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Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate.

ABSTRACT: 3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.

SUBMITTER: Schneider K

PROVIDER: S-EPMC3256647 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate.

Schneider Kathrin K Asao Marie M Carter Michael S MS Alber Birgit E BE

Journal of bacteriology 20111104 2

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed b ...[more]

PMID: 22056933

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Project description:UnlabelledPropionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR).ImportanceShort-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling expression of the gene encoding propionyl-CoA carboxylase, which is responsible for propionyl-CoA consumption by Rhodobacter sphaeroides. Many other Proteobacteria and Actinomycetales contain one or several PccR homologs that group into distinct clades on the basis of the pathway of acyl-CoA metabolism that they control. Furthermore, an upstream analysis of genes encoding PccR homologs allows the prediction of conserved binding motifs for these regulators. Overall, this study evaluates a single regulator of propionyl-CoA assimilation while expanding the knowledge of the regulation of short-chain acyl-CoAs in many bacterial species.

Project description:Rhodobacter sphaeroides is able to use 3-hydroxypropionate as the sole carbon source through the reductive conversion of 3-hydroxypropionate to propionyl coenzyme A (propionyl-CoA). The ethylmalonyl-CoA pathway is not required in this process because a crotonyl-CoA carboxylase/reductase (Ccr)-negative mutant still grew with 3-hydroxypropionate. Much to our surprise, a mutant defective for another specific enzyme of the ethylmalonyl-CoA pathway, mesaconyl-CoA hydratase (Mch), lost its ability for 3-hydroxypropionate-dependent growth. Interestingly, the Mch-deficient mutant was rescued either by introducing an additional ccr in-frame deletion that resulted in the blockage of an earlier step in the pathway or by heterologously expressing a gene encoding a thioesterase (YciA) that can act on several CoA intermediates of the ethylmalonyl-CoA pathway. The mch mutant expressing yciA metabolized only less than half of the 3-hydroxypropionate supplied, and over 50% of that carbon was recovered in the spent medium as free acids of the key intermediates mesaconyl-CoA and methylsuccinyl-CoA. A gradual increase in growth inhibition due to the blockage of consecutive steps of the ethylmalonyl-CoA pathway by gene deletions suggests that the growth defects were due to the titration of free CoA and depletion of the CoA pool in the cell rather than to detrimental effects arising from the accumulation of a specific metabolite. Recovery of carbon in mesaconate for the wild-type strain expressing yciA demonstrated that carbon flux through the ethylmalonyl-CoA pathway occurs during 3-hydroxypropionate-dependent growth. A possible role of the ethylmalonyl-CoA pathway is proposed that functions outside its known role in providing tricarboxylic acid intermediates during acetyl-CoA assimilation.IMPORTANCE Mutant analysis is an important tool utilized in metabolic studies to understand which role a particular pathway might have under certain growth conditions for a given organism. The importance of the enzyme and of the pathway in which it participates is discretely linked to the resulting phenotype observed after mutation of the corresponding gene. This work highlights the possibility of incorrectly interpreting mutant growth results that are based on studying a single unit (gene and encoded enzyme) of a metabolic pathway rather than the pathway in its entirety. This work also hints at the possibility of using an enzyme as a drug target although the enzyme may participate in a nonessential pathway and still be detrimental to the cell when inhibited.

Project description:BackgroundFarnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2).ResultsFOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain.ConclusionsThe original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy.

Project description:The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

Dataset Information

Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate.

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Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate.

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