Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae.

Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with in vivo continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. IMPORTANCE Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid in vivo mutagenesis. The combination of growth-based screening with in vivo mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis.

(p)ppGpp/GTP and Malonyl-CoA Modulate Staphylococcus aureus Adaptation to FASII Antibiotics and Provide a Basis for Synergistic Bi-Therapy.

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.

Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli.

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.

Modulation of the central carbon metabolism of Corynebacterium glutamicum improves malonyl-CoA availability and increases plant polyphenol synthesis.

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.

Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria.

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli, Pseudomonas putida, and Corynebacterium glutamicum RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA-derived chemicals.

Biosensor-aided high-throughput screening of hyper-producing cells for malonyl-CoA-derived products.

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.

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories.

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.

Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl-coenzyme A in breast cancer cell cultures applying online solid-phase extraction.

Cofactors such as coenzyme A and its derivatives acetyl-coenzyme A and malonyl-coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid-phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid-phase extraction was carried out by application of a weak anion-exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid-phase extraction significantly reduced matrix effects and increased the signal-to-noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF-7 breast cancer cell cultures, a commonly used model system, where acetyl-coenzyme A and malonyl-coenzyme A were determined using standard addition procedure in concentrations of 1.98 µM and 41 nM, respectively.

Development of a Synthetic Malonyl-CoA Sensor in Saccharomyces cerevisiae for Intracellular Metabolite Monitoring and Genetic Screening.

Genetic sensors capable of converting key metabolite levels to fluorescence signals enable the monitoring of intracellular compound concentrations in living cells, and emerge as an efficient tool in high-throughput genetic screening. However, the development of genetic sensors in yeasts lags far behind their development in bacteria. Here we report the design of a malonyl-CoA sensor in Saccharomyces cerevisiae using an adapted bacterial transcription factor FapR and its corresponding operator fapO to gauge intracellular malonyl-CoA levels. By combining this sensor with a genome-wide overexpression library, we identified two novel gene targets that improved intracellular malonyl-CoA concentration. We further utilized the resulting recombinant yeast strain to produce a valuable compound, 3-hydroxypropionic acid, from malonyl-CoA and enhanced its titer by 120%. Such a genetic sensor provides a powerful approach for genome-wide screening and could further improve the synthesis of a large range of chemicals derived from malonyl-CoA in yeast.

Negative feedback regulation of fatty acid production based on a malonyl-CoA sensor-actuator.

Engineering metabolic biosynthetic pathways has enabled the microbial production of many useful chemicals. However, pathway productivities and yields are often limited by metabolic imbalances. Synthetic regulatory circuits have been shown to be able to balance engineered pathways, improving titers and productivities. Here we developed a negative feedback regulatory circuit based on a malonyl-CoA-based sensor-actuator. Malonyl-CoA is biosynthesized from acetyl-CoA by the acetyl-CoA carboxylase, which is the rate-limiting step for fatty acid biosynthesis. Overexpression of acetyl-CoA carboxylase improves fatty acid production, but slows down cell growth. We have devised a malonyl-CoA sensor-actuator that controls gene expression levels based on intracellular malonyl-CoA concentrations. This sensor-actuator is used to construct a negative feedback circuit to regulate the expression of acetyl-CoA carboxylase. The negative feedback circuit is able to up-regulate acetyl-CoA carboxylase expression when the malonyl-CoA concentration is low and down-regulate acetyl-CoA carboxylase expression when excess amounts of malonyl-CoA have accumulated. We show that the regulatory circuit effectively alleviates the toxicity associated with acetyl-CoA carboxylase overexpression. When used to regulate the fatty acid pathway, the feedback circuit increases fatty acid titer and productivity by 34% and 33%, respectively.

Design and kinetic analysis of a hybrid promoter-regulator system for malonyl-CoA sensing in Escherichia coli.

Malonyl-CoA is the rate-limiting precursor involved in the chain elongation reaction of a range of value-added pharmaceuticals and biofuels. Development of malonyl-CoA responsive sensors holds great promise in overcoming critical pathway limitations and optimizing production titers and yields. By incorporating the Bacillus subtilis trans-regulatory protein FapR and the cis-regulatory element fapO, we constructed a hybrid promoter-regulatory system that responds to a broad range of intracellular malonyl-CoA concentrations (from 0.1 to 1.1 nmol/mgDW) in Escherichia coli. Elimination of regulatory protein and nonspecific DNA cross-communication leads to a sensor construct that exhibits malonyl-CoA-dependent linear phase kinetics with increased dynamic response range. The sensors reported in this study could potentially control and optimize carbon flux leading to robust biosynthetic pathways for the production of malonyl-CoA-derived compounds.

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues.

A highly sensitive and specific LC-MS/MS method was developed for simultaneous estimation of acetyl co-enzyme A (ACoA) and malonyl co-enzyme A (MCoA) in surrogate matrix using n-propionyl co-enzyme A as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. Simple acidification followed by dilution using an assay buffer process was used to extract ACoA, MCoA and IS from surrogate matrix and tissue samples. The total run time was 3 min and the elution of both analytes (ACoA, MCoA) and IS occurred at 1.28 min; this was achieved with a mobile phase consisting of 5 mM ammonium formate (pH 7.5)-acetonitrile (30:70, v/v) delivered at a flow rate of 1 mL/min on a monolithic RP-18e column. A linear response function was established for the range of concentrations 1.09-2187 and 1.09-2193 ng/mL for ACoA and MCoA, respectively. The intra- and inter-day precision values for ACoA and MCoA met the acceptance as per FDA guidelines. ACoA and MCoA were stable in a battery of stability studies viz. bench-top, auto-sampler and long-term. The developed assay was used to quantitate ACoA and MCoA levels in various tissues of rat.

Coenzyme A and its thioester pools in fasted and fed rat tissues.

Levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in fasted and fed rat tissues were analyzed by the acyl-CoA cycling method which makes detection possible at the pmol level. Malonyl-CoA in brain tissues readily increased with feeding, and inversely, acetyl-CoA decreased. This phenomenon occurred in the cerebral cortex, hippocampus, cerebellum, and medulla oblongata, as well as in the hypothalamus which controls energy balance by monitoring malonyl-CoA. In the non-brain tissues, the sizes of the acetyl-CoA, malonyl-CoA, and CoASH pools depended on the tissues. The total CoA pools consisting of the above three CoA species in the liver, heart, and brown adipose tissue were larger and those of the perirenal, epididymal, and ovarian adipose tissues were much smaller, compared with those of other tissues including brain tissues. In addition, the response of each CoA pool to feeding was not uniform, suggesting that the tissue-specific metabolism individually functions in the non-brain tissues. Thus, a comprehensive analysis of thirteen types of rat tissue revealed that CoA pools have different sizes and showed a different response to fasting and feeding depending on the tissue.

Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue.

The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoA concentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoA levels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extraction followed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoA in individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoA levels.

Decrease in malonyl-CoA and its background metabolic alterations in murine model of cancer cachexia.

The alterations of enzymatic activities involved in lipid degradation in cancer cachexia have not been fully elucidated. One of the two subclones of colon 26 adenocarcinoma, clone 20, with a potent ability to induce cachexia, or clone 5, without such an activity, was transplanted in to CDF-1 male mice. Murine livers were extirpated for analyses on the 14th day after tumor inoculation. The body weights and food intake of mice bearing clone 20 were all significantly lower than those of non-tumor bearing mice and mice bearing the clone 5 tumor. The decline of body weight was accompanied by a shrinkage of epididymal fat pads. Expression of spermidine/spermine N-1 acetyl transferase (SSAT) assessed by real-time PCR was significantly increased in cachectic mice. Conversely, acetyl-CoA carboxylase (ACC) measured by Western blotting and malonyl-CoA levels determined by malonyl-CoA:acetyl-CoA cycling procedures were decreased in cachectic mice. Indomethacin in drinking water reversed the clone 20 induced decrease in body and fat weight and food intake, and simultaneously negated the clone 20 induced increase of SSAT expressions and decrease of ACC and malonyl-CoA amounts. Because malonyl-CoA inhibits the rate-limiting step in the beta-oxidation of fatty acids, the decreased malonyl-CoA and the background metabolic alterations may contribute to the accelerated lipolysis of cancer cachexia.

Differences in concentrations of acetyl-CoA and malonyl-CoA in shoots and roots of Zea mays.

We measured the concentrations of acetyl-CoA and malonyl-CoA in shoots and roots of corn (Zea mays, L., cv. "Peter Corn"). Acetyl-CoA and malonyl-CoA concentrations were found to be relatively constant in shoots and in roots under a light-dark cycle. Acetyl-CoA concentrations were lower in shoots than in roots, whereas malonyl-CoA concentrations were higher in shoots than in roots.

Regulation of hypothalamic malonyl-CoA by central glucose and leptin.

Hypothalamic malonyl-CoA has been shown to function in global energy homeostasis by modulating food intake and energy expenditure. Little is known, however, about the regulation of malonyl-CoA concentration in the central nervous system. To address this issue we investigated the response of putative intermediates in the malonyl-CoA pathway to metabolic and endocrine cues, notably those provoked by glucose and leptin. Hypothalamic malonyl-CoA rises in proportion to the carbohydrate content of the diet consumed after food deprivation. Malonyl-CoA concentration peaks 1 h after refeeding or after peripheral glucose administration. This response depends on the dose of glucose administered and is blocked by the i.c.v. administration of an inhibitor of glucose metabolism, 2-deoxyglucose (2-DG). The kinetics of change in hypothalamic malonyl-CoA after glucose administration is coincident with the suppression of phosphorylation of AMP kinase and acetyl-CoA carboxylase. Blockade of glucose utilization in the CNS by i.c.v. 2-DG prevented the effects of glucose on 5'AMP-activated protein kinase, malonyl-CoA, hypothalamic neuropeptide expression, and food intake. Finally, we showed that leptin can increase hypothalamic malonyl-CoA and that the increase is additive with glucose administration. Leptin-deficient ob/ob mice, however, showed no defect in the glucose- or refeeding-induced rise in hypothalamic malonyl-CoA after food deprivation, demonstrating that leptin was not required for this effect. These studies show that hypothalamic malonyl-CoA responds to the level of circulating glucose and leptin, both of which affect energy homeostasis.

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS.

Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, beta-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.

Hepatic de novo lipogenesis is present in liver-specific ACC1-deficient mice.

Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes carboxylation of acetyl-CoA to form malonyl-CoA. In mammals, two isozymes exist with distinct physiological roles: cytosolic ACC1 participates in de novo lipogenesis (DNL), and mitochondrial ACC2 is involved in negative regulation of mitochondrial beta-oxidation. Since systemic ACC1 null mice were embryonic lethal, to clarify the physiological role of ACC1 in hepatic DNL, we generated the liver-specific ACC1 null mouse by crossbreeding of an Acc1(lox(ex46)) mouse, in which exon 46 of Acc1 was flanked by two loxP sequences and the liver-specific Cre transgenic mouse. In liver-specific ACC1 null mice, neither hepatic Acc1 mRNA nor protein was detected. However, to compensate for ACC1 function, hepatic ACC2 protein and activity were induced 1.4 and 2.2 times, respectively. Surprisingly, hepatic DNL and malonyl-CoA were maintained at the same physiological levels as in wild-type mice. Furthermore, hepatic DNL was completely inhibited by an ACC1/2 dual inhibitor, 5-tetradecyloxyl-2-furancarboxylic acid. These results strongly demonstrate that malonyl-CoA from ACC2 can access fatty acid synthase and become the substrate for the DNL pathway under the unphysiological circumstances that result with ACC1 disruption. Therefore, there does not appear to be strict compartmentalization of malonyl-CoA from either of the ACC isozymes in the liver.

Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects.

Increased accumulation of fatty acids and their derivatives can impair insulin-stimulated glucose disposal by skeletal muscle. To characterize the nature of the defects in lipid metabolism and to evaluate the effects of thiazolidinedione treatment, we analyzed the levels of triacylglycerol, long-chain fatty acyl-coA, malonyl-CoA, fatty acid oxidation, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), malonyl-CoA decarboxylase, and fatty acid transport proteins in muscle biopsies from nondiabetic lean, obese, and type 2 subjects before and after an euglycemic-hyperinsulinemic clamp as well as pre-and post-3-month rosiglitazone treatment. We observed that low AMPK and high ACC activities resulted in elevation of malonyl-CoA levels and lower fatty acid oxidation rates. These conditions, along with the basal higher expression levels of fatty acid transporters, led accumulation of long-chain fatty acyl-coA and triacylglycerol in insulin-resistant muscle. During the insulin infusion, muscle fatty acid oxidation was reduced to a greater extent in the lean compared with the insulin-resistant subjects. In contrast, isolated muscle mitochondria from the type 2 subjects exhibited a greater rate of fatty acid oxidation compared with the lean group. All of these abnormalities in the type 2 diabetic group were reversed by rosiglitazone treatment. In conclusion, these studies have shown that elevated malonyl-CoA levels and decreased fatty acid oxidation are key abnormalities in insulin-resistant muscle, and, in type 2 diabetic patients, thiazolidinedione treatment can reverse these abnormalities.