Acetate Assimilation Pathway Research Articles

Mesaconyl-Coenzyme A Hydratase, a New Enzyme of Two Central Carbon Metabolic Pathways in Bacteria

The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.

Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.

Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum

Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region.

Chloroflexus aurantiacus secretes 3-hydroxypropionate, a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 μM). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO2 fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using 13C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO2. The 3HP formed by cells grown on propionate and 13CO2 was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO2 fixation.

Evidence for algal heterotrophy in Lake Tahoe, California‐Nevada1

Significant differences in the uptake of 14C organic compounds between light and dark bottle incubations of water were recorded from Lake Tahoe. The response to light did not occur in situ below the maximum depth of inorganic carbon photoassimilation and was completely inhibited by a photosynthetic inhibitor. Dark acetate uptake in the deep euphotic zone (ca. 90–150 m) was strongly inhibited by a eucaryotic inhibitor. Microautoradiographic analysis showed that two species of green algae in Tahoe were capable of acetate transport at labeled substrate additions within previously determined ambient limits; in axenic culture these two species grew heterotrophically on acetate. Activities of key enzymes of the major inducible pathway for acetate assimilation were high per unit ATP in the region of the water column where acetate uptake was light stimulated. These data strongly support the hypothesis of heterotrophically active phytoplankton populations at the bottom of the euphotic zone.

Inhibition by itaconate of growth of methylotrophic bacteria

The effects of the isocitrate lyase-directed growth inhibitor itaconate on the growth of certain methylotrophic organisms was investigated. It was found that growth of those organisms possessing the Icl(+)-serine pathway of one-carbon metabolism was inhibited during growth on methylamine and on acetate, but not on glucose. Organisms possessing the Icl(-)-serine pathway pathway were unaffected. Organism PAR, an Icl(-)-serine pathway type, was not specifically inhibited during growth on acetate. This finding further substantiates previous reports of the lack of isocitrate lyase in this organism, indicating a totally new pathway for acetate assimilation.

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.