Reprogramming the Metabolism of Yeast for High-Level Production of Miltiradiene.

Miltiradiene serves as a crucial precursor in the synthesis of various high-value abietane-type diterpenes, exhibiting diverse pharmacological activities. Previous efforts to enhance miltiradiene production have primarily focused on the mevalonate acetate (MVA) pathway. However, limited emphasis has been placed on optimizing the supply of acetyl-CoA and NADPH. In this study, we constructed a platform yeast strain for miltiradiene production by reinforcing the biosynthetic pathway of geranylgeranyl diphosphate (GGPP) and acetyl-CoA, and addressing the imbalance between the supply and demand of the redox cofactor NADPH within the cytoplasm, resulting in an increase in miltiradiene yield to 1.31 g/L. Furthermore, we conducted modifications to the miltiradiene synthase fusion protein tSmKSL1-CfTPS1. Finally, the comprehensive engineering strategies and protein modification strategies culminated in 1.43 g/L miltiradiene in the engineered yeast under shake flask culture conditions. Overall, our work established efficient yeast cell factories for miltiradiene production, providing a foothold for heterologous biosynthesis of abietane-type diterpenes.

Transcriptomics aids in uncovering the metabolic shifts and molecular machinery of Schizochytrium limacinum during biotransformation of hydrophobic substrates to docosahexaenoic acid.

BACKGROUND: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA). RESULTS: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L- 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal ß-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation. CONCLUSIONS: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.

ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA.

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11C-acetate but less 18F-fluorodeoxyglucose (18F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients.

De Novo Synthesis of Chrysin in Saccharomyces cerevisiae.

Chrysin, a flavonoid, has been found to have been widely used in the health food field. But at present, chrysin production is hindered by the low availability of precursors and the lack of catalytic enzymes with high activity. Therefore, ZmPAL was initially screened to synthesize trans-cinnamic acid with high catalytic activity and specificity. To enhance the supply of precursors, the shikimic acid and chorismic acid pathway genes were overexpressed. Besides, the expression of the intracellular and mitochondrial carbon metabolism genes CIT, MAC1/3, CTP1, YHM2, RtME, and MDH was enhanced to increase the intracellular acetyl-CoA content. Chrysin was synthesized through a novel gene combination of ScCPR-EbFNSI-1 and PcFNSI. Finally, de novo synthesis of chrysin was achieved, reaching 41.9 mg/L, which is the highest reported concentration to date. In summary, we identified efficient enzymes for chrysin production and increased it by regulating acetyl-CoA metabolism in mitochondria and the cytoplasm, laying a foundation for future large-scale production.

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway.

BACKGROUND: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI. METHODS AND RESULTS: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1ß production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis. CONCLUSION: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.

Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase.

Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, Nα-acetyl transferase from Bacillus cereus (RimLBC), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimLBC belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimLBC. To gain further insight into RimLBC biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimLBC affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimLBC. Last, we advance a mechanistic model of RimLBC mode of recognition of its protein substrates. Taken together, our studies confirmed RimLBC as a new ribosomal Nα-acetyltransferase and provide structural and functional insights into substrate recognition by Nα-acetyltransferases and protein acetylation in bacteria.

An atypical 3-ketoacyl ACP synthase III required for acyl homoserine lactone synthesis in Pseudomonas syringae pv. syringae B728a.

The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from ß-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

Sulforaphane impedes mitochondrial reprogramming and histone acetylation in polarizing M1 (LPS) macrophages.

M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1ß, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.

Lysine acetylation of histone acetyltransferase adaptor protein ADA2 is a mechanism of metabolic control of chromatin modification in plants.

Histone acetylation is a predominant active chromatin mark deposited by histone acetyltransferases (HATs) that transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to lysine ε-amino groups in histones. GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) is one of the best-characterized HATs and functions in association with several adaptor proteins such as ADA2 within multiprotein HAT complexes. ADA2-GCN5 interaction increases GCN5 binding to acetyl-CoA and stimulates its HAT activity. It remains unclear whether the HAT activity of GCN5 (which acetylates not only histones but also cellular proteins) is regulated by acetyl-CoA levels, which vary greatly in cells under different metabolic and nutrition conditions. Here we show that the ADA2 protein itself is acetylated by GCN5 in rice cells. Lysine acetylation exposes ADA2 to a specific E3 ubiquitin ligase and reduces its protein stability. In rice plants, ADA2 protein accumulation reversely parallels its lysine acetylation and acetyl-CoA levels, both of which are dynamically regulated under varying growth conditions. Stress-induced ADA2 accumulation could stimulate GCN5 HAT activity to compensate for the reduced acetyl-CoA levels for histone acetylation. These results indicate that ADA2 lysine acetylation that senses cellular acetyl-CoA variations is a mechanism to regulate HAT activity and histone acetylation homeostasis in plants under changing environments.

ACETYL-COA PRODUCTION BY OCTANOIC ACID ALLEVIATES ACUTE COMPARTMENT SYNDROME-INDUCED SKELETAL MUSCLE INJURY THROUGH REGULATING MITOPHAGY.

ABSTRACT: Background: Treatment of acute compartment syndrome (ACS)-induced skeletal muscle injury remains a challenge. Previous studies have shown that octanoic acid is a promising treatment for ACS owing to its potential ability to regulate metabolic/epigenetic pathways in ischemic injury. The present study was designed to investigate the efficacy and underlying mechanism of octanoic acid in ACS-induced skeletal muscle injury. Methods: In this study, we established a saline infusion ACS rat model. Subsequently, we assessed the protective effects of sodium octanoate (NaO, sodium salt of octanoic acid) on ACS-induced skeletal muscle injury. Afterward, the level of acetyl-coenzyme A and histone acetylation in the skeletal muscle tissue were quantified. Moreover, we investigated the activation of the AMP-activated protein kinas pathway and the occurrence of mitophagy in the skeletal muscle tissue. Lastly, we scrutinized the expression of proteins associated with mitochondrial dynamics in the skeletal muscle tissue. Results: The administration of NaO attenuated muscle inflammation, alleviating oxidative stress and muscle edema. Moreover, NaO treatment enhanced muscle blood perfusion, leading to the inhibition of apoptosis-related skeletal muscle cell death after ACS. In addition, NaO demonstrated the ability to halt skeletal muscle fibrosis and enhance the functional recovery of muscle post-ACS. Further analysis indicates that NaO treatment increases the acetyl-CoA level in muscle and the process of histone acetylation by acetyl-CoA. Lastly, we found NaO treatment exerts a stimulatory impact on the activation of the AMPK pathway, thus promoting mitophagy and improving mitochondrial dynamics. Conclusion: Our findings indicate that octanoic acid may ameliorate skeletal muscle injury induced by ACS. Its protective effects may be attributed to the promotion of acetyl-CoA synthesis and histone acetylation within the muscular tissue, as well as its activation of the AMPK-related mitophagy pathway.

Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.

The central role of pyruvate metabolism on the epigenetic maturation and transcriptional profile of bovine oocytes.

In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.

MARCH5-mediated downregulation of ACC2 promotes fatty acid oxidation and tumor progression in ovarian cancer.

Lipid metabolic reprogramming has been recognized as a hallmark of human cancer. Acetyl-CoA Carboxylases (ACCs) are key rate-limiting enzymes involved in fatty acid metabolism regulation by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. Previously, most studies focused on the role of ACC1 in fatty acid metabolism in cancer, while the function of ACC2 remains largely uncharacterized in human cancers, especially in ovarian cancer (OC). Here, we show that ACC2 was significantly downregulated in cancerous tissue of OC, and the downregulation of ACC2 is closely associated with lager tumor size, metastases and worse prognosis in OC patients. Downregulation of ACC2 promoted proliferation and metastasis of OC both in vitro and in vivo by enhancing FAO. Notably, mitochondria-associated ubiquitin ligase (MARCH5) was identified to interact with and downregulate ACC2 by ubiquitination and degradation in OC. Moreover, ACC2 downregulation-enhanced FAO contributed to the progression of OC promoted by MARCH5. In conclusion, our findings demonstrate that MARCH5-mediated downregulation of ACC2 promotes FAO and tumorigenesis in OC, suggesting MARCH5-ACC2 axis as a potent candidate for the treatment and prevention of OC.

Efficient synthesis of squalene by cytoplasmic-peroxisomal engineering and regulating lipid metabolism in Yarrowia lipolytica.

Squalene, a high-value acyclic triterpenoid compound, is broadly used in the food and medical industries. Although the large acetyl-CoA pool and hydrophobic space of Yarrowia lipolytica are suitable for the accumulation of squalene, the current production level in Y. lipolytica is still not sufficient for industrial production. In this study, two rounds of multicopy integration of genes encoding key enzymes were performed to enhance squalene anabolic flux in the cytoplasm. Furthermore, the mevalonate pathway was imported into peroxisomes through the compartmentalization strategy, and the production of squalene was significantly increased. By augmenting the acetyl-CoA supply in peroxisomes and the cytoplasm, the squalene was boosted to 2549.1 mg/L. Finally, the squalene production reached 51.2 g/L by fed-batch fermentation in a 5-L bioreactor. This is the highest squalene production reported to date for microbial production, and this study lays the foundation for the synthesis of steroids and squalene derivatives.

NME4 mediates metabolic reprogramming and promotes nonalcoholic fatty liver disease progression.

Nonalcoholic fatty liver disease (NAFLD) is mainly characterized by excessive fat accumulation in the liver, and it is associated with liver-related complications and adverse systemic diseases. NAFLD has become the most prevalent liver disease; however, effective therapeutic agents for NAFLD are still lacking. We combined clinical data with proteomics and metabolomics data, and found that the mitochondrial nucleoside diphosphate kinase NME4 plays a central role in mitochondrial lipid metabolism. Nme4 is markedly upregulated in mice fed with high-fat diet, and its expression is positively correlated with the level of steatosis. Hepatic deletion of Nme4 suppresses the progression of hepatic steatosis. Further studies demonstrated that NME4 interacts with several key enzymes in coenzyme A (CoA) metabolism and increases the level of acetyl-CoA and malonyl-CoA, which are the major lipid components of the liver in NAFLD. Increased level of acetyl-CoA and malonyl-CoA  lead to increased triglyceride levels and lipid accumulation in the liver. Taken together, these findings reveal that NME4 is a critical regulator of NAFLD progression and a potential therapeutic target for NAFLD.

Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4 g/L of glucose, 17.3 g/L of beef extract, and 0.28 mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303 g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95 µg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.

Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.