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Metabolic pathways for the synthesis of n-alcohols and fatty acids. The elongated acyl-CoA is the critical metabolic node for the production of aldehydes, fatty acids, and n-alcohols. The elongated acyl-CoA can also enter the next elongation cycle, depending on the type of KAT
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Because of their function of catalyzing the rearrangement of the carbon chains, thiolases have attracted increasing attentions over the past decades. The 3-ketoacyl-CoA thiolase (KAT) is a member of the thiolase, which is capable of catalyzing the Claisen condensation reaction between the two acyl-CoAs, thereby achieving carbon chain elongation. In...
Citations
... The thiolase superfamily encompasses an array of enzymes including Archeal thiolase, thiolase I and II, HMG-CoA synthase (HMGS), β-ketoacyl-ACP synthase I, II and III (KAS I, II and III), β-ketoacyl-CoA synthase (KCS), and polyketide synthase I, II, and III (PKS I, II and III) ( Fig. 1) [3,7]. Notably, thiolase I is predisposed to the synthesis of medium-chain (C4-C8) [8] linear aliphatic compounds, which are commonly employed as precursors for fuels [9] and polymers [10,11]. Consequently, advancing our knowledge of the structural and biochemical foundations of thiolase I may enhance the versatility of these enzymes in the enzymatic and microbial production of desired chemicals. ...
... Purification via affinity chromatography followed by SDS-PAGE analysis revealed that Tfu_0875 possesses a molecular weight marginally above 40 kDa, aligning with the predicted molecular weight of 40.6 kDa (Fig. S1). Tfu_0875 belongs to the thiolase I which prefers medium-chain length substrates [8]. Therefore, we selected acetyl-CoA, malonyl-CoA, succinyl-CoA, and glutaryl-CoA as substrates to condensation with an extension unit acetyl-CoA by Tfu_0875 for evaluating the substrate specificity [14][15][16]. ...
... 3-Ketoacyl-CoA thiolase is attractive enzyme for the biosynthesis of products with varying chain lengths and functionalities [8,16,29]. However, the inherent promiscuity of their substrate specificity often results in the production of a mixture of products, which can split the metabolic flux and reduce productivity [32]. ...
The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production. However, the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application. Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875. Notably, Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA, a key precursor in adipic acid production. To enhance its performance, a deep learning approach (DLKcat) was employed to identify effective mutants, and a computational strategy, known as the greedy accumulated strategy for protein engineering (GRAPE), was used to accumulate these effective mutants. Among the mutants, Tfu_0875N249W/L163H/E217L exhibited the highest specific activity (320% of wild-type Tfu_0875), the greatest catalytic efficiency (kcat/KM = 1.00 min⁻¹mM⁻¹), the highest succinyl-CoA specificity (KM = 0.59 mM, 28.1% of Tfu_0875) and dramatically reduced substrate binding energy (−30.25 kcal mol⁻¹v.s. −15.94 kcal mol⁻¹). A structural comparison between Tfu_0875N249W/L163H/E217L and the wild type Tfu_0875 revealed that the increased interaction between the enzyme and succinyl-CoA was the primary reason for the enhanced enzyme activity. This interaction facilitated rapid substrate anchoring and stabilization. Furthermore, a reduced binding pocket volume improved substrate specificity by enhancing the complementarity between the binding pocket and the substrate in stereo conformation. Finally, our rationally designed mutant, Tfu_0875N249W/L163H/E217L, increased the adipic acid titer by 1.35-fold compared to the wild type Tfu_0875 in shake flask. The demonstrated enzymatic methods provide a promising enzyme variant for the adipic acid production. The above effective substrate binding pocket engineering strategy can be beneficial for the production of other industrially competitive biobased chemicals when be applied to other thiolases.
... The amino acid sequence of this gene has 95.7% similarity to the phaA gene in Cobetia marina DSM 4741 T strain and 42.4% to the phaA gene in Halomonas sp. SF2003 [48]. The reduction of acetoacetyl-CoA to (S)-3-hydroxybutyryl-CoA in the ES021 strain is catalyzed by fabG that can function as phaB encoding 3-oxoacyl-ACP reductase. ...
Background
Polyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production.
Results
Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC–MS), and nuclear magnetic resonance spectroscopy (NMR) analyses.
Conclusions
Herein,Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.
... The amino acid sequence of this gene has 95.7% similarity to the phaA gene in Cobetia marina DSM 4741 T strain and 42.4% to the phaA gene in Halomonas sp. SF2003 [40]. ...
Background
PHB is of significant concern due to its potential applications as green alternatives to traditional petrochemical-based plastics. In the present study, a new strain of Salinicola salarius, a halophilic bacterium, was isolated from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further analysis of the ES021 genome was conducted to identify and elucidate the genes involved in PHB production.
Results
Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. For PHB production from fatty acids, phaJ and fadB are involved. Environmental factors such as aeration and incubation temperature were optimized to attain high productivity of PHB. The results showed that growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h resulted in the highest PHB productivity. To maximize PHB production, different raw materials i.e., salted whey and molasses were examined as cheap carbon sources. The PHB productivity was increased two-fold (13.34 g/l) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/l, and PHB of 12.88 g/l. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR) and Gas chromatography-mass spectroscopy (GC-MS) analysis.
Conclusions
Herein, Salinicola salarius ES021 strain was demonstrated as a high natural producer of PHB from agro-industrial wastes. A detailed genome characterization of the ES021 strain showing the PHB-related genes was presented in this study. However, further metabolic engineering is required to confirm the gene networks required for PHB production in this strain.
... Structural superimposition of FadA2 with the PcaF showed that the region between tetramerization and covering loop of FadA2 was far from the tunnel than PcaF. The difference in the length of this region accounts for the substrate selectivity in Ralstonia eutropha thiolase BktB [28]. We also explored the residues which can point to the tunnel from other regions of proteins. ...
Tuberculosis (TB) is one of the leading causes of human death caused by Mycobacterium tuberculosis (Mtb). Mtb can enter into a long‐lasting persistence where it can utilize fatty acids as the carbon source. Hence, fatty acid metabolism pathway enzymes are considered promising and pertinent mycobacterial drug targets. FadA2 (thiolase) is one of the enzymes involved in Mtb's fatty acid metabolism pathway. FadA2 deletion construct (ΔL136‐S150) was designed to produce soluble protein. The crystal structure of FadA2 (ΔL136‐S150) at 2.9 Å resolution was solved and analysed for membrane‐anchoring region. The four catalytic residues of FadA2 are Cys99, His341, His390 and Cys427, and they belong to four loops with characteristic sequence motifs, i.e., CxT, HEAF, GHP and CxA. FadA2 is the only thiolase of Mtb which belongs to the CHH category containing the HEAF motif. Analysing the substrate‐binding channel, it has been suggested that FadA2 is involved in the β‐oxidation pathway, i.e., the degradative pathway, as the long‐chain fatty acid can be accommodated in the channel. The catalysed reaction is favoured by the presence of two oxyanion holes, i.e., OAH1 and OAH2. OAH1 formation is unique in FadA2, formed by the NE2 of His390 present in the GHP motif and NE2 of His341 present in the HEAF motif, whereas OAH2 formation is similar to CNH category thiolase. Sequence and structural comparison with the human trifunctional enzyme (HsTFE‐β) suggests the membrane‐anchoring region in FadA2. Molecular dynamics simulations of FadA2 with a membrane containing POPE lipid were conducted to understand the role of a long insertion sequence of FadA2 in membrane anchoring.
... The future prospects for KAT enzymes or the major isoforms like KAT2/KAT5 can be, post-translational modification, active site modification (Tan et. al. 2020), and protein engineering (Liu et. al. 2020) for drug development which could unravel the true potential of this enzyme for synthesizing important secondary metabolites. It's known that polyketide biosynthesis is performed by PKS via decarboxylative Claisen condensation reactions with possibly some additional modifications catalyzed by modifying domains of PKSs. Such modifications ...
Unlabelled:
Ginger is an important spice crop with medicinal values and gingerols are the most abundant pungent polyphenols present in ginger, responsible for most of its pharmacological properties. The present study focuses on the molecular mechanism of gingerol biosynthesis in ginger using transcriptome analysis. Suppression Subtractive Hybridization (SSH) was done in leaf and rhizome tissues using high gingerol-producing ginger somaclone B3 as the tester and parent cultivar Maran as the driver and generated high-quality leaf and rhizome Expressed Sequence Tags (ESTs). The Blast2GO annotations of the ESTs revealed the involvement of leaf ESTs in secondary metabolite production, identifying the peroxisomal KAT2 gene (Leaf EST 9) for the high gingerol production in ginger. Rhizome ESTs mostly coded for DNA metabolic processes and differential genes for high gingerol production were not observed in rhizomes. In the qRT-PCR analysis, somaclone B3 had shown high chalcone synthase (CHS: rate-limiting gene in gingerol biosynthetic pathway) activity (0.54 fold) in the leaves of rhizome sprouts. The presence of a high gingerol gene in leaf ESTs and high expression of CHS in leaves presumed that the site of synthesis of gingerols in ginger is the leaves. A modified pathway for gingerol/polyketide backbone formation has been constructed explaining the involvement of KAT gene isoforms KAT2 and KAT5 in gingerol/flavonoid biosynthesis, specifically the KAT2 gene which is otherwise thought to be involved mainly in β-oxidation. The results of the present investigations have the potential of utilizing KAT/thiolase superfamily enzymes for protein/metabolic pathway engineering in ginger for large-scale production of gingerols.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13562-022-00825-x.
... As base components of protein, amino acids also provide energy for metabolic pathways in insects (Parkhitko et al., 2016;Parkhitko et al., 2020). HADH and KAT, which we identified as upregulated DEGs, promote fatty acid metabolism, where OgdH is a key enzyme in the tricarboxylic acid cycle (Nakai et al., 1997;Liu et al., 2020). GUS and beta-gal, upregulated in the LC 25 group, are essential regulatory enzymes in carbohydrate-related metabolism, and the latter is also involved in lactose hydrolysis, glycoprotein modification and degradation in vivo (Bar et al., 2018;Yi et al., 2021). ...
Fipronil is widely used in the agricultural world as an efficient phenylpyrazole insecticide to control pests. Binodoxys communis is a key parasitic natural enemy of major homopteran pests and can successfully control the population of pests such as cotton aphids. It has not yet been studied what effects would sublethal doses of fipronil have on Binodoxys communis larvae. Here, this study evaluated the effect of fipronil on Binodoxys communis larvae and analyze the transcriptome results. The results showed that LC10 (1.19 mg/L) and LC25 (1.73 mg/L) had significant negative effects on the survival rate and parasitism rate of F0 generation. Moreover, exposure to high concentrations (LC25) of fipronil still had obvious passive effect on the F1 generation of Binodoxys communis. These results indicated that sublethal doses of fipronil have malignant effects on the biological functions of parasitoids and their offspring. The results of transcriptome analysis showed that differentially expressed genes (DEGs) of Binodoxys communis after LC10 treatment are mainly related to immunity and detoxification. LC25 treatment instead resulted in changes in the expression of genes related to nutrition, energy and metabolism reactions. Seven of the identified DEGs were selected for real-time fluorescence quantitative PCR analysis. To the best of our knowledge, this is the first report to evaluate the sublethal, intergenerational, and transcriptomic side effects of fipronil on larvae of parasitic natural pest enemies. Our findings provide data to accurately assess the risk of fipronil usage on Binodoxys communis larvae, and provide important theoretical support for the comprehensive prevention and control of natural enemies and pesticides.
... [16] The β-ketothiolase (Tfu_0875) in this pathway had a broad substrate spectrum, which could simultaneously catalyze multiple acyl-CoAs to produce different dicarboxylic acids. [17] Based on these, we inferred that by expressing the enzymes of RADP, using acetyl-CoA (C2) and glutaryl-CoA (C5) as substrates, pimelic acid (C7) could be synthesized ( Figure 1A). ...
Pimelic acid is an important seven‐carbon dicarboxylic acid, which is broadly applied in various fields. The industrial production of pimelic acid is mainly through a chemical method, which is complicated and environmentally unfriendly. Herein, we found that pimelic acid could be biosynthesized by the reverse adipate‐degradation pathway (RADP), a typical Claisen condensation reaction that could be applied to the arrangement of C−C bond. In order to strengthen the supply of glutaryl‐CoA precursor, PA5530 protein was used to transport glutaric acid. Subsequently, we discovered that the enzymes in the BIOZ pathway are isoenzyme of the RADP pathway enzymes. By combining the isoenzymes of the two pathways, the titer of pimelic acid reached 36.7 mg ⋅ L⁻¹ under the optimal combination, which was increased by 382.9 % compared with the control strain B‐3. It was also the highest titer of pimelic acid biosynthesized by Claisen condensation reaction, laying the foundation for the production of pimelic acid and its derivatives.
... On the other hand, KAT is capable of catalyzing the Claisen condensation reaction between the two acyl-CoA, thereby achieving carbon chain elongation (70) and lipid synthesis (71). ...
This study was the first to evaluate multiple hormonal manipulations to hepatopancreas over the ovarian development stages of the mud crab, Scylla paramamosain. A total of 1258 metabolites in 75 hepatopancreas explants from five female crabs were induced by juvenile hormone III (JH III), methyl farnesoate (MF), farnesoic acid (FA) and methoprene (Met), as identified from combined metabolomics and lipidomics (LC-MS/MS). 101 significant metabolites and 47 significant pathways were selected and compared for their comprehensive effects to ovarian maturation. While MF played an extensive role in lipid accumulation, JH III and Met shared similar effects, especially in the commonly and significantly elevated triglycerides and lysophospholipids (fold change≥2 and ≤0.5, VIP≥1). The significant upregulation of β-oxidation and key regulators in lipid degradation by FA (P ≤ 0.05) resulted in less lipid accumulation from this treatment, with a shift toward lipid export and energy consumption, unlike the effects of MF, JH III and Met. It was possible that MF and FA played their own unique roles and acted in synergy to modulate lipid metabolism during crab ovarian maturation. Our study yielded insights into the MF-related lipid metabolism in crustacean hepatopancreas for the overall regulation of ovarian maturation, and harbored the potential use of juvenoids to induce reproductive maturity of this economic crab species.
... First, one unit of acetyl-CoA and five units of malonyl-CoA could form the C12-fatty acid i with a double bond and a carbonyl group at α, β-and γ-position, respectively, which reaction resembled typical fatty acid biosynthesis. Then, oxidation of the terminal methyl group in i to carboxylic acid could afford dicarboxylic acid ii [40,41]. Geometrical isomerization could occur in ii that transforms the E-to Z-configuration of the double bond to yield iii. ...
... First, one unit of acetyl-CoA and five units of malonyl-CoA could form the C12-fatty acid i with a double bond and a carbonyl group at α, βand γ-position, respectively, which reaction resembled typical fatty acid biosynthesis. Then, oxidation of the terminal methyl group in i to carboxylic acid could afford dicarboxylic acid ii [40,41]. Geometrical isomerization could occur in ii that transforms the Eto Z-configuration of the double bond to yield iii. ...
Wasabi (Wasabia japonica (Miq.) Matsum.) is a pungent spice commonly consumed with sushi and sashimi. From the roots of this plant, a new 2-butenolide derivative (1) and 17 previously reported compounds (2–18) were isolated and structurally characterized. Their chemical structures were characterized based on the conventional NMR (1H and 13C, COSY, HSQC, and HMBC) and HRESIMS data analysis. All of these phytochemicals (1–18) were evaluated for their antiproliferative effects on the four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and MKN-1), for their inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, and for their nerve growth factor (NGF)-releasing effect from C6 glioma cells. Among the isolated compounds, compound 15 showed powerful antiproliferative activities against A549 and SK-MEL-2 cell lines with IC50 values of 2.10 and 9.08 μM, respectively. Moreover, the new compound 1 exhibited moderate NO inhibition activity with IC50 value of 45.3 μM.
... In the study, ACAA2 was the unique differentially acetylationassociated enzyme, which contributes to downregulation of acetylation in GAS. ACAA2 is a member of thiolases and capable of catalyzing the Claisen condensation reaction, which contributes to achieving carbon chain elongation, production of ketone bodies, fatty acid elongation, and degradation (34). However, ACAA2 expression and function in glioma with seizures remain unclear. ...
Objective: We test the hypothesis that lysine acetylation is involved in the metabolic process of glioma-associated seizures (GAS).
Methods: We used label-free mass spectrometry-based quantitative proteomics to quantify dynamic changes of protein acetylation between gliomas with seizure (CA1 group) and gliomas without seizure (CA2 group). Furthermore, differences of acetyltransferase and deacetylase expression between CA1 and CA2 groups were performed by a quantitative proteomic study. We further classified acetylated proteins into groups according to cell component, molecular function, and biological process. In addition, metabolic pathways and protein interaction networks were analyzed. Regulated acetyltransferases and acetylated profiles were validated by PRM and Western blot.
Results: We detected 169 downregulated lysine acetylation sites of 134 proteins and 39 upregulated lysine acetylation sites of 35 proteins in glioma with seizures based on acetylome. We detected 407 regulated proteins by proteomics, from which ACAT2 and ACAA2 were the differentially regulated enzymes in the acetylation of GAS. According to the KEGG analysis, the upregulated acetylated proteins within the PPIs were mapped to pathways involved in the TCA cycle, oxidative phosphorylation, biosynthesis of amino acids, and carbon metabolism. The downregulated acetylated proteins within the PPIs were mapped to pathways involved in fatty acid metabolism, oxidative phosphorylation, TCA cycle, and necroptosis. Regulated ACAT2 expression and acetylated profiles were validated by PRM and Western blot.
Conclusions: The data support the hypothesis that regulated protein acetylation is involved in the metabolic process of GAS, which may be induced by acetyl-CoA acetyltransferases.
