Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Hepatic malonyl-CoA synthesis restrains gluconeogenesis by suppressing fat oxidation, pyruvate carboxylation, and amino acid availability.

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.

Unique Behavior of Bacterially Expressed Rat Carnitine Palmitoyltransferase 2 and Its Catalytic Activity.

Mammalian type 2 carnitine parmitoyltransferase (EC 2.3.1.21), abbreviated as CPT2, is an enzyme involved in the translocation of fatty acid into the mitochondrial matrix space, and catalyzes the reaction acylcarnitine + CoA = acyl-CoA + carnitine. When rat CPT2 was expressed in Escherichia coli, its behavior was dependent on the presence or absence of i) its mitochondrial localization sequence and ii) a short amino acid sequence thought to anchor it to the mitochondrial inner membrane: CPT2 containing both sequences behaved as a hydrophobic protein, while recombinant CPT2 lacking both regions behaved as a water soluble protein; if only one region was present, the resultant proteins were observed in both fractions. Because relatively few protein species could be obtained from bacterial lysates as insoluble pellets under the experimental conditions used, selective enrichment of recombinant CPT2 protein containing both hydrophobic sequences was easily achieved. Furthermore, when CPT2 enriched in insoluble fraction was resuspended in an appropriate medium, it showed catalytic activity typical of CPT2: it was completely suppressed by the CPT2 inhibitor, ST1326, but not by the CPT1 inhibitor, malonyl-CoA. Therefore, we conclude that the bacterial expression system is an effective tool for characterization studies of mammalian CPT2.

Dynamic lid domain of Chloroflexus aurantiacus Malonyl-CoA reductase controls the reaction.

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.

Fatty acid elongation regulates mitochondrial ß-oxidation and cell viability in prostate cancer by controlling malonyl-CoA levels.

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 ß-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 ß-oxidation cycle. We hypothesized that changes in malonyl-CoA abundance following ELOVL5 knockdown could influence mitochondrial ß-oxidation rates in prostate cancer cells, and regulate cell viability. Accordingly, we find that ELOVL5 knockdown is associated with decreased mitochondrial ß-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.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the ß-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

Quantitative Analysis of Acetyl-CoA, Malonyl-CoA, and Succinyl-CoA in Myocytes.

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.

Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering.

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.

Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor.

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.

Structural basis of a bi-functional malonyl-CoA reductase (MCR) from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii.

Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

Direct Utilization of Peroxisomal Acetyl-CoA for the Synthesis of Polyketide Compounds in Saccharomyces cerevisiae.

Polyketides are a class of natural products with many applications but are mainly appealing as pharmaceuticals. Heterologous production of polyketides in the yeast Saccharomyces cerevisiae has been widely explored because of the many merits of this model eukaryotic microorganism. Although acetyl-CoA and malonyl-CoA, the precursors for polyketide synthesis, are distributed in several yeast subcellular organelles, only cytosolic synthesis of polyketides has been pursued in previous studies. In this study, we investigate polyketide synthesis by directly using acetyl-CoA in the peroxisomes of yeast strain CEN.PK2-1D. We first demonstrate that the polyketide flaviolin can be synthesized in this organelle upon peroxisomal colocalization of native acetyl-CoA carboxylase and 1,3,6,8-tetrahydroxynaphthalene synthase (a type III polyketide synthase). Next, using the synthesis of the polyketide triacetic acid lactone as an example, we show that (1) a new peroxisome targeting sequence, pPTS1, is more effective than the previously reported ePTS1 for peroxisomal polyketide synthesis; (2) engineering peroxisome proliferation is effective to boost polyketide production; and (3) peroxisomes provide an additional acetyl-CoA reservoir and extra space to accommodate enzymes so that utilizing the peroxisomal pathway plus the cytosolic pathway produces more polyketide than the cytosolic pathway alone. This research lays the groundwork for more efficient heterologous polyketide biosynthesis using acetyl-CoA pools in subcellular organelles.

Cholecalciferol affects cardiac proteins regulating malonyl-CoA availability and intracellular calcium level.

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.

A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis.

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.

SGLT2i alleviates epicardial adipose tissue inflammation by modulating ketone body-glyceraldehyde-3-phosphate dehydrogenase malonylation pathway.

AIMS: Inflammation in the epicardial adipose tissue (EAT) is a contributor to atrial fibrillation. Studies have reported that sodium glucose co-transporter 2 inhibitor (SGLT2i) can alleviate EAT inflammation. However, the mechanism remains elusive. This study aims to investigate the molecular mechanism of SGLT2i in reducing EAT inflammation and to explore the effects of SGLT2i on atrial fibrosis in atrial fibrillation. METHODS: Sprague-Dawley rats were injected with angiotensin II to induce atrial fibrillation and randomly assigned to receive SGLT2i ( n  = 6) or vehicle ( n  = 6). Macrophages (RAW264.7) were treated with ketone bodies; ACC1 knockdown/overexpression and malonyl-CoA overexpression were performed in vitro . The levels of inflammatory cytokines, ACC1, and malonyl-CoA were examined by ELISA. GAPDH malonylation was measured by co-immunoprecipitation. RESULTS: In atrial fibrillation rats, SGLT2i increased the ketone body levels and decreased the expression of ACC1 and alleviated EAT inflammation and atrial fibrosis. In RAW264.7 cells, ketone bodies decreased the levels of ACC1, malonyl-CoA, and GAPDH malonylation, accompanied by reduced inflammatory cytokines. ACC1 knockdown decreased the expression of malonyl-CoA and GAPDH malonylation and alleviated lipopolysaccharide (LPS)-induced macrophage inflammation; these effects were inhibited by malonyl-CoA overexpression. Furthermore, the protective effects of ketone bodies on macrophage inflammation were abrogated by ACC1 overexpression. CONCLUSION: SGLT2i alleviates EAT inflammation by reducing GAPDH malonylation via downregulating the expression of ACC1 through increasing ketone bodies, thus attenuating atrial fibrosis.

Malonyl-CoA Accumulation as a Compensatory Cytoprotective Mechanism in Cardiac Cells in Response to 7-Ketocholesterol-Induced Growth Retardation.

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 ß-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.

Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.

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 ß-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.

Characterization of Unexpected Self-Acylation Activity of Acyl Carrier Proteins in a Modular Type I Apicomplexan Polyketide Synthase.

Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylation─or substrate acylation without an AT domain─for three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2-4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.

Engineering a Novel Metabolic Pathway for Improving Cellular Malonyl-CoA Levels in Escherichia coli.

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.