Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.     

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20 g/L of glucose as a sole carbon source, P(3HB-co-2.3 mol% 3HV) was produced up to the polymer content of 61.7 wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5 mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.     

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY

The silent information regulator (Sir2) family proteins are NAD⁺‐dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2‐like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains ΔcobB, Δacs, Δ(cobB) Δ(acs), Δ(cheA) Δ(cheZ), Δ(cheA) Δ(cheZ) Δ(cobB) and Δ(cheA) Δ(cheZ) Δ(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the ΔcobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Δacs mutant was indistinguishable from the parental strain, (ii) CheY from the ΔcobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Δ(cheA) Δ(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.     

Pathways of acetate and propionate production in adult Fasciola hepatica

In adult F. hepatica pyruvate is decarboxylated via pyruvate dehydrogenase to acetyl-CoA; acetyl-CoA is then cleaved to acetate via three possible mechanisms (1) carnitine dependent hydrolysis, (2) CoA transferase, (3) reversal of a GTP dependent acyl-CoA synthetase. Of these three systems, CoA transferase has by far the greatest activity. Propionate production by F. hepatica is similar to the mammalian system, succinate being metabolized via succinic thiokinase, methylmalonyl-CoA isomerase, methyl-malonyl-CoA racemase and propionyl-CoA carboxylase to propionyl-CoA. Propionyl-CoA is then cleaved to propionate by the same three pathways as acetyl-CoA. No ATP or GTP production could be demonstrated when acetyl- or propionyl-CoA were incubated with homogenates of F. hepatica. This indicates that carnitine dependent hydrolysis or CoA transferase are the major pathways of acetyl- or propionyl-CoA breakdown. The CoA transferase reaction would result in the conservation of the bond energy although there is no net ATP synthesis.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Metabolic and pathway engineering to influence native and altered erythromycin production through E. coli

The heterologous production of the complex antibiotic erythromycin through Escherichia coli provides a unique challenge in metabolic engineering. In addition to introducing the 19 foreign genes needed for heterologous biosynthesis, E. coli metabolism must be engineered to provide the propionyl-CoA and (2S)-methylmalonyl-CoA substrates required to allow erythromycin formation. In this work, three different pathways to propionyl-CoA were compared in the context of supporting E. coli erythromycin biosynthesis. The comparison revealed that alternative citramalate and threonine metabolic pathways (both starting from exogenous glycerol) were capable of supporting final compound formation equal to a proven pathway reliant upon exogenous propionate. Furthermore, two pathways to (2S)-methylmalonyl-CoA were compared in the production of a novel benzyl-erythromycin analog. A pathway dependent upon exogenous methylmalonate improved selectivity and facilitated antibiotic assessment of this new analog.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

Analysis of a Streptomyces coelicolor A3(2) locus containing the nucleoside diphosphate kinase (ndk) and folylpolyglutamate synthetase (folC) genes

A 3.6-kb DNA fragment from Streptomyces coelicolor A3(2) with the genes valS probably encoding a valyl-tRNA synthetase, folC encoding folylpolyglutamate synthetase, and ndk encoding a nucleoside diphosphate kinase was analysed. folC and ndk are separated by a small open reading frame of unknown function, orfX. The deduced folC gene product is a protein of 46 677 Da whose sequence is similar to other folylpolyglutamate synthetases and folylpolyglutamate synthetase-dihydrofolate synthetases from both Gram-positive and Gram-negative bacteria. After cloning folC behind the lacZ promoter, the Streptomyces folC complemented a folC mutant of Escherichia coli. An essential function for Streptomyces folC was suggested by the fact that it could not be mutated using a conventional gene disruption technique.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Characterization of acetyl-CoA synthetase kinetics and ATP-binding

BackgroundThe superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo.MethodsIn this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry.ResultsThe results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although k_cat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding.ConclusionsThe differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated.General SignificanceThe results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.     

On the mechanism of action of the antifungal agent propionate.

Propionate is used to protect bread and animal feed from moulds. The mode of action of this short-chain fatty acid was studied using Aspergillus nidulans as a model organism. The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl-CoA via propionyl-CoA, methylcitrate and pyruvate. Propionate inhibits growth of A. nidulans on glucose but not on acetate; the latter was shown to inhibit propionate oxidation. When grown on glucose a methylcitrate synthase deletion mutant is much more sensitive towards the presence of propionate in the medium as compared to the wild-type and accumulates 10-fold higher levels of propionyl-CoA, which inhibits CoA-dependent enzymes such as pyruvate dehydrogenase, succinyl-CoA synthetase and ATP citrate lyase. The most important inhibition is that of pyruvate dehydrogenase, as this affects glucose and propionate metabolism directly. In contrast, the blocked succinyl-CoA synthetase can be circumvented by a succinyl-CoA:acetate/propionate CoA-transferase, whereas ATP citrate lyase is required only for biosynthetic purposes. In addition, data are presented that correlate inhibition of fungal polyketide synthesis by propionyl-CoA with the accumulation of this CoA-derivative. A possible toxicity of propionyl-CoA for humans in diseases such as propionic acidaemia and methylmalonic aciduria is also discussed.     

Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia

In propionic acidemia, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors. Little is known about the cerebral metabolism of propionate, although clinical effects of propionic acidemia are largely neurological. We found that propionate was metabolized oxidatively by glia: [3-(14)C]propionate injected into mouse striatum or cortex, gave a specific activity of glutamine that was 5-6 times that of glutamate, indicating metabolism in cells that express glutamine synthetase, i.e., glia. Further, cultured cerebellar astrocytes metabolized [3-(14)C]propionate; cultured neurons did not. However, both cultured cerebellar neurons and astrocytes took up [3H]propionate, and propionate exposure increased histone acetylation in cultured neurons and astrocytes as well as in hippocampal CA3 pyramidal neurons of wake mice. The inability of neurons to metabolize propionate may be due to lack of mitochondrial propionyl-CoA synthetase activity or transport of propionyl residues into mitochondria, as cultured neurons expressed propionyl-CoA carboxylase, a mitochondrial matrix enzyme, and oxidized isoleucine, which becomes converted into propionyl-CoA intramitochondrially. The glial metabolism of propionate suggests astrocytic vulnerability in propionic acidemia when intramitochondrial propionyl-CoA may accumulate. Propionic acidemia may alter both neuronal and glial gene expression by affecting histone acetylation.     

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO₂ fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min⁻¹mg protein⁻¹. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP_i. The enzyme was purified 95-fold to a specific activity of 18 μmol min⁻¹ mg protein⁻¹ from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO₂ fixation in this archaeon and possibly other members of the Sulfolobaceae family.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Connection of propionyl-CoA metabolism to polyketide biosynthesis in Aspergillus nidulans

Propionyl-CoA is an intermediate metabolite produced through a variety of pathways including thioesterification of propionate and catabolism of odd chain fatty acids and select amino acids. Previously, we found that disruption of the methylcitrate synthase gene, mcsA, which blocks propionyl-CoA utilization, as well as growth on propionate impaired production of several polyketides-molecules typically derived from acetyl-CoA and malonyl-CoA-including sterigmatocystin (ST), a potent carcinogen, and the conidiospore pigment. Here we describe three lines of evidence that demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. First, inactivation of a putative propionyl-CoA synthase, PcsA, which converts propionate to propionyl-CoA, restored polyketide production and reduced cellular propionyl-CoA content in a DeltamcsA background. Second, inactivation of the acetyl-CoA synthase, FacA, which is also involved in propionate utilization, restored polyketide production in the DeltamcsA background. Third, fungal growth on several compounds (e.g., heptadecanoic acid, isoleucine, and methionine) whose catabolism includes the formation of propionyl-CoA, were found to inhibit ST and conidiospore pigment production. These results demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis.     

Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering

A linker-based approach for combinatorial assembly of promoter and gene cassettes into a biochemical pathway is developed. A synthetic library containing 144 combinations, with 3 promoters and 4 gene variants, was constructed for the ackA and pta genes of the acetate utilization pathway in E. coli. The in vitro isothermal assembled library was then introduced into E. coli mutant (acs-, pta-, ackA-) and selected for restoration of acetate utilization. 81% of the colonies screened contained the complete functional pathway. Thirty positive clones were analyzed and accounted for 10% of the 144 promoter?Cgene combinations.     

Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent

Aspergillus nidulans was used as a model organism to investigate the fungal propionate metabolism and the mechanism of growth inhibition by propionate. The fungus is able to grow slowly on propionate as sole carbon and energy source. Propionate is oxidized to pyruvate via the methylcitrate cycle. The key enzyme methylcitrate synthase was purified and the corresponding gene mcsA, which contains two introns, was cloned, sequenced and overexpressed in A. nidulans. The derived amino acid sequence of the enzyme shows more than 50% identity to those of most eukaryotic citrate synthases, but only 14% identity to the sequence of the recently detected bacterial methylcitrate synthase from Escherichia coli. A mcsA deletion strain was unable to grow on propionate. The inhibitory growth effect of propionate on glucose medium was enhanced in this strain, which led to the assumption that trapping of the available CoA as propionyl-CoA and/or the accumulating propionyl-CoA itself interferes with other biosynthetic pathways such as fatty acid and polyketide syntheses. In the wild-type strain, however, the predominant inhibitor may be methylcitrate. Propionate (100 mM) not only impaired hyphal growth of A. nidulans but also synthesis of the green polyketide-derived pigment of the conidia, whereas in the mutant pigmentation was abolished with 20 mM propionate.     

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA^Asn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA^Asn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNA^Asn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.