acetate-CoA Ligase Research Articles

Ignisphaera cupida sp. nov., a hyperthermophilic hydrolytic archaeon from a hot spring of Uzon (Kamchatka), and emended description of the genus Ignisphaera

A novel strictly anaerobic hyperthermophilic archaeon, strain 4213-coT, was isolated from a terrestrial hot spring in the Uzon Caldera, Kamchatka (Russian Federation). Coccoid cells were present singly, in pairs, or aggregates, and occasionally were motile. The strain grew at 75–100 °C and within a pH range of 5.4–8.2 with the optimum at 92 °C and pH 6.4–6.7. Strain 4213-coT was a chemoorganoheterotroph, growing on proteinaceous substrates and mono-, di- and polysaccharides (starch, guar gum, xanthan gum). It did not require sodium chloride for growth. The complete genome of strain 4213-coT was 1.74 Mbp in size; its G+C content was 36.18 %. Genome analysis allowed to identify 25 genes encoding glycosidases involved in polysaccharide hydrolysis as well as genes of ADP-forming acetate-CoA ligase, lactate dehydrogenase and two [NiFe] hydrogenases responsible for acetate, lactate and hydrogen formation during fermentation. Moreover gene cluster encoding archaellum subunits was found. According to the phylogenomic analysis strain 4213-coT formed a species-level phylogenetic lineage within Ignisphaera genus. Our phylogenomic analysis also supports the delineation of the Ignisphaera genus into a separate family Ignisphaeraceae, as recently published. Here we propose a novel species Ignisphaera cupida, sp. nov. with type strain 4213-coT (=JCM 39446T=VKM B-3715T=UQM 41593T). Ecogenomic analysis showed that representatives of the Ignisphaera are thermophilic archaea, the majority of them were found in terrestrial hot springs and deep-sea hydrothermal vents. This study allowed a better understanding of physiology and ecology of Ignisphaeraceae – a rather understudied archaeal group.

Comparison of anaerobic co-digestion of vacuum toilet blackwater and kitchen waste under mesophilic and thermophilic conditions: Reactor performance, microbial response and metabolic pathway

Co-digestion of kitchen waste (KW) and black water (BW) can be considered as an attractive method to efficiently achieve the clean energy from waste. To find the optimal operation parameters for the co-digestion, the effects of different temperatures (35 and 55 °C) and BW:KW ratios on the reactor performances, microbial communities and metabolic pathways were studied. The results showed that the optimum BW:KW ratio was 1:3.6 and 1:4.5 for mesophilic and thermophilic optimal reactors, with methane production of 449.04 mL/g VS and 411.90 mL/g VS, respectively. Microbial communities showed significant differences between the reactors under different temperatures. For bacteria, increasing BW:KW ratio significantly promoted Defluviitoga enrichment (1.1%–9.5%) under thermophilic condition. For Archaea, the increase in BW:KW ratio promoted the enrichment of Methanosaeta (8.6%–56.4%) in the mesophilic reactor and Methanothermobacter (62.0%–89.2%) in the thermophilic reactor. The analysis of the key enzymes showed that, acetoclastic methanogenic pathway performed as the dominant under mesophilic condition, with high abundance of Acetate-CoA ligase (EC:6.2.1.1) and Pyruvate synthase (EC:1.2.7.1). Hydrogenotrophic methanogenic pathway was the main pathway in the thermophilic reactors, with high abundance of Formylmethanofuran dehydrogenase (EC:1.2.99.5).

Bioelectrochemical anaerobic digestion mitigates microplastic pollution and promotes methane recovery of wastewater treatment in biofilm system

Bioelectrochemical systems (BES) offer significant potential for treating refractory waste and recovering bioenergy. However, their ability to mitigate microplastic pollution in wastewater remains unexplored. This study showed that BES facilitated the treatment of polyethylene (PE), polyvinyl chloride (PVC), and Mix (PE+PVC) microplastic wastewater and the methane recovery (40.61%, 20.02%, 21.19%, respectively). The lactate dehydrogenase (LDH), adenosine triphosphate (ATP), cytochrome c, and nicotinamide adenine dinucleotide (NADH/NAD+) ratios were elevated with electrical stimulation. Moreover, the applied voltage improved the polysaccharides content of the extracellular polymeric substances (EPS) in the PE-BES but decreased in PVC-BES, while the proteins showed the opposite trend. Metatranscriptomic sequencing showed that the abundance of fermentation bacteria, acetogens, electrogens, and methanogens was greatly enhanced by applying voltage, especially at the anode. Methane metabolism was dominated by the acetoclastic methanogenic pathway, with the applied voltage promoting the enrichment of Methanothrix, resulting in the direct conversion of acetate to acetyl-CoA via acetate-CoA ligase (EC: 6.2.1.1), and increased metabolic activity in the anode. Moreover, applied voltage greatly boosted the function genes expression level related to energy metabolism, tricarboxylic acid (TCA) cycle, electron transport, and transporters on the anode biofilm. Overall, these results demonstrate that BES can mitigate microplastic pollution during wastewater treatment.

Biogas production characteristics of lignite and related metabolic functions with indigenous microbes from different coal seams

To investigate the effects of different exogenous microorganisms on coal biomethanation and its related microbial metabolic functions. Bacterial sources were obtained from the mine water of the Xinsheng, Qinan, Fangzhuang, and Tianyuan coal mines, and lignite was utilized for conducting experiments to produce biomethane. The biomethane yield and statistical analysis based on KEGG metabolic pathways were used to assess the biomethane production potential and the difference of metabolic functions during anaerobic fermentation. The results showed that the accumulative production of biomethane in the anaerobic fermentation system with microorganisms from Qinan mine was 1.80 times, 1.18 times and 1.13 times that of the fermentation systems with other microbes from the Xinsheng, Fangzhuang and Tianyuan mine, respectively. Based on the geological environment of groundwater, it was observed that the moderate water-rich fractures in the coal seam fractures of Qinan Mine are relatively developed, which facilitates the transport of organic matter during microbial metabolism. On the other hand, the Fangzhuang and Tianyuan coal mines are situated in the groundwater runoff zone, their fracture development characteristics are comparatively lower than those of the Qinan mine. In contrast, the coal seam roof and floor of Xinsheng mine are characterized by a stable aquifer comprising thick mudstone and claystone, which presents difficulties for hydrogen-producing bacteria and methanogenic bacteria in obtaining liquid nutrients. This leads to a lower potential for methane production in the Xinsheng mine as compared to the other coal mines. The prediction based on 16S rRNA gene function showed that the microorganisms in Qinan mine had the largest abundance of annotated homologous genes involved in glycolysis, fatty acid synthesis, and methane metabolism, which had a significant positive correlation with methane production. An important observation was the absence of acetate-CoA ligase in the glycolytic pathway, which rules out the acetate-trophic pathway as a significant contributor to coal biodegradation and methanogenesis. Moreover, the abundance of genes related to the hydrogenotrophic methanogenesis pathway was found to be the highest during methane metabolism, indicating that the CO2 reduction pathway plays a significant role during the anaerobic fermentation of coal to produce biomethane.

Bioelectrochemically assisting anaerobic digestion enhanced methane production under low-temperature

Bioelectrochemical systems (BES) with carbon brush electrode were developed to improve methanogenic performance of low-temperature (15 °C) anaerobic digestion (AD) in this study. It turned out that the BES system (1.0 V) could significantly improve the CH4 yield, and during the reaction process, propionic acid (∼618.73 mg/L) had a serious accumulation. Metatranscriptomic analysis revealed that methane was mainly produced by acetoclastic methanogenic pathway, and application of voltage could promote its improvement. On carbon brush electrodes, the enhancement of the acetoclastic pathway mainly depended on acetate kinase (Pta) and phosphate acetyltransferase (Ack) to convert acetate to acetyl-CoA. While it was mainly regulated by the acetate-CoA ligase (Acs) in fluid activated sludge. The activity of methanogenesis-related enzymes on the cathode was generally higher than that on the anode, while the enzyme activity on the fluid sludge anode was mostly inhibited. In addition, BES system significantly increased the abundance of methanogens and electrogens, and enhanced the activity of electron transfer related enzymes, which greatly promoted the direct interspecies electron transfer (DIET) of species. Therefore, the BES system could be an attractive alternative strategy to improve the performance of AD when treating low-temperature wastewater.

The changes of microbial abundance and functional genes in bioelectrochemistry at 15 ℃

Bioelectrochemistry system (BES) provides a new strategy for the improvement of low temperature anaerobic digestion (AD) for methane production. In this study, the BES with carbon brushes as electrodes was operated at 15 °C to analyze the changes of microbial abundance and functional genes. The microbial community structure showed that the application of voltage had no effect to type and relative abundance of bacteria based on 16S-rRNA sequencing. Metatranscriptomic analyses revealed that significantly enhanced the abundance of fermentation bacteria in the fluid activated sludge, and the relative abundance of biosynthesis of other secondary metabolites (61.56%) was also highly expressed. More than that, the abundance of electricigens (Pseudomonas, Syntrophomonas) was also enriched in the BES system. These electricigens could interact with methanogens to promote electron transfer by direct interspecies electron transfer (DIET). The expression level of functional enzyme related to electron transport was increased at the anode (30.60%), but decreased at the cathode (−33.84%). Analysis of methanogens found that the application of voltage decreased the relative abundance of Methanosarcina and promoted the enrichment of Methanothrix. At 15 ℃, acetoclastic was the mainly methanogenic pathway. What’s more, the application of voltage could enhance the acetoclastic and inhibited the hydrogenotrophic and the methylotrophic pathway. Further, the acetoclastic pathway was changed from acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8) to acetate-CoA ligase (EC: 6.2.1.1) by applied voltage. These results are conducive to the further development of low-temperature AD treatment technology.

CO2 improves the microalgal-bacterial granular sludge towards carbon-negative wastewater treatment

As a promising wastewater treatment technology, little is known about whether the greenhouse gas CO2 can be applied for microalgal-bacterial granular sludge (MBGS) process. This article applied CO2 for improving MBGS process. It was found that the physical structure of MBGS with no CO2 addition appeared to have a trend to be loose and disintegrated, with a sludge volume index at 5 min (SVI5) of over 150 mL/g and an average pore size of 35 nm in 60 d operation. However, CO2 could maintain the compact and integrated structure of MBGS with a SVI5 lower than 50 mL/g and an average pore size ranging from 10 to 13 nm. CO2 could enhance the production of extracellular polysaccharides and aromatic protein, thus favoring the granular stability of MBGS. CO2 could change the aqueous environment, e.g. lowering the pH values, which resulted in different microbial communities as well as metabolic potentials of MBGS. As for the reactor performance, CO2 could significantly improve the removals of organics and phosphorus, evidenced by the enhancement of genes encoding acetate-CoA ligase and ATPase, respectively. Although the mass ratio of algae to bacteria was elevated by CO2 addition, the ammonia removal related enzymes of glutamate dehydrogenase and glutamine synthetase could be negatively and positively impacted by CO2, respectively. Mass balance analysis of carbon indicated that CO2 could provide additional carbon source as well as enhance the buffering capacity for the MBGS system. Further estimations suggested that the MBGS process could achieve a carbon-negative objective for municipal wastewater treatment by supplying CO2 as additional carbon source. Hence, CO2 supply for MBGS process in municipal wastewater treatment can be deemed as a two-birds-one-stone strategy, i.e. maintaining the granular stability and eliminating the carbon emission. This article can advance our basic knowledge on MBGS process towards environment-sustainable wastewater treatment.

Sirtuin-Dependent Reversible Lysine Acetylation Controls the Activity of Acetyl Coenzyme A Synthetase in Campylobacter jejuni.

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group (Nε) of an active-site lysine of the AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, this active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N-acetyltransferases (GNATs). Acs activity is lost as a result of acetylation and is restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c, cj1715, and cj1050c loci, namely, the AMP-forming acetate-CoA ligase (CjAcs), a type IV GCN5-type lysine acetyltransferase (GNAT [CjLatA]), and a NAD+-dependent (class III) sirtuin deacylase (CjCobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of CjAcs. IMPORTANCE This work provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase (CjAcs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of CjAcs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

Dual-chamber differs from single-chamber microbial electrosynthesis in biogas production performance under low temperature (15℃)

In this study, single-chamber and dual-chamber Microbial electrosynthesis (MES) with carbon fiber brushes as electrodes were operated at 15°C to compare and analyze the difference in methanogenic performance. Metatranscriptomic analysis showed that the relative abundance of electroactive microorganisms Syntrophomonas, Pseudomonas and Bacteroides in each group exceeded 90%, while the abundance of Geobacter was less than 4%. Acetoclastic methanogens Methahnosarcina was more enriched in dual-chamber MES (61.74%~70.42%), and Methanothrix showed higher abundance in single-chamber MES (33.44%~51.71%). Methahnosarcina and Methanothrix could interact with electroactive microorganisms to improve the electron transfer efficiency through direct interspecies electron transfer (DIET). Analysis of the methane metabolic pathways of low-temperature MES found acetoclastic pathway was domination, and single-chamber MES achieved acetate to acetyl-CoA through acetate-CoA ligase (EC: 6.2.1.1), whereas dual-chamber MES was by acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8). These results are beneficial to further research on the treatment of low-temperature wastewater.

Fragmentation of acetate-CoA ligase gives a clue to understand domain rearrangement history of NDP-forming acyl-CoA synthetase superfamily proteins.

NDP-forming type acyl-CoA synthetase superfamily proteins are known to have six essential subdomains (1, 2, 3, a, b, c) of which partition and order are varied, suggesting yet-to-be-defined subdomain rearrangement happened in its evolution. Comparison in physicochemical and biochemical characteristics between the recombinant proteins which we made from fragmented subdomains and wild-type protein, acetate-CoA ligase in a hyperthermophilic archaeon, consisting of two distinct subunits (α1-2-3 and βa-b-c) provided a clue to the mystery of its molecular evolutionary passage. Although solubility and thermostability of each fragmented subdomain turned out to be lower than that of wild-type, mixture of the three synthetic subunits of α1-2, α3, and βa-b-c had quaternary structure, thermostability, and enzymatic activity comparable to those of the wild-type. This suggests that substantial independence and mobility of subdomain 3 have enabled rearrangement of the subdomains; and thermostability of the subdomains has constrained the composition of the subunits.

Genomic and enzymatic evidence of acetogenesis by anaerobic methanotrophic archaea

Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem.

Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60°C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

A Metagenomic Analysis of Bacterial Microbiota in the Digestive Tract of Triatomines.

The digestive tract of triatomines (DTT) is an ecological niche favored by microbiota whose enzymatic profile is adapted to the specific substrate availability in this medium. This report describes the molecular enzymatic properties that promote bacterial prominence in the DTT. The microbiota composition was assessed previously based on 16S ribosomal DNA, and whole sequenced genomes of bacteria from the same genera were used to calculate the GC level of rare and prominent bacterial species in the DTT. The enzymatic reactions encoded by coding sequences of both rare and common bacterial species were then compared and revealed key functions explaining why some genera outcompete others in the DTT. Representativeness of DTT microbiota was investigated by shotgun sequencing of DNA extracted from bacteria grown in liquid Luria-Bertani broth (LB) medium. Results showed that GC-rich bacteria outcompete GC-poor bacteria and are the dominant components of the DTT microbiota. In addition, oxidoreductases are the main enzymatic components of these bacteria. In particular, nitrate reductases (anaerobic respiration), oxygenases (catabolism of complex substrates), acetate-CoA ligase (tricarboxylic acid cycle and energy metabolism), and kinase (signaling pathway) were the major enzymatic determinants present together with a large group of minor enzymes including hydrogenases involved in energy and amino acid metabolism. In conclusion, despite their slower growth in liquid LB medium, bacteria from GC-rich genera outcompete the GC-poor bacteria because their specific enzymatic abilities impart a selective advantage in the DTT.

The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential.

BackgroundSynthetic biology allows the development of new biochemical pathways for the production of chemicals from renewable sources. One major challenge is the identification of suitable microorganisms to hold these pathways with sufficient robustness and high yield. In this work we analyzed the genome of the propionic acid producer Actinobacteria Propionibacterium acidipropionici (ATCC 4875).ResultsThe assembled P. acidipropionici genome has 3,656,170 base pairs (bp) with 68.8% G + C content and a low-copy plasmid of 6,868 bp. We identified 3,336 protein coding genes, approximately 1000 more than P. freudenreichii and P. acnes, with an increase in the number of genes putatively involved in maintenance of genome integrity, as well as the presence of an invertase and genes putatively involved in carbon catabolite repression. In addition, we made an experimental confirmation of the ability of P. acidipropionici to fix CO2, but no phosphoenolpyruvate carboxylase coding gene was found in the genome. Instead, we identified the pyruvate carboxylase gene and confirmed the presence of the corresponding enzyme in proteome analysis as a potential candidate for this activity. Similarly, the phosphate acetyltransferase and acetate kinase genes, which are considered responsible for acetate formation, were not present in the genome. In P. acidipropionici, a similar function seems to be performed by an ADP forming acetate-CoA ligase gene and its corresponding enzyme was confirmed in the proteome analysis.ConclusionsOur data shows that P. acidipropionici has several of the desired features that are required to become a platform for the production of chemical commodities: multiple pathways for efficient feedstock utilization, ability to fix CO2, robustness, and efficient production of propionic acid, a potential precursor for valuable 3-carbon compounds.

Synechococcus sp. Strain PCC 7002 nifJ Mutant Lacking Pyruvate:Ferredoxin Oxidoreductase

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.

Roseovariussp. strain 217: aerobic taurine dissimilation via acetate kinase and acetate-CoA ligase

The genome sequence of Roseovarius sp. strain 217 indicated that many pathway enzymes found in other organisms for the degradation of taurine are represented, but that a novel, apparently energy-dependent pathway is involved in the conversion of acetyl phosphate to acetyl CoA. Thus, an ABC transporter for taurine could be postulated, while inducible taurine: pyruvate aminotransferase, alanine dehydrogenase, sulfoacetaldehyde acetyltransferase and sulfite dehydrogenase could be assayed. Whereas phosphate acetyltransferase has been found in other organisms, none was indicated in the genome sequence and no activity was found in cell-free extracts. Instead, acetate kinase was active as was acetate-CoA ligase.

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [ 11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 μmol [ 11C]acetylcoenzyme A. (CoA-[ 11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125–180 μm particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 (μmol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 × 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.

Fatty acid synthesis in intestinal mucosa of guinea pig.

1. Acetate-CoA ligase, acetyl-CoA-carbon dioxide ligase and fatty acid synthetase were shown to be present in particle-free fractions of guinea-pig intestinal mucosa. 2. Each of these enzymes was partially purified by ammonium sulphate precipitation from the particle-free supernatant. 3. The incorporation of acetate and citrate into fatty acid was measured. 4. Gas-liquid radiochromatography was used to investigate the pattern of fatty acids synthesized. 5. The rate-limiting step in fatty acid synthesis was shown to be acetyl-CoA-carbon dioxide ligase.