Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection.

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

RATIONALE: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. CONCLUSIONS: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate.

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.

Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice.

Marked elevation in the brain concentration of N-acetyl-L-aspartate (NAA) is a characteristic feature of Canavan disease, a vacuolar leukodystrophy resulting from deficiency of the oligodendroglial NAA-cleaving enzyme aspartoacylase. We now demonstrate that inhibiting NAA synthesis by intracisternal administration of a locked nucleic acid antisense oligonucleotide to young-adult aspartoacylase-deficient mice reverses their pre-existing ataxia and diminishes cerebellar and thalamic vacuolation and Purkinje cell dendritic atrophy. Ann Neurol 2020;87:480-485.

Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria.

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative ß-alanine with high stoichiometric yield.

L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2 mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01 mol L-aspartate/mol glucose and 1.52 mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249 mM (33.1 g/L) L-aspartate or 424 mM (37.7 g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.

One-Pot Biosynthesis of l-Aspartate from Maleate via an Engineered Strain Containing a Dual-Enzyme System.

l-Aspartate has been widely used in medicine and the food and chemical industries. In this study, Serratia marcescens maleate cis-trans isomerase (MaiA) and Escherichia coli aspartase (AspA) were coupled and coexpressed in an engineered E. coli strain in which the byproduct metabolic pathway was inactivated. The engineered E. coli strain containing the dual-enzyme system (pMA) was employed to bioproduce l-aspartate from maleate with a conversion of 98%. We optimized the activity ratio of double enzymes through ribosome binding site (RBS) regulation and molecular modification of MaiA, resulting in an engineered strain: pMA-RBS4-G27A/G171A. The conversion of l-aspartate biotransformed from maleate using the pMA-RBS4-G27A/G171A strain was almost 100%. It required 40 min to complete the whole-cell catalysis, without the intermediate product and byproduct, compared to 120 min before optimization. The induction timing and the amount of inducer in a 5-liter fermentor were optimized for scale-up of the production of l-aspartate. The amount of produced l-aspartate using the cells obtained by fermentation reached 419.8 g/liter (3.15 M), and the conversion was 98.4%. Our study demonstrated an environmentally responsible and efficient method to bioproduce l-aspartate from maleate and provided an available pathway for the industrial production of l-aspartate. This work should greatly improve the economic benefits of l-aspartate, which can now be simply produced from maleate by the engineered strain constructed based on dual-enzyme coupling.IMPORTANCE l-Aspartate is currently produced from fumarate by biological methods, and fumarate is synthesized from maleate by chemical methods in industry. We established a biosynthesis method to produce l-aspartate from maleate that is environmentally responsible, convenient, and efficient. Compared to conventional l-aspartate production, no separation and purification of intermediate products is required, which could greatly improve production efficiency and reduce costs. As environmental issues are attracting increasing attention, conventional chemical methods gradually will be replaced by biological methods. Our results lay an important foundation for the industrialization of l-aspartate biosynthesis from maleate.

Suppressing N-Acetyl-l-Aspartate Synthesis Prevents Loss of Neurons in a Murine Model of Canavan Leukodystrophy.

Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in AspaNur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like. SIGNIFICANCE STATEMENT: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.

ß-Hydroxyaspartic acid in siderophores: biosynthesis and reactivity.

A growing number of siderophores are found to contain ß-hydroxyaspartic acid (ß-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic ß-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl ß-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of ß-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for ß-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Aspartate Glycosylation Triggers Isomerization to Isoaspartate.

O-Linked ß-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate.

The gut microbiome has been shown to regulate the development and functions of the enteric and central nervous systems. Its involvement in the regulation of behavior has attracted particular attention because of its potential translational importance in clinical disorders, however little is known about the pathways involved. We previously have demonstrated that administration of Lactobacillus rhamnosus (JB-1) to healthy male BALB/c mice, promotes consistent changes in GABA-A and -B receptor sub-types in specific brain regions, accompanied by reductions in anxiety and depression-related behaviors. In the present study, using magnetic resonance spectroscopy (MRS), we quantitatively assessed two clinically validated biomarkers of brain activity and function, glutamate+glutamine (Glx) and total N-acetyl aspartate+N-acetyl aspartyl glutamic acid (tNAA), as well as GABA, the chief brain inhibitory neurotransmitter. Mice received 1×10(9) cfu of JB-1 per day for 4weeks and were subjected to MRS weekly and again 4weeks after cessation of treatment to ascertain temporal changes in these neurometabolites. Baseline concentrations for Glx, tNAA and GABA were equal to 10.4±0.3mM, 8.7±0.1mM, and 1.2±0.1mM, respectively. Delayed increases were first seen for Glx (~10%) and NAA (~37%) at 2weeks which persisted only to the end of treatment. However, Glx was still elevated 4weeks after treatment had ceased. Significantly elevated GABA (~25%) was only seen at 4weeks. These results suggest specific metabolic pathways in our pursuit of mechanisms of action of psychoactive bacteria. They also offer through application of standard clinical neurodiagnostic techniques, translational opportunities to assess biomarkers accompanying behavioral changes induced by alterations in the gut microbiome.

Identification and characterization of natural microbial products that alter the free d-aspartate content of mammalian cells.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.