Acs is essential for propionate utilization in Escherichia coli

Bacteria like Escherichia coli can use propionate as sole carbon and energy source. All pathways for degradation of propionate start with propionyl-CoA. However, pathways of propionyl-CoA synthesis from propionate and their regulation mechanisms have not been carefully examined in E. coli. In this study, roles of the acetyl-CoA synthetase encoding gene acs and the NAD⁺-dependent protein deacetylase encoding gene cobB on propionate utilization in E. coli were investigated. Results from biochemical analysis showed that, reversible acetylation also modulates the propionyl-CoA synthetase activity of Acs. Subsequent genetic analysis revealed that, deletion of acs in E. coli results in blockage of propionate utilization, suggesting that acs is essential for propionate utilization in E. coli. Besides, deletion of cobB in E. coli also results in growth defect, but only under lower concentrations of propionate (5 mM and 10 mM propionate), suggesting the existence of other propionyl-CoA synthesis pathways. In combination with previous observations, our data implies that, for propionate utilization in E. coli, a primary amount of propionyl-CoA seems to be required, which is synthesized by Acs.     

Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N₂H₄). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.     

Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering

The overexpression of acetyl-CoA (CoA) synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation. It also greatly enhanced acetate assimilation when acetate was used as a carbon source. These features are ideal for applications in metabolic engineering. ACS overexpression can be strategically applied to reduce acetate byproduct, recover wasted carbon, and redirect carbon flux toward more favorable pathways. The native acs gene was cloned and overexpressed in E. coli. Studies showed significant effects on acetate production and assimilation in cultures grown in minimal and complex media with glucose or acetate as the carbon source.     

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.     

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.Subject terms: Environmental microbiology, Metabolism     

ACR1, a gene encoding a protein related to mitochondrial carriers,is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

Acetate scavenging activity in <Emphasis Type="Italic">Escherichia coli</Emphasis>: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl−CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl−CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl−CoA synthetase are upregulated. A mutant in acs (encoding acetyl−CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl−CoA synthetase by cAMP−CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl−CoA and acetyl−phosphate pools, although establishing an energy-dissipating substrate cycle.     

Spinach leaf acetyl-coenzyme a synthetase: purification and characterization

Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M_r value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K_i for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA.     

Acetyl-CoA synthetase (ADP forming) in archaea,a novel enzyme involved in acetate formation and ATP synthesis

The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated:

1. Cell extracts of all species, both archaea and bacteria, catalyzed the coenzyme A-dependent oxidative decarboxylation of pyruvate with viologen dyes or with Clostridium pasteurianum ferredoxin as electron acceptors indicating a pyruvate: ferredoxin oxidoreductase to be operative in acetyl-CoA formation from pyruvate.
2. Cell extracts of all archaeal species, both hyperthermophiles (D. amylolyticus, H. butylicus, T. celer, P. woesei) and the mesophile H. saccharovorum, contained an acetyl-CoA synthetase (ADP forming), which catalyzes both acetate formation from acetyl-CoA and ATP synthesis from ADP and phosphate (P_i): Acetyl-CoA+ADP+P_i?Acetate + ATP+CoA. Phosphate acetyltransferase and acetate kinase could not be detected.
3. Cell extracts of the hyperthermophilic (eu)bacteria T. maritima and C. thermohydrosulfuricum contained phosphate acetyltransferase and acetate kinase rather than acetyl-CoA synthetase (ADP forming).

These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and P_i are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies

When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.     

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.     

Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α₂β₂ structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS III_Tk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICS_Tk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICS_Tk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

A dual-specific Glu-tRNA and Asp-tRNA amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA^Gln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA^Gln to Gln-tRNA^Gln and Asp-tRNA^Asn to Asn-tRNA^Asn. Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.     

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PP_i). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.     

Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.     

Microbial production of ethanol from acetate by engineered <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

This study was performed to produce ethanol from acetate using a genetically engineered Ralstonia eutropha. In order to genetically modify R. eutropha H16, phaCAB operon encoding metabolic pathway genes from acetyl-CoA to polyhydroxybutyrate (PHB) was deleted and adhE encoding an alcohol dehydrogenase from Escherichia coli was overexpressed for conversion of acetyl-CoA to ethanol. The resulting strain produced ethanol up to 170 mg/L when cultivated in minimal media supplemented with 5 g/L of acetate as a sole carbon source. Growth and ethanol production were optimized by adjusting nitrogen source (NH₄Cl) content and repetitive feeding of acetate into the bacterial culture, by which the ethanol production was reached to approximately 350 mg/L for 84 h.