Search Results (22)

Pentadecanoic acid (C15:0) is an odd-chain fatty acid (OCFA) with significant health benefits, mainly produced by microbial fermentation. To improve C15:0 production, this study compared the effects of different alcohols on C15:0 production in Yarrowia lipolytica CICC1778, identified 1-propanol as the most effective precursor, assessed its optimal concentration, and employed transcriptomic and metabolomic analyses to elucidate the regulatory mechanisms. The results showed that supplementation with 0.5% 1-propanol resulted in a total lipid production of 1.54 g/L in Y. lipolytica CICC1778, showing no differences compared with the negative control (NC) group, while C15:0 production increased to 76.68 mg/L, representing a 794.7% increase compared with the NC group. Integrated omics analysis showed that propionylcarnitine was positively correlated with ADH2, ADH1, ACADSB, ALDH6A1, and CAT2; O-methylmalonylcarnitine was positively correlated with IVD, MCCC2, ACADSB, and ALDH6A1; and (R)-leucic acid and 2-hydroxy-3-methylbutyric acid were positively correlated with IVD, BAT2, MCCC2, and ACADSB and ALDH6A1 and BAT2, respectively. All of these DEGs and DEMs were upregulated in the alcohol-treated (ALC; supplementation with 0.5% 1-propanol) group. Taken together, supplementation with 0.5% 1-propanol was an effective strategy for enhancing C15:0 production in Y. lipolytica CICC1778; 1-propanol underwent dehydrogenation-oxidation and promoted branched-chain amino acid degradation to expand the propionyl-CoA pool, thereby facilitating C15:0 synthesis. Full article

Androstenedione (AD) is a vital intermediate in the synthesis of steroid drugs, making its efficient production critical in the steroid drug industry. Acetyl-CoA acetyltransferase (FadA5), a thiolase enzyme, plays an important role in the metabolic process of degrading phytosterol side chains in Mycolicibacterium to produce AD. This work is the first systematic analysis of the role of FadA5 in the transformation of phytosterols by Mycolicibacterium to produce AD. The relationship between redox potential and AD production was examined using resting cells, and it was confirmed that FadA5 is a key enzyme for AD production. Mutating the 87th cysteine of FadA5 to alanine reduced its redox effect, enhancing the substrate tolerance and biotransformation capacity of the strain. Co-expressing Vitreoscilla hemoglobin (VHb) and propionyl-CoA metabolized the transcription activator (PrpR), decreased intracellular reactive oxygen species levels, and improved cell viability. The AD yield of MSP-fA5C87A-VP/ΔfA5 was 2.541 g/L, an increase of 16.83% over the control strain. Using a repeated batch fermentation process, the production efficiency of the MSP-fA5C87A-VP/ΔfA5 strain was 0.658 g/L/d, which was 1.82 times higher than that of the control strain. These findings provide a theoretical basis for understanding and regulating steroid side-chain catabolism in Mycolicibacterium and offer support for the rational modification of industrial strains for steroidal drug precursor production. Full article

Androstenedione (AD) is an important intermediate for the production of steroidal drugs. The process of transforming phytosterols into AD by Mycolicibacterium is mainly the degradation process of the phytosterol side chain, and the excessive accumulation of propionyl-CoA produced by Mycobacterium will produce toxic effects, which seriously restricts the transformation performance of strains. In this study, Mycolicibacterium sp. LZ2 (Msp) was used as the research object to study the transcription factor PccD of the TetR family, which has the role of propionyl-CoA metabolism regulation. By constructing overexpression and deletion strains of pccD, it was confirmed that pccD had an inhibitory effect on the transcription of propionyl-CoA carboxylase genes (pccA and pccB). Electrophoretic Mobility Shift Assay (EMSA) and DNase I footprint analysis demonstrated that PccD is directly involved in the transcriptional regulation of pccA and pccB and is a negative transcriptional regulator of the pcc operon. In the study of phytosterol transformation, the growth rate and bacterial viability of Msp-ΔpccD were higher than Msp, but the growth of Msp-pccD was inhibited. As a result of testing of intracellular propionyl-CoA levels and AD production yields, it was found that lower propionyl-CoA levels and higher AD production yields were observed in Msp-ΔpccD. The results expand the cognition of propionyl-CoA metabolism regulation and provide a theoretical basis and reference for the rational transformation of phytosterol transformation strains and secondary metabolite synthesis strains with propionyl-CoA as a substrate, which has important research significance. Full article

Methylmalonyl-CoA epimerase enzyme (MCEE) is responsible for catalyzing the isomeric conversion between D- and L-methylmalonyl-CoA, an intermediate along the conversion of propionyl-CoA to succinyl-CoA. A dedicated test for MCEE deficiency is not included in the newborn screening (NBS) panels but it can be incidentally identified when investigating methylmalonic acidemia and propionic acidemia. Here, we report for the first time the biochemical description of a case detected by NBS. The NBS results showed increased levels of propionylcarnitine (C3) and 2-methylcitric acid (MCA), while methylmalonic acid (MMA) and homocysteine (Hcy) were within the reference limits. Confirmatory analyses revealed altered levels of metabolites, including MCA and MMA, suggesting a block in the propionate degradation pathway. The analysis of methylmalonic pathway genes by next-generation sequencing (NGS) allowed the identification of the known homozygous nonsense variation c.139C>T (p.R47X) in exon 2 of the MCE gene. Conclusions: Elevated concentrations of C3 with a slight increase in MCA and normal MMA and Hcy during NBS should prompt the consideration of MCEE deficiency in differential diagnosis. Increased MMA levels may be negligible at NBS as they may reach relevant values beyond the first days of life and thus could be identified only in confirmatory analyses. Full article

In fungi, the methylcitrate cycle converts cytotoxic propionyl-coenzyme A (CoA) to pyruvate, which enters gluconeogenesis. The glyoxylate cycle converts acetyl-CoA to succinate, which enters gluconeogenesis. The tricarboxylic acid cycle is a central carbon metabolic pathway that connects the methylcitrate cycle, the glyoxylate cycle, and other metabolisms for lipids, carbohydrates, and amino acids. Fungal citrate synthase and 2-methylcitrate synthase as well as isocitrate lyase and 2-methylisocitrate lyase, each evolved from a common ancestral protein. Impairment of the methylcitrate cycle leads to the accumulation of toxic intermediates such as propionyl-CoA, 2-methylcitrate, and 2-methylisocitrate in fungal cells, which in turn inhibits the activity of many enzymes such as dehydrogenases and remodels cellular carbon metabolic processes. The methylcitrate cycle and the glyoxylate cycle synergistically regulate carbon source utilization as well as fungal growth, development, and pathogenic process in pathogenic fungi. Full article

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-¹⁴C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources. Full article

Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione (9-OHAD), which belong to C-19 steroids, are critical steroid-based drug intermediates. The biotransformation of phytosterols into C-19 steroids by Mycolicibacterium cell factories is the core step in the synthesis of steroid-based drugs. The production performance of engineered mycolicibacterial strains has been effectively enhanced by sterol core metabolic modification. In recent years, research on the non-core metabolic pathway of steroids (NCMS) in mycolicibacterial strains has made significant progress. This review discusses the molecular mechanisms and metabolic modifications of NCMS for accelerating sterol uptake, regulating coenzyme I balance, promoting propionyl-CoA metabolism, reducing reactive oxygen species, and regulating energy metabolism. In addition, the recent applications of biotechnology in steroid intermediate production are summarized and compared, and the future development trend of NCMS research is discussed. This review provides powerful theoretical support for metabolic regulation in the biotransformation of phytosterols. Full article

Liver cancers are rising worldwide. Between molecular and epidemiological studies, a research gap has emerged which might be amenable to the technique of metabolomics. This review investigates the current understanding of liver cancer’s trends, etiology and its correlates with existing literature for hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) and hepatoblastoma (HB). Among additional factors, the literature reports dysfunction in the tricarboxylic acid metabolism, primarily for HB and HCC, and point mutations and signaling for CCA. All cases require further investigation of upstream and downstream events. All liver cancers reported dysfunction in the WNT/β-catenin and P13K/AKT/mTOR pathways as well as changes in FGFR. Metabolites of IHD1, IDH2, miRNA, purine, Q10, lipids, phosphatidylcholine, phosphatidylethanolamine, acylcarnitine, 2-HG and propionyl-CoA emerged as crucial and there was an attempt to elucidate the WNT/β-catenin and P13K/AKT/mTOR pathways metabolomically. Full article

Background: Paracoccidioidomycosis is a systemic mycosis caused by the inhalation of conidia of the genus Paracoccidioides. During the infectious process, fungal cells use several carbon sources, leading to the production of propionyl-CoA. The latter is metabolized by the methylcitrate synthase, a key enzyme of the methylcitrate cycle. We identified an inhibitor compound (ZINC08964784) that showed antifungal activity against P. brasiliensis. Methods: This work aimed to understand the fungal metabolic response of P. brasiliensis cells exposed to ZINC08964784 through a proteomics approach. We used a glucose-free medium supplemented with propionate in order to simulate the environment found by the pathogen during the infection. We performed pyruvate dosage, proteolytic assay, dosage of intracellular lipids and quantification of reactive oxygen species in order to validate the proteomic results. Results: The proteomic analysis indicated that the fungal cells undergo a metabolic shift due to the inhibition of the methylcitrate cycle and the generation of reactive species. Proteolytic enzymes were induced, driving amino acids into degradation for energy production. In addition, glycolysis and the citric acid cycle were down-regulated while ß-oxidation was up-regulated. The accumulation of pyruvate and propionyl-CoA led the cells to a state of oxidative stress in the presence of ZINC08964784. Conclusions: The inhibition of methylcitrate synthase caused by the compound promoted a metabolic shift in P. brasiliensis damaging energy production and generating oxidative stress. Hence, the compound is a promising alternative for developing new strategies of therapies against paracoccidioidomycosis. Full article

Propionic acidaemia (PA) is an innate error of metabolism involving a deficiency in the enzyme propionyl-CoA carboxylase. Better control of acute decompensation episodes together with better treatment and monitoring have improved the prognosis of patients with this problem. However, long-term complications can arise in those in whom good metabolic control is achieved, the result of mitochondrial dysfunction caused by deficient anaplerosis, increased oxidative stress, and reduced antioxidative capacity. Coenzyme Q10 (CoQ10) is a nutritional supplement that has a notable antioxidative effect and has been shown to improve mitochondrial function. The present prospective, interventional study examines the plasma concentration of CoQ10 in patients with PA, their tolerance of such supplementation with ubiquinol, and its benefits. Seven patients with PA (aged 2.5 to 20 years, 4 males) received supplements of CoQ10 in the form of ubiquinol (10 mg/kg/day for 6 months). A total of 6/7 patients showed reduced plasma CoQ10 concentrations that normalized after supplementation with ubiquinol (p-value < 0.001), which was well tolerated. Urinary citrate levels markedly increased during the study (p-value: 0.001), together with elevation of citrate/methlycitrate ratio (p-value: 0.03). No other significant changes were seen in plasma or urine biomarkers of PA. PA patients showed a deficiency of plasma CoQ10, which supplementation with ubiquinol corrected. The urinary excretion of Krebs cycle intermediate citrate and the citrate/methylcitrate ratio significantly increased compared to the baseline, suggesting improvement in anaplerosis. This treatment was well tolerated and should be further investigated as a means of preventing the chronic complications associated with likely multifactorial mitochondrial dysfunction in PA. Full article

The α-proteobacterium Caenibius tardaugens can use estrogens and androgens as the sole carbon source. These compounds are steroidal endocrine disruptors that are found contaminating soil and aquatic ecosystems. Here, we show that C. tardaugens, which has been considered as a valuable biocatalyst for aerobic steroidal hormone decontamination, is also able to produce polyhydroxyalkanoates (PHA), biodegradable and biocompatible polyesters of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers. Steroid catabolism yields a significant amount of propionyl-CoA that is metabolically directed towards PHA production through condensation into 3-ketovaleryl-CoA, rendering a PHA rich in 3-hydroxyvalerate. To the best of our knowledge, this is the first report where PHAs are produced from steroids as carbon sources. Full article

The oleaginous fungus Mortierella alpina is a safe source of polyunsaturated fatty acids (PUFA) in industrial food and feed production. Besides PUFA production, pharmaceutically relevant surface-active and antimicrobial oligopeptides were isolated from this basal fungus. Both production of fatty acids and oligopeptides rely on the biosynthesis and high turnover of branched-chain-amino acids (BCAA), especially l-leucine. However, the regulation of BCAA biosynthesis in basal fungi is largely unknown. Here, we report on the regulation of the leucine, isoleucine, and valine metabolism in M. alpina. In contrast to higher fungi, the biosynthetic genes for BCAA are hardly transcriptionally regulated, as shown by qRT-PCR analysis, which suggests a constant production of BCAAs. However, the enzymes of the leucine metabolism are tightly metabolically regulated. Three enzymes of the leucine metabolism were heterologously produced in Escherichia coli, one of which is inhibited by allosteric feedback loops: The key regulator is the α-isopropylmalate synthase LeuA1, which is strongly disabled by l-leucine, α-ketoisocaproate, and propionyl-CoA, the precursor of the odd-chain fatty acid catabolism. Its gene is not related to homologs from higher fungi, but it has been inherited from a phototrophic ancestor by horizontal gene transfer. Full article

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products. Full article

Propionic Acidemia (PA) is a rare inherited metabolic disorder caused by the enzymatic block of propionyl-CoA carboxylase with the consequent accumulation of propionic acid, which is toxic for the brain and cardiac cells. Since a considerable amount of propionate is produced by intestinal bacteria, interest arose in the attempt to reduce propionate-producing bacteria through a monthly antibiotic treatment of metronidazole. In the present study, we investigated the gut microbiota structure of an infant diagnosed at 4 days of life through Expanded Newborn Screening (NBS) and treated the child following international guidelines with a special low-protein diet, specific medications and strict biochemical monitoring. Microbiota composition was assessed during the first month of life, and the presence of Bacteroides fragilis, known to be associated with propionate production, was effectively decreased by metronidazole treatment. After five antibiotic therapy cycles, at 4 months of age, the infant was supplemented with a daily mixture of three bifidobacterial strains, known not to be propionate producers. The supplementation increased the population of bifidobacteria, with Bifidobacterium breve as the dominating species; Ruminococcus gnavus, an acetate and formate producer, was also identified. Metabarcoding analysis, compared with low coverage whole metagenome sequencing, proved to capture all the microbial biodiversity and could be the elected tool for fast and cost-effective monitoring protocols to be implemented in the follow up of rare metabolic disorders such as PA. Data obtained could be a possible starting point to set up tailored microbiota modification treatment studies in the attempt to improve the quality of life of people affected by propionic acidemia. Full article

Propionic acidemia (PA) is a disorder of organic acid metabolism which typically presents with acute encephalopathy-like symptoms associated with metabolic acidosis and hyperammonemia during the neonatal period. The estimated incidence of symptomatic PA in Japan is 1/400,000. The introduction of neonatal screening using tandem mass spectrometry has revealed a far higher disease frequency of approximately 1/45,000 live births due to a prevalent variant of c.1304T>C (p.Y435C) in PCCB, which codes β-subunit of propionyl-CoA carboxylase. Our questionnaire-based follow-up study reveals that most of these patients remain asymptomatic. However, reports on symptomatic patients exhibiting cardiac complications such as cardiomyopathy and QT prolongation have been increasing. Moreover, there were even cases in which these cardiac complications were the only symptoms related to PA. A currently ongoing study is investigating the risk of cardiac complications in patients with neonatal screening-detected PA caused by this common variant. Full article