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Isoflavones are predominantly found in legumes and play roles in plant defense and prevention of estrogen-related diseases. Genistein is an important isoflavone backbone with various biological activities. In this paper, we describe how a cell factory that can de novo synthesize genistein was constructed in Saccharomyces cerevisiae. Different combinations of isoflavone synthase, cytochrome P450 reductase, and 2-hydroxyisoflavone dehydratase were tested, followed by pathway multicopy integration, to stably de novo synthesize genistein. The catalytic activity of isoflavone synthase was enhanced by heme supply and an increased intracellular NADPH/NADP+ ratio. Redistribution of the malonyl-CoA flow and balance of metabolic fluxes were achieved by adjusting the fatty acid synthesis pathway, yielding 23.33 mg/L genistein. Finally, isoflavone glycosyltransferases were introduced into S. cerevisiae, and the optimized strain produced 15.80 mg/L of genistin or 10.03 mg/L of genistein-8-C-glucoside. This is the first de novo synthesis of genistein-8-C-glucoside in S. cerevisiae, which is advantageous for the green industrial production of isoflavone compounds. [ABSTRACT FROM AUTHOR]

Liver-targeted acetyl-coenzyme A (CoA) carboxylase (ACC) inhibitors in metabolic dysfunction-associated steatotic liver disease (MASLD) trials reveal notable secondary effects: hypertriglyceridemia and altered glucose metabolism, paradoxically with reduced hepatic steatosis. In their study, Deja et al. explored how hepatic ACC influences metabolism using different pharmacological and genetic methods, coupled with targeted metabolomics and stable isotope-based tracing techniques. [ABSTRACT FROM AUTHOR]

Malonyl-CoA is a precursor of fatty acids, polyketides, and bio-based chemicals with potential applications in medicine, antibiotics, and fuels. However, its low intracellular concentration and high cost have led to difficulties in research and production. To develop an efficient method for producing malonyl-CoA, we screened the acyl-CoA synthetase (ACS) gene from Streptomyces sp. using sequence-structure alignment. This protein contains conserved sequences and active sites for malonyl-CoA synthetases. The purified recombinant enzyme ACS was heterologously expressed in Escherichia coli BL21 and characterised. The results showed that it converted the substrates malonate and CoA into malonyl-CoA. Under the optimal conditions, the specific activity of the purified ACS was 32.3 U·mg−1 and the conversion rate reached 98.8%. In addition, when the cell-free extracts were used as catalysts, the highest yield of malonyl-CoA was obtained after 4 h, yielding 24.2 g·L−1 with a conversion rate of 90.3%. After the product was purified and vacuum freeze-dried, a solid powder of malonyl-CoA was obtained. This study characterised and identified a new ACS and optimised the reaction conditions to efficiently synthesise pure malonyl-CoA in vitro in high yield using enzyme-mediated methods. [Display omitted] • One new acyl-CoA synthetase (ACS) was mined and characterized. • ACS catalyze the synthesis of malonyl-CoA. • Malonyl-CoA received a high conversion rate (90.3%) and a high yield (24.2 g/L) with cell-free extracts catalyzed reaction. • Efficient malonyl-CoA synthesis on a 100-mL scale. [ABSTRACT FROM AUTHOR]

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism. • A platform strain with an easily swappable malonyl-CoA biosynthetic pathway. • Development of a colorimetric reporter for malonyl-CoA. • A framework to extensively rewrite and monitor malonyl-CoA biosynthetic pathway. • Multiple superior enzyme mutants for malonyl-CoA production were identified. • Pathway-scale engineering boosted flaviolin, β-carotene and betaxanthin production. [ABSTRACT FROM AUTHOR]

DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l -lysine, l -leucine, and l -isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l -lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica , whose synthesis is limited by the availability of CoA ester precursors. • Multi-omics insights into the DHA-producer Yarrowia lipolytica Af4. • DHA production is limited by CoA-precursor availability. • Enhanced ketogenic amino acid catabolism boosts DHA production. • Lysine-supplementation provides DHA at the gram scale with high selectivity. [ABSTRACT FROM AUTHOR]

An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica , which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Long-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), are essential for human health and have been widely used in the food and pharmaceutical industries. However, the limited availability of natural sources, such as oily fish, has led to the pursuit of microbial production as a promising alternative. Yarrowia lipolytica can produce various PUFAs via genetic modification. A recent study upgraded Y. lipolytica for DHA production by expressing a four-gene cluster encoding a myxobacterial PKS-like PUFA synthase, reducing the demand for redox power. However, the genetic architecture of gene expression in Y. lipolytica is complex and involves various control elements, offering space for additional improvement of DHA production. This study was designed to optimize the expression of the PUFA cluster using a modular cloning approach. Results: Expression of the monocistronic cluster with each gene under the control of the constitutive TEF promoter led to low-level DHA production. By using the minLEU2 promoter instead and incorporating additional upstream activating UAS1B4 sequences, 5' promoter introns, and intergenic spacers, DHA production was increased by 16-fold. The producers remained stable over 185 h of cultivation. Beneficially, the different genetic control elements acted synergistically: UAS1B elements generally increased expression, while the intron caused gene-specific effects. Mutants with UAS1B16 sequences within 2–8 kb distance, however, were found to be genetically unstable, which limited production performance over time, suggesting the avoidance of long repetitive sequence blocks in synthetic multigene clusters and careful monitoring of genetic stability in producing strains. Conclusions: Overall, the results demonstrate the effectiveness of synthetic heterologous gene clusters to drive DHA production in Y. lipolytica. The combinatorial exploration of different genetic control elements allowed the optimization of DHA production. These findings have important implications for developing Y. lipolytica strains for the industrial-scale production of valuable polyunsaturated fatty acids. [ABSTRACT FROM AUTHOR]

Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media. [ABSTRACT FROM AUTHOR]

3-Hydroxypropionic acid (3-HP) is an important intermediate compound in the chemical industry. Green and environmentally friendly microbial synthesis methods are becoming increasingly popular in a range of industries. Compared to other chassis cells, Yarrowia lipolytica possesses advantages, such as high tolerance to organic acid and a sufficient precursor required to synthesize 3-HP. In this study, gene manipulations, including the overexpression of genes MCR-N C a , MCR-C C a , GAPN S m , ACC1 and ACS S e L 641 P and knocking out bypass genes MLS1 and CIT2, leading to the glyoxylate cycle, were performed to construct a recombinant strain. Based on this, the degradation pathway of 3-HP in Y. lipolytica was discovered, and relevant genes MMSDH and HPDH were knocked out. To our knowledge, this study is the first to produce 3-HP in Y. lipolytica. The yield of 3-HP in recombinant strain Po1f-NC-14 in shake flask fermentation reached 1.128 g·L−1, and the yield in fed-batch fermentation reached 16.23 g·L−1. These results are highly competitive compared to other yeast chassis cells. This study creates the foundation for the production of 3-HP in Y. lipolytica and also provides a reference for further research in the future. [ABSTRACT FROM AUTHOR]

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L−1 resveratrol from supplemented p -coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas. [Display omitted] • Pseudomonas taiwanensis has a high tolerance towards the stilbenoid pinosylvin. • Heterologous synthesis of the polyketides pinosylvin, resveratrol and flaviolin. • Drain in fatty acid biosynthesis limits malonyl-CoA availability in Pseudomonas. • Engineered P. taiwanensis GRC3Δ6 MC-III platform for secondary metabolite synthesis. [ABSTRACT FROM AUTHOR]

The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-13C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of β-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene (Mlycd−/−) alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells. [ABSTRACT FROM AUTHOR]

The molecule (2S)-naringenin is a scaffold molecule with several nutraceutical properties. Currently, (2S)-naringenin is obtained through chemical synthesis and plant isolation. However, these methods have several drawbacks. Thus, heterologous biosynthesis has emerged as a viable alternative to its production. Recently, (2S)-naringenin production studies in Escherichia coli have used different tools to increase its yield up to 588 mg/L. In this study, we designed and assembled a bio-factory for (2S)-naringenin production. Firstly, we used several parametrized algorithms to identify the shortest pathway for producing (2S)-naringenin in E. coli, selecting the genes phenylalanine ammonia lipase (pal), 4-coumarate: CoA ligase (4cl), chalcone synthase (chs), and chalcone isomerase (chi) for the biosynthetic pathway. Then, we evaluated the effect of oxygen transfer on the production of (2S)-naringenin at flask (50 mL) and bench (4 L culture) scales. At the flask scale, the agitation rate varied between 50 rpm and 250 rpm. At the bench scale, the dissolved oxygen was kept constant at 5% DO (dissolved oxygen) and 40% DO, obtaining the highest (2S)-naringenin titer (3.11 ± 0.14 g/L). Using genome-scale modeling, gene expression analysis (RT-qPCR) of oxygen-sensitive genes was obtained. [ABSTRACT FROM AUTHOR]

Carminic acid is a natural red dye extracted from the insect Dactylopius coccus. Due to its ideal dying effect and high safety, it is widely used in food and cosmetics industries. Previous study showed that introduction of polyketide synthase (OKS) from Aloe arborescens , cyclase (ZhuI) and aromatase (ZhuJ) from Streptomyces sp. R1128, and C-glucosyltransferase (UGT2) from D. coccus into Aspergillus nidulans could achieve trace amounts of de novo production. These four genes were introduced into Saccharomyces cerevisiae , but carminic acid was not detected. Analysis of the genome of A. nidulans revealed that 4′-phosphopantetheinyl transferase (NpgA) and monooxygenase (AptC) are essential for de novo biosynthesis of carminic acid in S. cerevisiae. Additionally, endogenous hydroxylase (Cat5) from S. cerevisiae was found to be responsible for hydroxylation of flavokermesic acid to kermesic acid. Therefore, all enzymes and their functions in the biosynthesis of carminic acid were explored and reconstructed in S. cerevisiae. Through systematic pathway engineering, including regulating enzyme expression, enhancing precursor supply, and modifying the β-oxidation pathway, the carminic acid titer in a 5 L bioreactor reached 7580.9 μg/L, the highest yet reported for a microorganism. Heterologous reconstruction of the carminic acid biosynthetic pathway in S. cerevisiae has great potential for de novo biosynthesis of anthraquinone dye. • Biosynthesis pathway of carminic acid has been reconstructed in S. cerevisiae. • 4′-Phosphopantetheinyl transferase is essential for functional expression of OKS. • Monooxygenase (AptC) is responsible for oxidation. • Endogenous hydroxylase (Cat5) is responsible for hydroxylation. • The highest production of carminic acid in microorganisms has been achieved. [ABSTRACT FROM AUTHOR]

Abstract Background Isopropanol is widely used as a biofuel and a disinfectant. Chemical preparation of isopropanol destroys the environment, which makes biological preparation of isopropanol necessary. Previous studies focused on the use of expensive glucose as raw material. Therefore, the microbial cell factory that ferments isopropanol with cheap raw materials will provide a greener way to produce isopropanol. Results This study converted crude glycerol into isopropanol using Y. lipolytica. As a microbial factory, the active natural lipid and fatty acid synthesis pathway endows Y. lipolytica with high malonyl-CoA production capacity. Acetoacetyl-CoA synthase (nphT7) and isopropanol synthesis genes are integrated into the Y. lipolytica genome. The nphT7 gene uses the accumulated malonyl-CoA to synthesize acetoacetyl-CoA, which increases isopropanol production. After medium optimization, the best glycerol medium was found and resulted in a 4.47-fold increase in isopropanol production. Fermenter cultivation with pure glycerol medium resulted in a maximum isopropanol production of 1.94 g/L. In a crude glycerol fermenter, 1.60 g/L isopropanol was obtained, 82.53% of that achieved with pure glycerol. The engineered Y. lipolytica in this study has the highest isopropanol titer reported. Conclusions The engineered Y. lipolytica successfully produced isopropanol by using crude glycerol as a cheap carbon source. This is the first study demonstrating the use of Y. lipolytica as a cell factory to produce isopropanol. In addition, this is also a new attempt to accumulate lipid synthesis precursors to synthesize other useful chemicals by integrating exogenous genes in Y. lipolytica.

Hypoketotic hypoglycaemia is a biochemical hallmark of glycogen storage disease type 1 (GSD1). This is due to inhibition of carnitine-palmitoyl transferase 1 by malonyl-CoA. This inhibits the influx of long-chain fatty acids into the mitochondrial matrix for fatty acid oxidation. This leads to reduced hepatic ketogenesis and impaired energy production in the liver and kidney. Hypoketotic hypoglycaemia may result in CNS symptoms due to energy depletion. Recently, it was reported that enzymes involved in mitochondrial long-chain fatty acid oxidation are upregulated in PBMC from patients suffering from GSD1. I suggest that administration of the prodrug bempedoic acid results in reduced production of malonyl-CoA by inhibiting the ATP-citrate lyase, thus releasing the block of mitochondrial long-chain fatty acid influx. These fatty acids could make use of the increased capacity of fatty acid oxidation as observed in PBMC recently. In the liver, ketogenesis is activated, and energy production is increased in both the liver and kidney. This could result in improved metabolic control and avoidance of cerebral energy depletion. Bempedoic acid is approved as medication in adult patients with hypercholesterolaemia and mixed dyslipidaemia. Repurposing bempedoic acid for the use in GSD1 may improve metabolic control in GSD1. [ABSTRACT FROM AUTHOR]

Biosensors can be used for high-throughput screening, real-time monitoring of metabolites, and dynamic regulation of metabolic processes, which have been a popular research direction in recent years. Here, five promoters from Saccharomyces cerevisiae were selected to construct Malonyl-CoA sensors with the fapO/fapR system derived from Bacillus subtilis, and pCCW12 was finally selected for further optimization. Based on pCCW12, a series of sensors with different response sensitivities were obtained by selecting different fapO insertion sites and combining the best two or three of them. Then, through a combination of promoter hybrid, intron insertion, and transcription factor modification strategies, we obtained sensors with different effects, one of which, the H-pCCW12(TFBS)-Cti6~fapR sensor, had the lowest background noise, doubled response range and higher response sensitivity compared to the original sensor. Sensors with different characteristics constructed in this study, can be applied to Malonyl-CoA related high-throughput screening and finer regulation of metabolism. It also proves that the combined application of different promoter engineering strategies is a feasible idea for the precise construction and regulation of biosensors. [ABSTRACT FROM AUTHOR]

Olivetolic acid (OLA), an initial precursor of cannabinoids, is catalyzed by type III polyketide synthase, which has a wide range of pharmacological activities, such as antimicrobial and cytotoxic effects. Here, we applied systematic metabolic engineering to develop a multienzyme cascade system to produce OLA via two low-cost inputs. The polyketide synthase (OLS) and cyclase enzymes (OAC), along with the best combination of hexanoyl-CoA and malonyl-CoA synthetases (AEE3 and MatB), were first introduced into the biocatalytic system to increase the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units. To drive the catalysis smoothly, an ATP regeneration system and a CoA-sufficient supply system were incorporated into the biocatalysts to provide enough cofactors. Furthermore, malonyl-CoA flux was redirected to OLA biosynthesis through delicate control of the fatty acid biosynthesis (FAB) pathway via promoter engineering. Collectively, these strategies have led us to produce OLA at a titer of 102.1 mg/L with a productivity of 25.5 mg/L/h by using malonate and hexanoate as direct substrates. Our biocatalytic system provides an effective platform for the production of the cannabinoid precursor OLA in Escherichia coli and may be a valuable reference for the development of microbial cell factories that use hexanoyl-CoA and malonyl-CoA as important intermediates.

Malonic acid (MA) is a high-value-added chemical with significant applications in the polymers, pharmaceutical, and food industries. Microbial production of MA presents enzyme inefficiencies, competitive metabolic pathways, and dispersive carbon flux, which collectively limit its biosynthesis. Here, the non-conventional oleaginous yeast Yarrowia lipolytica is genetically engineered to enhance MA production. Initially, the malonyl-CoA pathway, comprising a malonyl-CoA hydrolase from Saccharomyces cerevisiae, is confirmed as the most efficient for MA production in Y. lipolytica. To further enhance MA production, two novel malonyl-CoA hydrolases exhibiting higher activity than the hydrolase from S. cerevisiae, are identified from Y. lipolytica and Fusarium oxysporum, respectively. The introduction of the malonyl-CoA hydrolase from F. oxysporum increases the MA titer to 6.3 g L -1 . Subsequently, advanced metabolic engineering strategies are performed to ensure a sufficient flux of the precursors acetyl-CoA and malonyl-CoA for MA production, resulting in a production of 13.8 g L -1 MA in shaking-flasks. Finally, by employing the fermentation conditions and feeding strategies, a maximum concentration of 63.6 g L -1 of MA is achieved at 156 h with a productivity of 0.41 g L -1  h -1 in fed-batch fermentation. This study provides a new way for engineering Y. lipolytica to enhance MA production at high titer., (© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Malonyl-CoA is an energy-rich molecule formed by the ATP-dependent carboxylation of acetyl coenzyme A catalyzed by acetyl-CoA carboxylase. This molecule is an important precursor for many biotechnologically interesting compounds such as flavonoids, polyketides, and fatty acids. The yeast Saccharomyces cerevisiae remains one of the preferred cell factories, but has a limited capacity to produce malonyl-CoA compared to oleaginous organisms. We developed a new S. cerevisiae strain with a conditional allele of ACC1, the essential acetyl-CoA carboxylase (ACC) gene, as a tool to test heterologous genes for complementation. Yarrowia lipolytica is an oleaginous yeast with a higher capacity for lipid production than S. cerevisiae, possibly due to a higher capacity to produce malonyl-CoA. Measuring relative intracellular malonyl-CoA levels with an in-vivo biosensor confirmed that expression of Y. lipolytica ACC in S. cerevisiae leads to a higher accumulation of malonyl-CoA compared with overexpression of the native gene from an otherwise identical vector. The higher accumulation was generally accompanied by a decreased growth rate. Concomitant expression of both the homologous and heterologous ACC1 genes eliminated the growth defect, with a marginal reduction of malonyl-CoA accumulation.

Previous fibroblast and recombinant enzyme studies showed a markedly thermolabile p.Ser113Leu variant compared to the wild-type (WT) in muscle carnitine palmitoyltransferase II (CPT II) deficiency. Additionally, it has been shown that cardiolipin (CLP) stimulated or inhibited the p.Ser113Leu recombinant variant depending on the pre-incubation temperatures. In this study, the thermolabilities of mitochondrial enzyme CPT II in muscle homogenates of patients with the p.Ser113Leu (n = 3) and p.Arg631Cys (n = 2) variants were identified to be similar to that of WT. Pre-incubation with CLP on ice stimulated the WT enzyme more than both variants. However, CLP stimulated the variants and WT at 46 °C to about 6–18-fold. The present data indicate that the thermostability of CPT II variant in muscle homogenate is similar to that of WT. This is in contrast to the increased thermolability of enzymes derived from fibroblast and that of recombinant enzymes. Hence, it can be speculated that the disruption of the compartmentation in muscle homogenate mediates a protective effect on the thermolability of the native variant. However, the exact mechanism remains unclear. However, the activating effect of CLP on CPT II in muscle homogenate seems to align with those on recombinant enzymes. [ABSTRACT FROM AUTHOR]

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation. • A malonyl-CoA pathway was constructed with mutant Ehd3 and Acc1. • The catalytic activity of Ehd3 was significantly influenced with E124 site. • A fluorescence screening method was built to screen high-copy integration strains. • Pathway and fermentation optimization improved malonate titer by 14-fold. [ABSTRACT FROM AUTHOR]

Background: Isopropanol is widely used as a biofuel and a disinfectant. Chemical preparation of isopropanol destroys the environment, which makes biological preparation of isopropanol necessary. Previous studies focused on the use of expensive glucose as raw material. Therefore, the microbial cell factory that ferments isopropanol with cheap raw materials will provide a greener way to produce isopropanol. Results: This study converted crude glycerol into isopropanol using Y. lipolytica. As a microbial factory, the active natural lipid and fatty acid synthesis pathway endows Y. lipolytica with high malonyl-CoA production capacity. Acetoacetyl-CoA synthase (nphT7) and isopropanol synthesis genes are integrated into the Y. lipolytica genome. The nphT7 gene uses the accumulated malonyl-CoA to synthesize acetoacetyl-CoA, which increases isopropanol production. After medium optimization, the best glycerol medium was found and resulted in a 4.47-fold increase in isopropanol production. Fermenter cultivation with pure glycerol medium resulted in a maximum isopropanol production of 1.94 g/L. In a crude glycerol fermenter, 1.60 g/L isopropanol was obtained, 82.53% of that achieved with pure glycerol. The engineered Y. lipolytica in this study has the highest isopropanol titer reported. Conclusions: The engineered Y. lipolytica successfully produced isopropanol by using crude glycerol as a cheap carbon source. This is the first study demonstrating the use of Y. lipolytica as a cell factory to produce isopropanol. In addition, this is also a new attempt to accumulate lipid synthesis precursors to synthesize other useful chemicals by integrating exogenous genes in Y. lipolytica. [ABSTRACT FROM AUTHOR]

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1S1132A) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L−1 of 3-HP at 0.712 g L−1 h−1 with a final product yield on glycerol of 0.194 Cmol−1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

Raspberry ketone (RK), a natural product derived from raspberry fruit, is commonly utilized as a flavoring agent in foods and as an active component for weight loss. Metabolic engineering has enabled microorganisms to produce RK more efficiently and cost-effectively. However, the biosynthesis of RK is hindered by an unbalanced synthetic pathway and a deficiency of precursors, including tyrosine and malonyl-CoA. In this study, we constructed and optimized the RK synthetic pathway in Escherichia coli using a static metabolic engineering strategy to enhance the biosynthesis of tyrosine from glucose, thereby achieving the de novo production of RK. Additionally, the synthetic and consumption pathways of malonyl-CoA were dynamically regulated by p -coumaric acid-responsive biosensor to balance the metabolic flux distribution between cell growth and RK biosynthesis. Following pathway optimization, the medium components and fermentation conditions were further refined, resulting in a significant increase in the RK titer to 415.56 mg/L. The optimized strain demonstrated a 32.4-fold increase in the RK titer while maintaining a comparable final OD 600 to the initial strain. Overall, the implemented static and dynamic regulatory strategies provide a novel approach for the efficient production of RK, taking into account cell viability and growth.

Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on dynamic changes in malonyl-CoA and fatty acid biosynthesis that could be occurring within a single cell. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu ( mal onyl-CoA i ntracellular b iosensor to u nderstand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in E. coli , observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to sensitively study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis., Competing Interests: The authors declare they have no competing interests.

Background: Endurance is an important capacity to sustain healthy lifestyles. Aged garlic extract (AGE) has been reported to exert an endurance-enhancing effect in clinical and animal studies, although little is known about its active ingredients and mechanism of action., Objectives: This study investigated the potential effect of S-1-propenylcysteine (S1PC), a characteristic sulfur amino acid in AGE, on the swimming endurance of mice, and examined its mechanism of action by a metabolomics-based approach., Methods: Male Institute of Cancer Research (ICR) mice (6 wk old) were orally administered either water (control) or S1PC (6.5 mg/kg/d) for 2 wk. The swimming duration to exhaustion was measured at 24 h after the final administration. Nontargeted metabolomic analysis was conducted on the plasma samples obtained from mice after 40-min submaximal swimming bouts. Subsequently, the enzyme activity of carnitine acyltransferase-1 (CPT-1) and the content of malonyl-coenzyme A (CoA), acetyl-CoA, and adenosine triphosphate (ATP) were quantified in heart, skeletal muscles, and liver of mice., Results: The duration time of swimming was substantially increased in the S1PC-treated mice as compared with the control group. Metabolomic analysis revealed significant alterations in the plasma concentration of the metabolites involved in fatty acid metabolism, in particular medium- or long-chain acylcarnitines in the mice treated with S1PC. Moreover, the administration of S1PC significantly enhanced the CPT-1 activity with the concomitant decrease in the malonyl-CoA content in the heart and skeletal muscles. These effects of S1PC were accompanied by the elevation of the acetyl-CoA and ATP levels to enhance the energy production in those tissues., Conclusions: S1PC is a key constituent responsible for the endurance-enhancing effect of AGE. This study suggests that S1PC helps provide energy during endurance exercise by increasing fatty acid metabolism via CPT-1 activation in the heart and skeletal muscles., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2 S)-naringenin and p -coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH , acpS , acpT , acs , and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2 S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production. ∙ Three-layered dynamic regulation network for the production of (2S)-naringenin ∙ Acyl carrier protein was used as a novel critical node for malonyl-CoA balancing ∙ Biosensor-based directed evolution to optimize dynamic regulation network ∙ Growth coupled dynamic regulation network increases cellular production [ABSTRACT FROM AUTHOR]

Abstract Background In the last years, different biotechnologically relevant microorganisms have been engineered for the synthesis of plant polyphenols such as flavonoids and stilbenes. However, low intracellular availability of malonyl-CoA as essential precursor for most plant polyphenols of interest is regarded as the decisive bottleneck preventing high product titers. Results In this study, Corynebacterium glutamicum, which emerged as promising cell factory for plant polyphenol production, was tailored by rational metabolic engineering towards providing significantly more malonyl-CoA for product synthesis. This was achieved by improving carbon source uptake, transcriptional deregulation of accBC and accD1 encoding the two subunits of the acetyl-CoA carboxylase (ACC), reduced flux into the tricarboxylic acid (TCA) cycle, and elimination of anaplerotic carboxylation of pyruvate. The constructed strains were used for the synthesis of the pharmacologically interesting plant pentaketide noreugenin, which is produced by plants such as Aloe arborescens from five molecules of malonyl-CoA. In this context, accumulation of the C1/C6 cyclized intermediate 1-(2,4,6-trihydroxyphenyl)butane-1,3-dione (TPBD) was observed, which could be fully cyclized to the bicyclic product noreugenin by acidification. Conclusion The best strain C. glutamicum Nor2 C5 mufasO BCD1 PO6-iolT1 ∆pyc allowed for synthesis of 53.32 mg/L (0.278 mM) noreugenin in CGXII medium supplemented with casamino acids within 24 h.

Previous fibroblast and recombinant enzyme studies showed a markedly thermolabile p.Ser113Leu variant compared to the wild-type (WT) in muscle carnitine palmitoyltransferase II (CPT II) deficiency. Additionally, it has been shown that cardiolipin (CLP) stimulated or inhibited the p.Ser113Leu recombinant variant depending on the pre-incubation temperatures. In this study, the thermolabilities of mitochondrial enzyme CPT II in muscle homogenates of patients with the p.Ser113Leu (n = 3) and p.Arg631Cys (n = 2) variants were identified to be similar to that of WT. Pre-incubation with CLP on ice stimulated the WT enzyme more than both variants. However, CLP stimulated the variants and WT at 46 °C to about 6–18-fold. The present data indicate that the thermostability of CPT II variant in muscle homogenate is similar to that of WT. This is in contrast to the increased thermolability of enzymes derived from fibroblast and that of recombinant enzymes. Hence, it can be speculated that the disruption of the compartmentation in muscle homogenate mediates a protective effect on the thermolability of the native variant. However, the exact mechanism remains unclear. However, the activating effect of CLP on CPT II in muscle homogenate seems to align with those on recombinant enzymes.

Background: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. Results: Supplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L−1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. Conclusions: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further. [ABSTRACT FROM AUTHOR]

Background and Purpose: The enzyme α/β-hydrolase domain containing 6 (ABHD6), a new member of the endocannabinoid system, is a promising therapeutic target against neuronal-related diseases. However, how ABHD6 activity is regulated is not known. ABHD6 coexists in protein complexes with the brain-specific carnitine palmitoyltransferase 1C (CPT1C). CPT1C is involved in neuro-metabolic functions, depending on brain malonyl-CoA levels. Our aim was to study CPT1C-ABHD6 interaction and determine whether CPT1C is a key regulator of ABHD6 activity depending on nutritional status.Experimental Approach: Co-immunoprecipitation and FRET assays were used to explore ABHD6 interaction with CPT1C or modified malonyl-CoA-insensitive or C-terminal truncated CPT1C forms. Cannabinoid CB1 receptor-mediated signalling was investigated by determining cAMP levels. A novel highly sensitive fluorescent method was optimized to measure ABHD6 activity in non-neuronal and neuronal cells and in brain tissues from wild-type (WT) and CPT1C-KO mice.Key Results: CPT1C interacted with ABHD6 and negatively regulated its hydrolase activity, thereby regulating 2-AG downstream signalling. Accordingly, brain tissues of CPT1C-KO mice showed increased ABHD6 activity. CPT1C malonyl-CoA sensing was key to the regulatory role on ABHD6 activity and CB1 receptor signalling. Fasting, which attenuates brain malonyl-CoA, significantly increased ABHD6 activity in hypothalamus from WT, but not CPT1C-KO, mice.Conclusions and Implications: Our finding that negative regulation of ABHD6 activity, particularly in the hypothalamus, is sensitive to nutritional status throws new light on the characterization and the importance of the proteins involved as potential targets against diseases affecting the CNS. [ABSTRACT FROM AUTHOR]

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract Background Economical production of value-added chemicals from renewable biomass is a promising path to sustainability. 3-Hydroxypropionic acid (3-HP) is an important chemical for building a bio-sustainable society. Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources. Results Here, we describe metabolic engineering of the fission yeast Schizosaccharomyces pombe to enhance 3-HP production from glucose and cellobiose via the malonyl-CoA pathway. The mcr gene, encoding the malonyl-CoA reductase of Chloroflexus aurantiacus, was dissected into two functionally distinct fragments, and the activities of the encoded protein were balanced. To increase the cellular supply of malonyl-CoA and acetyl-CoA, we introduced genes encoding endogenous aldehyde dehydrogenase, acetyl-CoA synthase from Salmonella enterica, and endogenous pantothenate kinase. The resulting strain produced 3-HP at 1.0 g/L from a culture starting at a glucose concentration of 50 g/L. We also engineered the sugar supply by displaying beta-glucosidase (BGL) on the yeast cell surface. When grown on 50 g/L cellobiose, the beta-glucosidase-displaying strain consumed cellobiose efficiently and produced 3-HP at 3.5 g/L. Under fed-batch conditions starting from cellobiose, this strain produced 3-HP at up to 11.4 g/L, corresponding to a yield of 11.2% (g-3-HP/g-glucose; given that 1 g cellobiose corresponds to 1.1 g glucose upon digestion). Conclusions In this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.

Objective(s): This study intended to investigate the effects of Ginsenoside-Rbl (Gs-Rbl) on fatty acid β-oxidation (FAO) in rat failing heart and to identify potential mechanisms of Gs-Rbl improving heart failure (HF) by FAO pathway dependent on AMP-activated protein kinase (AMPK). Materials and Methods: Rats with chronic HF, induced by adriamycin (Adr), were randomly grouped into 7 groups. Gs-Rb1, adenine 9-β-D-arabinofuranoside (Ara A, specific AMPK inhibitor), and 5'-aminoimidazole-4-carboxamide riboside (Aicar, specific AMPK activator) were administered to rats with HF, singly and/or combinedly. Myocardial high-energy phosphate (such as phosphocreatine, ADP, and ATP), free L-Carnitine, malonyl-CoA, and the activity of FAO-related enzymes in left ventricle from different groups were measured by using the corresponding molecular biological techniques. Results: Gs-Rb1 improved HF significantly, accompanied by a significant increase in phosphocreatine (PCr), ADP, ATP, PCr/ATP ratio, free carnitine, malonyl-CoA, mRNA, activity of carnitine palmitoyltransferase (Cpt), medium-chain Acyl-CoA Dehydrogenase (MCAD) and long-chain acyl-CoA Synthetase (ACSL) and a significant decrease of the ADP/ATP ratio in the left ventricular myocardium. However, all those effects were almost abolished by Ara A and were not further improved by Aicar. Conclusion: Taken together, it suggests that Gs-Rb1 may modulate cardiac metabolic remodeling by improving myocardial fatty acid β-oxidation in failing heart. In addition, the effects of Gs-Rb1 may be mediated via activating AMPK.

• Malonyl-CoA is a key metabolic intermediate for many useful compounds. • The productivity of malonyl-CoA derivatives is restricted by the low cellular level of malonyl-CoA. • At present, different metabolic engineering strategies have been taken to increase the intracellular malonyl-CoA levels. To reduce dependence on petroleum, the biosynthesis of important chemicals from simple substrates using industrial microorganisms has attracted increased attention. Metabolic engineering of Saccharomyces cerevisiae offers a sustainable and flexible alternative for the production of various chemicals. As a key metabolic intermediate, malonyl-CoA is a precursor for many useful compounds. However, the productivity of malonyl-CoA derivatives is restricted by the low cellular level of malonyl-CoA and enzymatic performance. In this review, we focused on how to increase the intracellular malonyl-CoA level and summarize the recent advances in different metabolic engineering strategies for directing intracellular malonyl-CoA to the desired malonyl-CoA derivatives, including strengthening the malonyl-CoA supply, reducing malonyl-CoA consumption, and precisely controlling the intracellular malonyl-CoA level. These strategies provided new insights for further improving the synthesis of malonyl-CoA derivatives in microorganisms. [ABSTRACT FROM AUTHOR]

• A synthetic malonyl-CoA independent module allowed continuous polyketide synthesis. • A synthetic malonyl-CoA dependent module dynamically regulated fatty acid synthesis. • An integrated biosystem redirected malonyl-CoA flux to improve polyketide synthesis. Malonyl-CoA is a basic but limited precursor for the biosynthesis of various bioactive compounds and life-supporting fatty acids in cells. This study develops a biosynthetic system to dynamically redirect malonyl-CoA flux and improve production of malonyl-CoA derived polyketide (6-MSA) in Komagataella phaffii. A synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to work with a hybrid promoter (-7) fapO -cP AOX1 and activate gene expression. Expression mode by the Prm1-FapR/(-7) fapO -cP AOX1 device was not affected by intracellular malonyl-CoA levels. Further, 9 promoter variants of P GAP with insertion of fapO at various sites were tested with the Prm1-FapR. It generated a biosensor of Prm1-FapR/P GAP -(+2) fapO with regulation behavior of malonyl-CoA-low-level repression/high-level derepression. Both devices were subsequently integrated into a single cell, for which fatty acid synthesis module was driven by Prm1-FapR/P GAP -(+2) fapO but 6-MSA synthesis module was expressed by Prm1-FapR/(-7) fapO -cP AOX1. The integrated system allowed continuous polyketide synthesis but malonyl-CoA-high-level "on"/low-level "off" fatty acid synthesis. This design finally increased 6-MSA production capacity by 260 %, proving the positive effects of dynamic malonyl-CoA distribution to the target compounds. It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts. [ABSTRACT FROM AUTHOR]

Malonyl-CoA is an important central metabolite serving as the basic building block for the microbial synthesis of many pharmaceutically interesting polyketides, but also fatty acid–derived compounds including biofuels. Especially Saccharomyces cerevisiae, Escherichia coli, and Corynebacterium glutamicum have been engineered towards microbial synthesis of such compounds in recent years. However, developed strains and processes often suffer from insufficient productivity. Usually, tightly regulated intracellular malonyl-CoA availability is regarded as the decisive bottleneck limiting overall product formation. Therefore, metabolic engineering towards improved malonyl-CoA availability is essential to design efficient microbial cell factories for the production of polyketides and fatty acid derivatives. This review article summarizes metabolic engineering strategies to improve intracellular malonyl-CoA formation in industrially relevant microorganisms and its impact on productivity and product range, with a focus on polyketides and other malonyl-CoA-dependent products. Key Points • Malonyl-CoA is the central building block of polyketide synthesis. • Increasing acetyl-CoA supply is pivotal to improve malonyl-CoA availability. • Improved acetyl-CoA carboxylase activity increases availability of malonyl-CoA. • Fatty acid synthesis as an ambivalent target to improve malonyl-CoA supply. [ABSTRACT FROM AUTHOR]

Glutaric acid is an important organic acid applied widely in different fields. Most previous researches have focused on the production of glutaric acid in various strains using the 5-aminovaleric acid (AMV) or pentenoic acid synthesis pathways. We previously utilized a five-step reversed adipic acid degradation pathway (RADP) in Escherichia coli BL21 (DE3) to construct strain Bgl146. Herein, we found that malonyl-CoA was strictly limited in this strain, and increasing its abundance could improve glutaric acid production. We, therefore, constructed a malonic acid uptake pathway in E. coli using matB (malonic acid synthetase) and matC (malonic acid carrier protein) from Clover rhizobia. The titer of glutaric acid was improved by 2.1-fold and 1.45-fold, respectively, reaching 0.56 g/L and 4.35 g/L in shake flask and batch fermentation following addition of malonic acid. Finally, the highest titer of glutaric acid was 6.3 g/L in fed-batch fermentation at optimized fermentation conditions. [ABSTRACT FROM AUTHOR]

Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition. [Display omitted] • ACC inhibition increases gluconeogenesis in fed mice by activating CPT-1 and PC flux • ACC inhibition unexpectedly increases anaplerosis and gluconeogenesis in fasted mice • Adaptive changes in proteostasis mediate ACC's unexpected effects during fasting • Hepatic Nrf2 is activated by ACC inhibition and may play a role in its adaptive effects Hepatic acetyl-CoA carboxylase produces malonyl-CoA during feeding to fuel lipogenesis and inhibit fat oxidation. The authors report that malonyl-CoA synthesis is also necessary to inhibit gluconeogenesis by limiting anaplerosis and TCA cycle metabolism. Surprisingly, chronic loss of malonyl-CoA altered fasting metabolism by activating Nrf2, altering proteostasis, and increasing amino acid availability. [ABSTRACT FROM AUTHOR]

The Laiwu pig is an indigenous fatty pig breed distributed in North China, characterized by an extremely high level of intramuscular fat (IMF) content (9% ∼ 12%), but the regulatory mechanism underlying intramuscular fat deposition in skeletal muscle is still unknown. In this study, the TMT-labeled quantitative malonylome of the longissimus dorsi muscle in Laiwu pigs at the fastest IMF deposition stage (240 d vs 120 d) was compared to analyze the molecular mechanism of IMF variation in pigs. In Laiwu pigs aged 240 days/120 days, we identified 291 malonylated lysine sites across 188 proteins in the longissimus dorsi muscle. Among these, 38 sites across 31 proteins exhibited differential malonylation. Annotation analysis and enrichment analysis were performed for differentially malonylated proteins (DMPs). These DMPs were mainly clustered into 12 GO functional categories accounting for 5 biological processes, 4 cellular components and 3 molecular functions, and 2 signaling pathways by KEGG enrichment analysis. The function of differentially malonylated protein ACOT7 in the process of fat deposition was further investigated during the differentiation of 3 T3-L1 cells. The results showed that the protein level of ACOT7 in 3 T3-L1 cells decreased but the malonylated level of ACOT7 increased significantly. The malonyl-CoA that is synthesized by ACSF3 affected the malonylation level of ACOT7 in 3 T3-L1 cells. The intramuscular fat (IMF) content, by affecting sensory quality traits of meat, such as tenderness, flavor and juiciness, plays an important role in meat quality. Using TMT-based quantitative malonylated proteome analysis, we identified malonylated proteins in LD muscle samples in two stages (120 d and 240 d) of development and further identified differentially malonylated proteins, such as SLC25A4, ANXA5, TPM3 and ACOT7, that are associated with intramuscular fat deposition and fat metabolism in pigs. These differentially malonylated proteins could serve as candidates for elucidating the molecular mechanism of IMF deposition in pigs. In addition, we found that the malonyl-CoA in 3 T3-L1 cells is mainly synthesized by ACSF3, affecting the malonylated level of ACOT7. The study provides some data concerning the role of protein malonylation in regulating the variation in porcine IMF content. [Display omitted] • Malonylation is a notable post-translational modification during IMF deposition from 120 d to 240 d in Laiwu pigs. • Malonylation level of 31 proteins including ACOT7 increased during IMF deposition from 120 d to 240 d in Laiwu pigs. • The level of malonylated ACOT7 increased along with fat deposition in 3 T3-L1 cells. • The activity of malonyl CoA was regulated primarily by ACSF3 in 3 T3-L1 cells. [ABSTRACT FROM AUTHOR]

Abstract Background Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts. Results Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch. Conclusions In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.

Abstract Background Malonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low. Results To identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations. The biosensor was successfully applied as a high-throughput screening tool for identifying targets at a genome-wide scale that could be critical for improving the malonyl-CoA biosynthesis in vivo. The mutant strains selected synthesized significantly higher titers of the type III polyketide triacetic acid lactone (TAL), phloroglucinol, and free fatty acids compared to the wild-type strain, using malonyl-CoA as a precursor. Conclusion These results validated this novel whole-cell biosensor as a rapid and sensitive malonyl-CoA high-throughput screening tool. Further analysis of the mutant strains showed that the iron ion concentration is closely related to the intracellular malonyl-CoA biosynthesis.

Anterograde transport of late endosomes or lysosomes (LE/Lys) is crucial for proper axon growth. However, the role of energetic nutrients has been poorly explored. Malonyl-CoA is a precursor of fatty acids, and its intracellular levels highly fluctuate depending on glucose availability or the energy sensor AMP-activated protein kinase (AMPK). We demonstrate in HeLa cells that carnitine palmitoyltransferase 1C (CPT1C) senses malonyl-CoA and enhances LE/Lys anterograde transport by interacting with the endoplasmic reticulum protein protrudin and facilitating the transfer of Kinesin-1 from protrudin to LE/Lys. In cultured mouse cortical neurons, glucose deprivation, pharmacological activation of AMPK or inhibition of malonyl-CoA synthesis decreases LE/Lys abundance at the axon terminal, and shortens axon length in a CPT1C-dependent manner. These results identify CPT1C as a new regulator of anterograde LE/Lys transport in response to malonyl-CoA changes, and give insight into how axon growth is controlled by nutrients.