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The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Malonyl-coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value-added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl-CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome-reduced Pseudomonas putida strain to increase acetyl-CoA carboxylation while limiting malonyl-CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked-out in specific combinations towards increasing the malonyl-CoA pool. An enzyme-coupled biosensor, based on the rppA gene, was employed to monitor malonyl-CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl-CoA into flaviolin, a red-colored polyketide. We isolated strains displaying enhanced malonyl-CoA availability via a colorimetric screening method based on the RppA-dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl-CoA levels. We further modified these strains by adding a non-canonical pathway that uses malonyl-CoA as precursor for poly(3-hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl-CoA-dependent pathways in P. putida., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

STING (stimulator of interferon genes) is a critical immunoregulatory protein in sepsis and is regulated by various mechanisms, especially palmitoylation. FASN (fatty acid synthase) is the rate-limiting enzyme to generate cellular palmitic acid (PA) via acetyl-CoA and malonyl-CoA and participates in protein palmitoylation. However, the mechanisms underlying the interaction between STING and FASN have not been completely understood. In this study, STING-knockout mice were used to confirm the pivotal role of STING in sepsis-induced liver injury. Metabolomics confirmed the dyslipidemia in septic mice and patients. The compounds library was screened, revealing that FASN inhibitors exerted a significant inhibitory effect on the STING pathway. Mechanically, the regulatory effect of FASN on the STING pathway was dependent on palmitoylation. Further experiments indicated that the upstream of FASN, malonyl-CoA inhibited STING pathway possibly due to C91 (palmitoylated residue) of STING. Overall, this study reveals a novel paradigm of STING regulation and provides a new perspective on immunity and metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP + are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pK a value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of interests J.D.H. is a consultant for Merck, Pfizer, and Regeneron., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and this is further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Recently, the fatty acid elongation enzyme ELOVL5 was identified as a critical pro-metastatic factor in prostate cancer, required for cell growth and mitochondrial homeostasis. The fatty acid elongation reaction catalyzed by ELOVL5 utilizes malonyl-CoA as the carbon donor. Here, we demonstrate that ELOVL5 knockdown causes malonyl-CoA accumulation. Malonyl-CoA is a cellular substrate that can inhibit fatty acid β-oxidation in the mitochondria through allosteric inhibition of carnitine palmitoyltransferase 1A (CPT1A), the enzyme that controls the rate-limiting step of the long chain fatty acid β-oxidation cycle. We hypothesized that changes in malonyl-CoA abundance following ELOVL5 knockdown could influence mitochondrial β-oxidation rates in prostate cancer cells, and regulate cell viability. Accordingly, we find that ELOVL5 knockdown is associated with decreased mitochondrial β-oxidation in prostate cancer cells. Combining ELOVL5 knockdown with FASN inhibition to increase malonyl-CoA abundance endogenously enhances the effect of ELOVL5 knockdown on prostate cancer cell viability, while preventing malonyl-CoA production rescues the cells from the effect of ELOVL5 knockdown. Our findings indicate an additional role for fatty acid elongation, in the control of malonyl-CoA homeostasis, alongside its established role in the production of long-chain fatty acid species, to explain the importance of fatty acid elongation for cell viability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding., (© 2023. The Author(s).)

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Cholecalciferol improves insulin signaling and glucose metabolism in the heart and reduces circulating non-esterified fatty acids. Cholecalciferol effects on the cardiac fatty acid (FA) metabolism and the consequences on calcium handling were examined. Blood lipid profile was determined. Western blot and qRT-PCR were used to examine protein and mRNA expression. Cholecalciferoltreated rats had increased acetyl CoA carboxylase 2 protein expression and decreased expression of malonyl CoA decarboxylase. In addition, the expression of uncoupling protein 3 was elevated. Also, the level of peroxisome proliferator-activated receptor-gamma coactivator in the nucleus of heart cells was increased along with the level of sarcoplasmic/endoplasmic reticulum Ca2+ATPase in the microsomal fraction. In parallel, the L-type calcium channel and ryanodine receptor expression was reduced. In the heart of healthy rats, cholecalciferol affects proteins regulating malonyl CoA availability and intracellular Ca2+ handling proteins.

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., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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- 13 C] 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.

The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae ( Schizochytrium ) and marine bacteria ( Moritella marina , Photobacterium profundum , and Shewanella ). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium . Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 C β -O γ bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (N ε2 ) and Ser154 (O γ ), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.

3-Hydroxypropionate (3-HP) is a platform chemical for production of acrylic acid, acrylamide and biodegradable polymers. Several microbial cell factories have been constructed for production of 3-HP from malonyl-CoA by using a malonyl-CoA reductase, which however suffer from inadequate supply of precursor and cofactor. Here 3-HP biosynthesis was optimized in a super yeast chassis with sufficient supply of precursor malonyl-CoA and cofactor NADPH, which had a 3-fold higher 3-HP (1.4 g/L) than that of wild-type background. The instability of the engineered strain was observed in fed-batch fermentation due to the plasmid loss, which may be caused by the toxic intermediate malonate semialdehyde. Genome integration of MCR-C encoding C-terminal of MCR enabled stable gene expression and much higher 3-HP production of 4.4 g/L under batch fermentation and 56.5 g/L under fed-batch fermentation with a yield of 0.31 g/g glucose. This was the highest 3-HP production reported from glucose in engineered microbes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The expression of exogenous genes encoding acetyl-CoA carboxylase (Acc) and pantothenate kinase (CoaA) in Escherichia coli enable highly effective fatty acid production. Acc-only strains grown at 37 °C or 23 °C produced an approximately twofold increase in fatty acid content, and additional expression of CoaA achieved a further twofold accumulation. In the presence of pantothenate, which is the starting material for the CoA biosynthetic pathway, the size of the intracellular CoA pool at 23 °C was comparable to that at 30 °C during cultivation, and more than 500 mg/L of culture containing cellular fatty acids was produced, even at 23 °C. However, the highest yield of cellular fatty acids (1100 mg/L of culture) was produced in cells possessing the gene encoding type I bacterial fatty acid synthase (FasA) along with the acc and coaA, when the transformant was cultivated at 30 °C in M9 minimal salt medium without pantothenate or IPTG. This E. coli transformant contained 141 mg/L of oleic acid attributed to FasA under noninducible conditions. The increased fatty acid content was brought about by a greatly improved specific productivity of 289 mg/g of dry cell weight. Thus, the effectiveness of the foreign acc and coaA in fatty acid production was unambiguously confirmed at culture temperatures of 23 °C to 37 °C. Cofactor engineering in E. coli using the exogenous coaA and acc genes resulted in fatty acid production over 1 g/L of culture and could effectively function at 23 °C., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed acc sr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the acc sr gene and by the concentration of malonyl-CoA, the acc sr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed acc sr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by acc sr gene over-expression led to the improvement in rimocidin production., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nutrients, hormones and the energy sensor AMP-activated protein kinase (AMPK) tightly regulate the intracellular levels of the metabolic intermediary malonyl-CoA, which is a precursor of fatty acid synthesis and a negative regulator of fatty acid oxidation. In the brain, the involvement of malonyl-CoA in the control of food intake and energy homeostasis has been known for decades. However, recent data uncover a new role in cognition and brain development. The sensing of malonyl-CoA by carnitine palmitoyltransferase 1 (CPT1) proteins regulates a variety of functions, such as the fate of neuronal stem cell precursors, the motility of lysosomes in developing axons, the trafficking of glutamate receptors to the neuron surface (necessary for proper synaptic function) and the metabolic coupling between astrocytes and neurons. We discuss the relevance of those recent findings evidencing how nutrients and metabolic disorders impact cognition. We also enumerate all nutritional and hormonal conditions that are known to regulate malonyl-CoA levels in the brain, reflect on protein malonylation as a new post-translational modification, and give a reasoned vision of the opportunities and challenges that future research in the field could address., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l -1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

A highly oxygenated phenethyl derivative ustethylin A was isolated from Aspergillus ustus . Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one O -methyltransferase lead to the final product, ustethylin A.

We recently developed an Aspergillus oryzae strain in which malonyl-coenzyme A (CoA) supply is strengthened by the deletion of snfA and SCAP as an efficient host to produce a plant polyketide, curcumin. Here, we examined the effectiveness of this strain in producing another polyketide, atrochrysone carboxylic acid (ACA), which is synthesized from eight molecules of malonyl-CoA using an iterative type I polyketide synthase, ACA synthase (ACAS), and atrochrysone carboxyl ACP thioesterase (ACTE) in Aspergillus terreus. When ACAS and ACTE were introduced, the A. oryzae ΔsnfAΔSCAP strain produced approximately four times more ACA-related polyketides than did the control strain expressing both genes. This result further demonstrated the availability of the A. oryzae ΔsnfAΔSCAP strain for heterologous polyketide production.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gap junctions are ubiquitous in metazoans and play critical roles in important biological processes, including electrical conduction and development. Yet, only a few defined molecules passing through gap junction channels have been linked to specific functions. We isolated gap junction channel mutants that reduce coupling between the soma and germ cells in the Caenorhabditis elegans gonad. We provide evidence that malonyl-CoA, the rate-limiting substrate for fatty acid synthesis (FAS), is produced in the soma and delivered through gap junctions to the germline; there it is used in fatty acid synthesis to critically support embryonic development. Separation of malonyl-CoA production from its site of utilization facilitates somatic control of germline development. Additionally, we demonstrate that loss of malonyl-CoA production in the intestine negatively impacts germline development independently of FAS. Our results suggest that metabolic outsourcing of malonyl-CoA may be a strategy by which the soma communicates nutritional status to the germline., Competing Interests: TS, XB, DG No competing interests declared

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.

There has been recent interest in creating an efficient microbial production route for 3-hydroxypropionic acid, an important platform chemical. We develop and solve a mathematical model for the time-dependent metabolite concentrations in the malonyl-CoA pathway for 3-hydroxypropionic acid production in microbes, using a combination of numerical and asymptotic methods. This allows us to identify the most important targets for enzyme regulation therein under conditions of plentiful and sparse pyruvate, and to quantify their relative importance. In our model, we account for sinks of acetyl-CoA and malonyl-CoA to, for example, the citric acid cycle and fatty acid biosynthesis, respectively. Notably, in the plentiful pyruvate case we determine that there is a bifurcation in the asymptotic structure of the system, the crossing of which corresponds to a significant increase in 3-hydroxypropionic acid production. Moreover, we deduce that the most significant increases to 3-hydroxypropionic acid production can be obtained by up-regulating two specific enzymes in tandem, as the inherent nonlinearity of the system means that a solo up-regulation of either does not result in large increases in production. The types of issue arising here are prevalent in synthetic biology applications, and it is hoped that the system considered provides an instructive exemplar for broader applications.

Feeding behavior is closely related to hypothalamic malonyl-CoA level in the brain and diet-induced obesity affects total CoA pools in liver. Herein, we performed a comprehensive analysis of the CoA pools formed in thirteen tissues of Zucker and Zucker diabetic fatty (ZDF) rats. Hypothalamic malonyl-CoA levels in obese rats remained low and were almost the same as those of lean rats, despite obese rats having much higher content of leptin, insulin, and glucose in their sera. Regardless of the fa -genotypes, larger total CoA pools were formed in the livers of ZDF rats and the size of hepatic total CoA pools in Zucker rats showed almost one tenth of the size of ZDF rats. The decreased total CoA pool sizes in Zucker rats was observed in the brown adipose tissues, while ZDF-fatty rats possessed 6% of total CoA pool in the lean rats in response to fa deficiency. This substantially lower CoA content in the obese rats would be disadvantageous to non-shivering thermogenesis. Thus, comparing the intracellular CoA behaviors between Zucker and ZDF rats, as well as the lean and fatty rats of each strain would help to elucidate features of obesity and type 2 diabetes in combination with result (s) of differential gene expression analysis and/or comparative genomics.

The bi-functional malonyl-CoA reductase is a key enzyme of the 3-hydroxypropionate bi-cycle for bacterial CO 2 fixation, catalysing the reduction of malonyl-CoA to malonate semialdehyde and further reduction to 3-hydroxypropionate. Here, we report the crystal structure and the full-length architecture of malonyl-CoA reductase from Porphyrobacter dokdonensis. The malonyl-CoA reductase monomer of 1230 amino acids consists of four tandemly arranged short-chain dehydrogenases/reductases, with two catalytic and two non-catalytic short-chain dehydrogenases/reductases, and forms a homodimer through paring contact of two malonyl-CoA reductase monomers. The complex structures with its cofactors and substrates revealed that the malonyl-CoA substrate site is formed by the cooperation of two short-chain dehydrogenases/reductases and one novel extra domain, while only one catalytic short-chain dehydrogenase/reductase contributes to the formation of the malonic semialdehyde-binding site. The phylogenetic and structural analyses also suggest that the bacterial bi-functional malonyl-CoA has a structural origin that is completely different from the archaeal mono-functional malonyl-CoA and malonic semialdehyde reductase, and thereby constitute an efficient enzyme., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Significant advances have been made in deciphering the mechanisms underlying fuel-stimulated insulin secretion by pancreatic beta cells. The contribution of the triggering/ATP-sensitive potassium (K ATP )-dependent Ca 2+ signalling and K ATP -independent amplification pathways, that include anaplerosis and lipid signalling of glucose-stimulated insulin secretion (GSIS), are well established. A proposed model included a key role for a metabolic partitioning 'switch', the acetyl-CoA carboxylase (ACC)/malonyl-CoA/carnitine palmitoyltransferase-1 (CPT-1) axis, in beta cell glucose and fatty acid signalling for insulin secretion. This model has gained overwhelming support from a number of studies in recent years and is now refined through its link to the glycerolipid/NEFA cycle that provides lipid signals through its lipolysis arm. Furthermore, acetyl-CoA carboxylase may also control beta cell growth. Here we review the evidence supporting a role for the ACC/malonyl-CoA/CPT-1 axis in the control of GSIS and its particular importance under conditions of elevated fatty acids (e.g. fasting, excess nutrients, hyperlipidaemia and diabetes). We also document how it is linked to a more global lipid signalling system that includes the glycerolipid/NEFA cycle.

Curcuminoids are natural phenylpropanoids that are biosynthesized via an L-phenylalanine metabolism pathway in turmeric (Curcuma longa L.). Curcuminoids have various chemopreventive activities and pharmaceutical applications in human life. In this study, we synthesized dicinnamoylmethane, one principal component of curcuminoids, from cinnamic acid by means of co-expression of Oryza sativa curcuminoid synthase and Petroselinum crispum 4-coumarate-CoA ligase in Escherichia coli BL21 (DE3). Moreover, we used CRISPRi systems to knock down the genes in a tricarboxylic acid cycle and fatty acid biosynthesis pathway. The repression of target genes led to an increase of up to 0.236 μmol g -1 DCW of malonyl-CoA in cytosol-engineered E. coli and subsequently increased the biosynthesis of dicinnamoylmethane. We found that the S10 strain containing a CRISPRi repression for three genes, fabF, fabD, and mdh, showed the highest amount of dicinnamoylmethane of 7.54 μM, which is 5.76-fold higher than that of the wild-type strain. Finally, 41.94 μM (~ 11.6 mg) of dicinnamoylmethane was obtained in a 3-L fermenter.

Deuterated water ( 2 H 2 O) is widely used for measuring de novo lipogenesis (DNL). 2 H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2 H enrichment requires correcting. In mice, we measured the 2 H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U- 2 H 7 ]glucose to determine 2 H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2 H enrichments were compared with 13 C enrichment of the same sites from an equivalent amount of [U- 13 C 6 ]glucose provided alongside the [U- 2 H 7 ]glucose tracer. Transfer of glucose 2 H to fatty acid position 2 and methyl sites was low ( 2 H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13 C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2 H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13 C, P < 10 -5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U- 13 C 6 ]glucose and 2 H 2 O had equivalent 2 H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2 H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2 H enrichment from 2 H 2 O prior to DNL. Under such conditions, 2 H enrichment of fatty acids from 2 H 2 O does not need correction., (Copyright © 2019 Belew et al.)

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae . Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 μg/plate) on an agar medium containing feruloyl- N -acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP ; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus . This study provided useful information for heterologous polyketide production in A. oryzae .

The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription-translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse α-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L -1 (0.088 mM) naringenin or 112 mg·L -1 (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis., (© 2019 Wiley Periodicals, Inc.)

Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace&#95;4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE&#95;4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace&#95;4237 is essential for S. erythraea survival as determined by the construction of a sace&#95;4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.

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 C 1 /C 6 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 P O6 -iolT1 ∆pyc allowed for synthesis of 53.32 mg/L (0.278 mM) noreugenin in CGXII medium supplemented with casamino acids within 24 h.

Malonyl-CoA is one of the key metabolic intermediates in fatty acid metabolism as well as a key player in protein post-translational modifications. Detection of malonyl-CoA in live cells is challenging because of the lack of effective measuring tools. Here we developed a genetically encoded biosensor, FapR-NLuc, by combining a malonyl-CoA responsive bacterial transcriptional factor, FapR, with an engineered luciferase, NanoLuciferase (NLuc). FapR-NLuc specifically responds to malonyl-CoA and enables the rapid detection of malonyl-CoA at the micromolar level. More importantly, it is reflective of the fluctuations of malonyl-CoA in live cells. Upon being targeted to subcellular compartments, this biosensor can detect the changes of malonyl-CoA in situ within organelles. Thus, FapR-NLuc can potentially be used as a tool to study the kinetics of malonyl-CoA in live cells, which will shed light on the underlying mechanisms of malonyl-CoA-mediated biological processes.

Starvation of essential nutrients, such as nitrogen, sulfur, magnesium, and phosphorus, leads cells into stationary phase and potentially enhances target metabolite production because cells do not consume carbon for the biomass synthesis. The overall metabolic behavior changes depend on the type of nutrient starvation in Escherichia coli. In the present study, we determined the optimum nutrient starvation type for producing malonyl-CoA-derived metabolites such as 3-hydroxypropionic acid (3HP) and naringenin in E. coli. For 3HP production, high production titer (2.3 or 2.0 mM) and high specific production rate (0.14 or 0.28 mmol gCDW -1 h -1 ) was observed under sulfur or magnesium starvation, whereas almost no 3HP production was detected under nitrogen or phosphorus starvation. Metabolic profiling analysis revealed that the intracellular malonyl-CoA concentration was significantly increased under the 3HP producing conditions. This accumulation should contribute to the 3HP production because malonyl-CoA is a precursor of 3HP. Strong positive correlation (r = 0.95) between intracellular concentrations of ATP and malonyl-CoA indicates that the ATP level is important for malonyl-CoA synthesis due to the ATP requirement by acetyl-CoA carboxylase. For naringenin production, magnesium starvation led to the highest production titer (144 ± 15 μM) and specific productivity (127 ± 21 μmol gCDW -1 ). These results demonstrated that magnesium starvation is a useful approach to improve the metabolic state of strains engineered for the production of malonyl-CoA derivatives., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Malonyl-CoA is a central metabolite in fatty acid biochemistry. It is the rate-determining intermediate in fatty acid synthesis but is also an allosteric inhibitor of the rate-setting step in mitochondrial long-chain fatty acid oxidation. While these canonical cytoplasmic roles of malonyl-CoA have been well described, malonyl-CoA can also be generated within the mitochondrial matrix by an alternative pathway: the ATP-dependent ligation of malonate to Coenzyme A by the malonyl-CoA synthetase ACSF3. Malonate, a competitive inhibitor of succinate dehydrogenase of the TCA cycle, is a potent inhibitor of mitochondrial respiration. A major role for ACSF3 is to provide a metabolic pathway for the clearance of malonate by the generation of malonyl-CoA, which can then be decarboxylated to acetyl-CoA by malonyl-CoA decarboxylase. Additionally, ACSF3-derived malonyl-CoA can be used to malonylate lysine residues on proteins within the matrix of mitochondria, possibly adding another regulatory layer to post-translational control of mitochondrial metabolism. The discovery of ACSF3-mediated generation of malonyl-CoA defines a new mitochondrial metabolic pathway and raises new questions about how the metabolic fates of this multifunctional metabolite intersect with mitochondrial metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN KD ) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN KD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN KD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs., (© 2018 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Omega-3 polyunsaturated fatty acids (PUFA) are produced in some unicellular organisms, such as marine gammaproteobacteria, myxobacteria, and thraustochytrids, by large enzyme complexes called PUFA synthases. These enzymatic complexes resemble bacterial antibiotic-producing proteins known as polyketide synthases (PKS). One of the PUFA synthase subunits is a conserved large protein (PfaA in marine proteobacteria) that contains three to nine tandem acyl carrier protein (ACP) domains as well as condensation and modification domains. In this work, a study of the PfaA architecture and its ability to initiate the synthesis by selecting malonyl units has been carried out. As a result, we have observed a self-acylation ability in tandem ACPs whose biochemical mechanism differ from the previously described for type II PKS. The acyltransferase domain of PfaA showed a high selectivity for malonyl-CoA that efficiently loads onto the ACPs domains. These results, together with the structural organization predicted for PfaA, suggest that this protein plays a key role at early stages of the anaerobic pathway of PUFA synthesis., (© 2018 Santín and Moncalián.)

Aims: Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism., Methods and Results: We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis., Conclusions: Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

3-Hydroxypropionate (3HP) is an attractive platform chemical, serving as a precursor to a variety of commodity chemicals like acrylate and acrylamide, as well as a monomer of a biodegradable plastic. To establish a sustainable way to produce these commercially important chemicals and materials, fermentative production of 3HP is widely investigated in recent years. It is reported that 3HP can be produced from several intermediates, such as glycerol, malonyl-CoA, and β-alanine. Among all these biosynthetic routes, the malonyl-CoA pathway has some distinct advantages, including a broad feedstock spectrum, thermodynamic feasibility, and redox neutrality. To date, this pathway has been successfully constructed in various species including Escherichia coli, yeast and cyanobacteria, and optimized through carbon flux redirection, enzyme screening and engineering, and an increasing supply of energy and cofactors, resulting in significantly enhanced 3HP titer up to 40 g/L. These results show the feasibility of commercial manufacturing of 3HP and its derivatives in the future.

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)