Hyaluronic Acid: A Powerful Biomolecule with Wide-Ranging Applications-A Comprehensive Review.

Hyaluronic acid (HA) is a glycosaminoglycan widely distributed in the human body, especially in body fluids and the extracellular matrix of tissues. It plays a crucial role not only in maintaining tissue hydration but also in cellular processes such as proliferation, differentiation, and the inflammatory response. HA has demonstrated its efficacy as a powerful bioactive molecule not only for skin antiaging but also in atherosclerosis, cancer, and other pathological conditions. Due to its biocompatibility, biodegradability, non-toxicity, and non-immunogenicity, several HA-based biomedical products have been developed. There is an increasing focus on optimizing HA production processes to achieve high-quality, efficient, and cost-effective products. This review discusses HA's structure, properties, and production through microbial fermentation. Furthermore, it highlights the bioactive applications of HA in emerging sectors of biomedicine.

A Picrocrocin-Enriched Fraction from a Saffron Extract Affects Lipid Homeostasis in HepG2 Cells through a Non-Statin-like Mode.

Dyslipidemia is a lipid metabolism disorder associated with the loss of the physiological homeostasis that ensures safe levels of lipids in the organism. This metabolic disorder can trigger pathological conditions such as atherosclerosis and cardiovascular diseases. In this regard, statins currently represent the main pharmacological therapy, but their contraindications and side effects limit their use. This is stimulating the search for new therapeutic strategies. In this work, we investigated in HepG2 cells the hypolipidemic potential of a picrocrocin-enriched fraction, analyzed by high-resolution 1H NMR and obtained from a saffron extract, the stigmas of Crocus sativus L., a precious spice that has already displayed interesting biological properties. Spectrophotometric assays, as well as expression level of the main enzymes involved in lipid metabolism, have highlighted the interesting hypolipidemic effects of this natural compound; they seem to be exerted through a non-statin-like mechanism. Overall, this work provides new insights into the metabolic effects of picrocrocin, thus confirming the biological potential of saffron and paving the way for in vivo studies that could validate this spice or its phytocomplexes as useful adjuvants in balancing blood lipid homeostasis.

Mitochondrial transport and metabolism of the vitamin B-derived cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ , and related diseases: A review.

Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD+ , which are all structurally similar to nucleotides and derived from different B-vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease-causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor-dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ are the focus of this review.

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.

ISCA1 mutation in a patient with infantile-onset leukodystrophy causes defects in mitochondrial [4Fe-4S] proteins.

Multiple mitochondrial dysfunction syndromes (MMDS) comprise a group of severe autosomal recessive diseases characterized by impaired respiration and lipoic acid metabolism, resulting in infantile-onset mitochondrial encephalopathy, non-ketotic hyperglycinemia, myopathy, lactic acidosis and early death. Four different MMDS have been analyzed in detail according to the genes involved in the disease, MMDS1 (NFU1), MMDS2 (BOLA3), MMDS3 (IBA57) and MMDS4 (ISCA2). MMDS5 has recently been described in a clinical case report of patients carrying a mutation in ISCA1, but with no further functional analysis. ISCA1 encodes a mitochondrial protein essential for the assembly of [4Fe-4S] clusters in key metabolic and respiratory enzymes. Here, we describe a patient with a severe early onset leukodystrophy, multiple defects of respiratory complexes and a severe impairment of lipoic acid synthesis. A homozygous missense mutation in ISCA1 (c.29T>G; p.V10G) identified by targeted MitoExome sequencing resulted in dramatic reduction of ISCA1 protein level. The mutation located in the uncleaved presequence severely affected both mitochondrial import and stability of ISCA1. Down-regulation of ISCA1 in HeLa cells by RNAi impaired the biogenesis of mitochondrial [4Fe-4S] proteins, yet could be complemented by expression of wild-type ISCA1. In contrast, the ISCA1 p.V10G mutant protein only partially complemented the defects, closely resembling the biochemical phenotypes observed for ISCA1 patient fibroblasts. Collectively, our comprehensive clinical and biochemical investigations show that the ISCA1 p.V10G mutation functionally impaired mitochondrial [4Fe-4S] protein assembly and hence was causative for the observed clinical defects.

Drosophila melanogaster Mitochondrial Carriers: Similarities and Differences with the Human Carriers.

Mitochondrial carriers are a family of structurally related proteins responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. The in silico analysis of the Drosophila melanogaster genome has highlighted the presence of 48 genes encoding putative mitochondrial carriers, but only 20 have been functionally characterized. Despite most Drosophila mitochondrial carrier genes having human homologs and sharing with them 50% or higher sequence identity, D. melanogaster genes display peculiar differences from their human counterparts: (1) in the fruit fly, many genes encode more transcript isoforms or are duplicated, resulting in the presence of numerous subfamilies in the genome; (2) the expression of the energy-producing genes in D. melanogaster is coordinated from a motif known as Nuclear Respiratory Gene (NRG), a palindromic 8-bp sequence; (3) fruit-fly duplicated genes encoding mitochondrial carriers show a testis-biased expression pattern, probably in order to keep a duplicate copy in the genome. Here, we review the main features, biological activities and role in the metabolism of the D. melanogaster mitochondrial carriers characterized to date, highlighting similarities and differences with their human counterparts. Such knowledge is very important for obtaining an integrated view of mitochondrial function in D. melanogaster metabolism.

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster.

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.

New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

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.

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.

KEY POINTS: Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. ABSTRACT: Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

The mitochondrial aspartate/glutamate carrier does not transport GABA.

É£-aminobutyric acid (GABA) is a four­carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown.

Human mitochondrial uncoupling protein 3 functions as a metabolite transporter.

Since its discovery, a major debate about mitochondrial uncoupling protein 3 (UCP3) has been whether its metabolic actions result primarily from mitochondrial inner membrane proton transport, a process that decreases respiratory efficiency and ATP synthesis. However, UCP3 expression and activity are induced by conditions that would seem at odds with inefficient 'uncoupled' respiration, including fasting and exercise. Here, we demonstrate that the bacterially expressed human UCP3, reconstituted into liposomes, catalyses a strict exchange of aspartate, malate, sulphate and phosphate. The R282Q mutation abolishes the transport activity of the protein. Although the substrate specificity and inhibitor sensitivity of UCP3 display similarity with that of its close homolog UCP2, the two proteins significantly differ in their transport mode and kinetic constants.

An in vivo "turning model" reveals new RanBP9 interactions in lung macrophages.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

The mitochondrial oxoglutarate carrier: from identification to mechanism.

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carriers.

Mutant deoxynucleotide carrier is associated with congenital microcephaly.

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.

Role of Mitochondrial Transporters on Metabolic Rewiring of Pancreatic Adenocarcinoma: A Comprehensive Review.

Pancreatic cancer is among the deadliest cancers worldwide and commonly presents as pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming is a hallmark of PDAC. Glucose and glutamine metabolism are extensively rewired in order to fulfil both energetic and synthetic demands of this aggressive tumour and maintain favorable redox homeostasis. The mitochondrial pyruvate carrier (MPC), the glutamine carrier (SLC1A5_Var), the glutamate carrier (GC), the aspartate/glutamate carrier (AGC), and the uncoupling protein 2 (UCP2) have all been shown to influence PDAC cell growth and progression. The expression of MPC is downregulated in PDAC and its overexpression reduces cell growth rate, whereas the other four transporters are usually overexpressed and the loss of one or more of them renders PDAC cells unable to grow and proliferate by altering the levels of crucial metabolites such as aspartate. The aim of this review is to comprehensively evaluate the current experimental evidence about the function of these carriers in PDAC metabolic rewiring. Dissecting the precise role of these transporters in the context of the tumour microenvironment is necessary for targeted drug development.

Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N-Acetylglutamate Transporter.

The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.