosphatase ◽

Central Carbon ◽

Metabolite Repair ◽

By Products

ABSTRACT Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparum pgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite. IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.

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The metabolic repair enzyme phosphoglycolate phosphatase regulates central carbon metabolism and fosmidomycin sensitivity in Plasmodium falciparum

10.1101/415505 ◽

2018 ◽

Cited By ~ 1

Author(s):

Laure Dumont ◽

Mark B Richardson ◽

Phillip van der Peet ◽

Matthew WA Dixon ◽

Spencer J Williams ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Carbon Metabolism ◽

Blood Cells ◽

Malaria Parasite ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Phosphoglycolate Phosphatase ◽

End Products ◽

Central Carbon

AbstractThe asexual blood stages of the malaria parasite, Plasmodium falciparum are highly dependent on glycolysis for ATP synthesis, redox balance and provision of essential anabolic precursors. Recent studies have suggested that members of the haloacid dehalogenase (HAD) family of metabolite phosphatases may play an important role in regulating multiple pathways in P. falciparum central carbon metabolism. Here, we show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PfPGP), which is homologous to yeast Pho13 and mammalian PGP, regulates glycolysis in asexual blood stages by controlling intracellular levels of several intermediates and novel end-products of this pathway. Deletion of the P. falciparum pgp gene significantly attenuated asexual parasite growth in red blood cells, while comprehensive metabolomic analysis revealed the accumulation of two previously uncharacterized metabolites, as well as changes in a number of intermediates in glycolysis and the pentose phosphate pathway. The two unknown metabolites were assigned as 2-phospho-lactate and 4-phosphoerythronate by comparison of their mass spectra with synthetic standards. 2-Phospho-lactate was significantly elevated in wildtype and ΔPfPGP parasites cultivated in the presence of methylglyoxal and D-lactate, but not L-lactate, indicating that it is a novel end-product of the methylglyoxal pathway. 4-Phosphoerythronate is a putative side product of the glycolytic enzyme, glyceraldehyde dehydrogenase and the accumulation of both 4-phosphoerythronate and 2-phospho-D-lactate were associated with changes in glycolytic and the pentose phosphate pathway fluxes as shown by 13C-glucose labelling studies and increased sensitivity of the ΔPfPGP parasites to the drug fosmidomycin. Our results suggest that PfPGP contributes to a novel futile metabolic cycle involving the phosphorylation/dephosphorylation of D-lactate as well as detoxification of metabolites, such as 4-phosphoerythronate, and both may have important roles in regulating P. falciparum central carbon metabolism.Author summaryThe major pathogenic stages of the malaria parasite, Plasmodium falciparum, develop in red blood cells where they have access to an abundant supply of glucose. Unsurprisingly these parasite stages are addicted to using glucose, which is catabolized in the glycolytic and the pentose phosphate pathways. While these pathways also exist in host cells, there is increasing evidence that P. falciparum has evolved novel ways for regulating glucose metabolism that could be targeted by next-generation of anti-malarial drugs. In this study, we show the red blood cell stages of P. falciparum express an enzyme that is specifically involved in regulating the intracellular levels of two metabolites that are novel end-products or side products of glycolysis. Parasite mutants lacking this enzyme are viable but exhibit diminished growth rates in red blood cells. These mutant lines accumulate the two metabolites, and exhibit global changes in central carbon metabolism. Our findings suggest that metabolic end/side products of glycolysis directly regulate the metabolism of these parasites, and that the intracellular levels of these are tightly controlled by previously uncharacterized metabolite phosphatases.

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Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.02429-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 174-183 ◽

Cited By ~ 22

Author(s):

Audrey Bloem ◽

Isabelle Sanchez ◽

Sylvie Dequin ◽

Carole Camarasa

Keyword(s):

Saccharomyces Cerevisiae ◽

Volatile Compounds ◽

Carbon Metabolism ◽

Isoamyl Alcohol ◽

Redox Status ◽

Central Carbon Metabolism ◽

Ethyl Esters ◽

Content Type ◽

Central Carbon ◽

The Impact

ABSTRACTRedox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth inSaccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD+/NADH or, to a lesser extent, NADP+/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation.

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Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

Applied and Environmental Microbiology ◽

10.1128/aem.03653-14 ◽

2015 ◽

Vol 81 (10) ◽

pp. 3349-3356 ◽

Cited By ~ 11

Author(s):

Susan Maleki ◽

Mali Mærk ◽

Svein Valla ◽

Helga Ertesvåg

Keyword(s):

Pseudomonas Fluorescens ◽

Glucose Dehydrogenase ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Content Type ◽

Alginate Production ◽

Metabolism Enzymes ◽

Pentose Phosphate Pathways ◽

Glucose 6 Phosphate Dehydrogenase ◽

The Impact

ABSTRACTThe biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacteriumPseudomonas fluorescensrelies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated inP. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption ofzwf-1resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption ofgcdincreased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

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The genome of a nonphotosynthetic diatom provides insights into the metabolic shift to heterotrophy and constraints on the loss of photosynthesis

10.1101/2020.05.28.115543 ◽

2020 ◽

Author(s):

Anastasiia Pendergrass ◽

Wade R. Roberts ◽

Elizabeth C. Ruck ◽

Jeffrey A. Lewis ◽

Andrew J. Alverson

Keyword(s):

Carbon Metabolism ◽

Plastid Genome ◽

Nuclear Genome ◽

Primary Source ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Diatom Species ◽

Apicomplexan Parasites ◽

Central Carbon ◽

Trophic Shift

AbstractAlthough most of the tens of thousands of diatom species are obligate photoautotrophs, many mixotrophic species can also use extracellular organic carbon for growth, and a small number of obligate heterotrophs have lost photosynthesis entirely. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia sp. strain Nitz4, to determine how carbon metabolism was altered in the wake of this rare and radical trophic shift in diatoms. Like other groups that have lost photosynthesis, the genomic consequences were most evident in the plastid genome, which is exceptionally AT-rich and missing photosynthesis-related genes. The relatively small (27 Mb) nuclear genome did not differ dramatically from photosynthetic diatoms in gene or intron density. Genome-based models suggest that central carbon metabolism, including a central role for the plastid, remains relatively intact in the absence of photosynthesis. All diatom plastids lack an oxidative pentose phosphate pathway (PPP), leaving photosynthesis as the main source of plastid NADPH. Consequently, nonphotosynthetic diatoms lack the primary source of NADPH required for essential biosynthetic pathways that remain in the plastid. Genomic models highlighted similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids. The ancestral absence of a plastid PPP might constrain loss of photosynthesis in diatoms compared to Archaeplastida, whose plastid PPP continues to produce reducing cofactors following loss of photosynthesis. Finally, Nitzschia possesses a complete β-ketoadipate pathway. Previously known only from fungi and bacteria, this pathway may allow mixotrophic and heterotrophic diatoms to obtain energy through the degradation of abundant plant-derived aromatic compounds.

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Characterization of Mutations That Affect the Nonoxidative Pentose Phosphate Pathway in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00436-17 ◽

2017 ◽

Vol 200 (2) ◽

Cited By ~ 3

Author(s):

Justin P. Hawkins ◽

Patricia A. Ordonez ◽

Ivan J. Oresnik

Keyword(s):

Nitrogen Fixation ◽

Mutant Strain ◽

Pentose Phosphate Pathway ◽

Carbon Metabolism ◽

Sinorhizobium Meliloti ◽

Iron Acquisition ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Content Type ◽

Central Carbon

ABSTRACTSinorhizobium melilotiis a Gram-negative alphaproteobacterium that can enter into a symbiotic relationship withMedicago sativaandMedicago truncatula. Previous work determined that a mutation in thetkt2gene, which encodes a putative transketolase, could prevent medium acidification associated with a mutant strain unable to metabolize galactose. Since the pentose phosphate pathway inS. melilotiis not well studied, strains carrying mutations in eithertkt2andtal, which encodes a putative transaldolase, were characterized. Carbon metabolism phenotypes revealed that both mutants were impaired in growth on erythritol and ribose. This phenotype was more pronounced for thetkt2mutant strain, which also displayed auxotrophy for aromatic amino acids. Changes in pentose phosphate pathway metabolite concentrations were also consistent with a mutation in eithertkt2ortal. The concentrations of metabolites in central carbon metabolism were also found to shift dramatically in strains carrying atkt2mutation. While the concentrations of proteins involved in central carbon metabolism did not change significantly under any conditions, the levels of those associated with iron acquisition increased in the wild-type strain with erythritol induction. These proteins were not detected in either mutant, resulting in less observable rhizobactin production in thetkt2mutant. While both mutants were impaired in succinoglycan synthesis, only thetkt2mutant strain was unable to establish symbiosis with alfalfa. These results suggest thattkt2andtalplay central roles in regulating the carbon flow necessary for carbon metabolism and the establishment of symbiosis.IMPORTANCESinorhizobium melilotiis a model organism for the study of plant-microbe interactions and metabolism, especially because it effects nitrogen fixation. The ability to derive the energy necessary for nitrogen fixation is dependent on an organism's ability to metabolize carbon efficiently. The pentose phosphate pathway is central in the interconversion of hexoses and pentoses. This study characterizes the key enzymes of the nonoxidative branch of the pentose phosphate pathway by using defined genetic mutations and shows the effects the mutations have on the metabolite profile and on physiological processes such as the biosynthesis of exopolysaccharide, as well as the ability to regulate iron acquisition.

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Lighting Up Live-Cell and In Vivo Central Carbon Metabolism with Genetically Encoded Fluorescent Sensors

Annual Review of Analytical Chemistry ◽

10.1146/annurev-anchem-091619-091306 ◽

2020 ◽

Vol 13 (1) ◽

pp. 293-314 ◽

Cited By ~ 1

Author(s):

Zhuo Zhang ◽

Xiawei Cheng ◽

Yuzheng Zhao ◽

Yi Yang

Keyword(s):

Carbon Metabolism ◽

Tricarboxylic Acid Cycle ◽

Cell Metabolism ◽

Central Carbon Metabolism ◽

Fluorescent Sensors ◽

Pentose Phosphate ◽

Spatiotemporal Information ◽

Extant Species ◽

Central Carbon

As the core component of cell metabolism, central carbon metabolism, consisting of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle converts nutrients into metabolic precursors for biomass and energy to sustain the life of virtually all extant species. The metabolite levels or distributions in central carbon metabolism often change dynamically with cell fates, development, and disease progression. However, traditional biochemical methods require cell lysis, making it challenging to obtain spatiotemporal information about metabolites in living cells and in vivo. Genetically encoded fluorescent sensors allow the rapid, sensitive, specific, and real-time readout of metabolite dynamics in living organisms, thereby offering the potential to fill the gap in current techniques. In this review, we introduce recent progress made in the development of genetically encoded fluorescent sensors for central carbon metabolism and discuss their advantages, disadvantages, and applications. Moreover, several future directions of metabolite sensors are also proposed.

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Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

10.1101/2021.01.08.426012 ◽

2021 ◽

Author(s):

Daniel C. Volke ◽

Karel Olavarria ◽

Pablo Ivan Nikel

Keyword(s):

Pseudomonas Putida ◽

Carbon Metabolism ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Biochemical Network ◽

Evolutionary Strategy ◽

Cofactor Specificity ◽

Central Carbon ◽

Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic 'gatekeepers' for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH.

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