The aminosalicylate - folate connection.

Two aminosalicylate isomers have been found to possess useful pharmacological behavior: p-aminosalicylate (PAS, 4AS) is an anti-tubercular agent that targets M. tuberculosis, and 5-aminosalicylate (5AS, mesalamine, mesalazine) is used in the treatment of ulcerative colitis (UC) and other inflammatory bowel diseases (IBD). PAS, a structural analog of pABA, is biosynthetically incorporated by bacterial dihydropteroate synthase (DHPS), ultimately yielding a dihydrofolate (DHF) analog containing an additional hydroxyl group in the pABA ring: 2'-hydroxy-7,8-dihydrofolate. It has been reported to perturb folate metabolism in M. tuberculosis, and to selectively target M. tuberculosis dihydrofolate reductase (mtDHFR). Studies of PAS metabolism are reviewed, and possible mechanisms for its mtDHFR inhibition are considered. Although 5AS is a more distant structural relative of pABA, multiple lines of evidence suggest a related role as a pABA antagonist that inhibits bacterial folate biosynthesis. Structural data support the likelihood that 5AS is recognized by the DHPS pABA binding site, and its effects probably range from blocking pABA binding to formation of a dead-end dihydropterin-5AS adduct. These studies suggest that mesalamine acts as a gut bacteria-directed antifolate, that selectively targets faster growing, more folate-dependent species.

p-Aminobenzoic acid inhibits the growth of soybean pathogen Xanthomonas axonopodis pv. glycines by altering outer membrane integrity.

BACKGROUND: p-Aminobenzoic acid (pABA) is an environmentally friendly bioactive metabolite synthesized by Lysobacter antibioticus. This compound showed an unusual antifungal mode of action based on cytokinesis inhibition. However, the potential antibacterial properties of pABA remain unexplored. RESULTS: In this study, pABA showed antibacterial activity against Gram-negative bacteria. This metabolite inhibited growth (EC50 = 4.02 mM), and reduced swimming motility, extracellular protease activity, and biofilm formation in the soybean pathogen Xanthomonas axonopodis pv. glycines (Xag). Although pABA was previously reported to inhibit fungal cell division, no apparent effect was observed on Xag cell division genes. Instead, pABA reduced the expression of various membrane integrity-related genes, such as cirA, czcA, czcB, emrE, and tolC. Consistently, scanning electron microscopy observations revealed that pABA caused major alternations in Xag morphology and blocked the formation of bacterial consortiums. In addition, pABA reduced the content and profile of outer membrane proteins and lipopolysaccharides in Xag, which may explain the observed effects. Preventive and curative applications of 10 mM pABA reduced Xag symptoms in soybean plants by 52.1% and 75.2%, respectively. CONCLUSIONS: The antibacterial properties of pABA were studied for the first time, revealing new insights into its potential application for the management of bacterial pathogens. Although pABA was previously reported to show an antifungal mode of action based on cytokinesis inhibition, this compound inhibited Xag growth by altering the outer membrane's integrity. © 2023 Society of Chemical Industry.

Molecular docking and antimalarial evaluation of hybrid para-aminobenzoic acid 1,3,5 triazine derivatives via inhibition of Pf-DHFR.

In this study, a structurally guided pharmacophore hybridization strategy is used to combine the two key structural scaffolds, para-aminobenzoic acid (PABA), and 1,3,5 triazine in search of new series of antimalarial agents. A combinatorial library of 100 compounds was prepared in five different series as [4A (1-22), 4B (1-21), 4 C (1-20), 4D (1-19) and 4E (1-18)] using different primary and secondary amines, from where 10 compounds were finally screened out through molecular property filter analysis and molecular docking study as promising PABA substituted 1,3,5-triazine scaffold as an antimalarial agent. The docking results showed that compounds 4A12 and 4A20 exhibited good binding interaction with Phe58, IIe164, Ser111, Arg122, Asp54 (-424.19 to -360.34 kcal/mol) and Arg122, Phe116, Ser111, Phe58 (-506.29 to -431.75 kcal/mol) against wild (1J3I) and quadruple mutant (1J3K) type of Pf-DHFR. These compounds were synthesized by conventional as well as microwave-assisted synthesis and characterized by different spectroscopic methods. In-vitro antimalarial activity results indicated that two compounds 4A12 and 4A20 showed promising antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of Plasmodium falciparum with IC50 (1.24-4.77 µg mL-1) and (2.11-3.60 µg mL-1). These hybrid PABA substituted 1,3,5-triazine derivatives might be used in the lead discovery towards a new class of Pf-DHFR inhibitors.Communicated by Ramaswamy H. Sarma.

Discovery of a Colon-Targeted Azo Prodrug of Tofacitinib through the Establishment of Colon-Specific Delivery Systems Constructed by 5-ASA-PABA-MAC and 5-ASA-PABA-Diamine for the Treatment of Ulcerative Colitis.

To mitigate the systemic adverse effects of tofacitinib, 5-ASA-PABA-MAC and 5-ASA-PABA-diamine colon-specific delivery systems were constructed, and tofacitinib azo prodrugs 9 and 20a-20g were synthesized accordingly. The release studies suggested that these systems could effectively release tofacitinib in vitro, and the 5-ASA-PABA-diamine system could successfully realize the colon targeting of tofacitinib in vivo. Specifically, compound 20g displayed a 3.67-fold decrease of plasma AUC(tofacitinib, 0-∞) and a 9.61-fold increase of colonic AUC(tofacitinib, 0-12h), compared with tofacitinib at a molar equivalent oral dose. Moreover, mouse models suggested that compound 20g (1.5 mg/kg) could achieve roughly the same efficacy against ulcerative colitis compared with tofacitinib (10 mg/kg) and did not impair natural killer cells. These results demonstrated the feasibility of compound 20g as an effective alternative to mitigate the systemic adverse effects of tofacitinib, and 5-ASA-PABA-MAC and 5-ASA-PABA-diamine systems were proven to be effective for colon-specific drug delivery.

Plant Growth Hormones Increase the Stimulation Efficiency of Seedlings Development for Spring Wheat Seeds upon Pre-sowing Treatment.

The influence of pre-sowing treatment of spring wheat seeds with combined use of plant growth hormones and sorption preparations based on bentonite-humate mixtures on seeds germination and their development in soils was studied. In some cases, the combined use of plant growth hormones and the sorption preparation (CB-H-BYA) that can decrease the intake of allelotoxins from soil to seeds allows noticeably increasing the efficiency of plant growth hormones used for pre-sowing treatment. The inclusion of cytokinins (6-benzylaminopurine, kinetin, and forchlorophenuron) into the sorption preparation (CB-H-BYA) had markedly different effects on seeds germination. The addition of Polysorbate 20 to the sorption preparation (CB-H-BYA) leads to an increase in the effectiveness of its action on seed germination.

Synthesis and evaluation of novel peptidomimetics bearing p-aminobenzoic acid moiety as potential antidiabetic agents.

Aim: Search for a new class of potential antidiabetic agents. Methodology: A series of novel peptidomimetics bearing the p-aminobenzoic acid moiety (TM3-TM6) were designed and synthesized. For all synthetic target molecules, the peroxisome proliferator response element (PPRE) activated activities have been evaluated and the toxicity were computed. Results & discussion: 46 new p-aminobenzoic acid derivatives have been characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS). The results of in vitro PPRE-activated activity, molecular docking study and toxicity prediction revealed that these compounds had potential antidiabetic activities and low toxicity. In particular compound 3b had up to 87% PPRE-activated activity compared with pioglitazone. This discovery may provide new insights for finding novel PPRE lead compound.

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress.

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l-carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

4-Aminobenzoic Acid Derivatives: Converting Folate Precursor to Antimicrobial and Cytotoxic Agents.

4-aminobenzoic acid (PABA), an essential nutrient for many human pathogens, but dispensable for humans, and its derivatives have exhibited various biological activities. In this study, we combined two pharmacophores using a molecular hybridization approach: this vitamin-like molecule and various aromatic aldehydes, including salicylaldehydes and 5-nitrofurfural, via imine bond in one-step reaction. Resulting Schiff bases were screened as potential antimicrobial and cytotoxic agents. The simple chemical modification of non-toxic PABA resulted in constitution of antibacterial activity including inhibition of methicillin-resistant Staphylococcus aureus (minimum inhibitory concentrations, MIC, from 15.62 µM), moderate antimycobacterial activity (MIC ≥ 62.5 µM) and potent broad-spectrum antifungal properties (MIC of ≥ 7.81 µM). Some of the Schiff bases also exhibited notable cytotoxicity for cancer HepG2 cell line (IC50 ≥ 15.0 µM). Regarding aldehyde used for the derivatization of PABA, it is possible to tune up the particular activities and obtain derivatives with promising bioactivities.

Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents.

The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.

Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers.

BACKGROUND: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1'-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20⁻100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2⁻20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug⁻target interactions.

Antifungal Metabolite p-Aminobenzoic Acid (pABA): Mechanism of Action and Efficacy for the Biocontrol of Pear Bitter Rot Disease.

Colletotrichum fructicola, a fungal pathogen that causes bitter rot disease in pears, has recently emerged in Eastern Asia and caused enormous economic losses and crop penalties. For this reason, new strategies for the management of bitter rot disease are greatly needed and can have a great impact on the field. In this regard, our research group recently reported that p-aminobenzoic acid (pABA), which was found in the secretions of rhizobacterium Lysobacter antibioticus OH13, showed a broad spectrum of antifungal activities. Following this project, the antifungal mode of action of pABA has been elucidated in this work indicating that pABA affects the fungal cell cycle of C. fructicola by inhibiting septation during cell division. pABA stability and diffusion screening revealed that pABA degrades after 15 days and is able to cross the pear skin into the external parts of the mesocarp. In vivo studies demonstrated that pABA shows high curative ability against the infection of C. fructicola in pears. To show the efficacy of OH13 for the biocontrol of bitter rot disease, cultures of OH13 containing 379.4 mg/L pABA were sprayed on inoculated pears, significantly reducing the symptoms of the pathogen.

Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis.

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as, para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter, designated MetM, play crucial roles in this process. Disruption of metM by transposon insertion resulted in a ≥30-fold decrease in uptake of methionine in M. bovis BCG, indicating that metM is the major facilitator of methionine transport. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.

Positron Emission Tomography Imaging with 2-[18F]F- p-Aminobenzoic Acid Detects Staphylococcus aureus Infections and Monitors Drug Response.

Staphylococcus aureus is the leading cause of life-threatening infections, frequently originating from unknown or deep-seated foci. Source control and institution of appropriate antibiotics remain challenges, especially with infections due to methicillin-resistant S. aureus (MRSA). In this study, we developed a radiofluorinated analog of para-aminobenzoic acid (2-[18F]F-PABA) and demonstrate that it is an efficient alternative substrate for the S. aureus dihydropteroate synthase (DHPS). 2-[18F]F-PABA rapidly accumulated in vitro within laboratory and clinical (including MRSA) strains of S. aureus but not in mammalian cells. Biodistribution in murine and rat models demonstrated localization at infection sites and rapid renal elimination. In a rat model, 2-[18F]F-PABA positron emission tomography (PET) rapidly differentiated S. aureus infection from sterile inflammation and could also detect therapeutic failures associated with MRSA. These data suggest that 2-[18F]F-PABA has the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify, localize, and monitor S. aureus infections.

Antitumor activity of the polo-like kinase inhibitor, TAK-960, against preclinical models of colorectal cancer.

BACKGROUND: Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is a key regulator of multiple stages of mitotic progression. Plk1 is upregulated in many tumor types including colorectal cancer (CRC) and portends a poor prognosis. TAK-960 is an ATP-competitive Plk1 inhibitor that has demonstrated efficacy across a broad range of cancer cell lines, including CRC. In this study, we investigated the activity of TAK-960 against a large collection of CRC models including 55 cell lines and 18 patient-derived xenografts. METHODS: Fifty-five CRC cell lines and 18 PDX models were exposed to TAK-960 and evaluated for proliferation (IC50) and Tumor Growth Inhibition Index, respectively. Additionally, 2 KRAS wild type and 2 KRAS mutant PDX models were treated with TAK-960 as single agent or in combination with cetuximab or irinotecan. TAK-960 mechanism of action was elucidated through immunoblotting and cell cycle analysis. RESULTS: CRC cell lines demonstrated a variable anti-proliferative response to TAK-960 with IC50 values ranging from 0.001 to > 0.75 µmol/L. Anti-proliferative effects were sustained after removal of drug. Following TAK-960 treatment a highly variable accumulation of mitotic (indicating cell cycle arrest) and apoptotic markers was observed. Cell cycle analysis demonstrated that TAK-960 treatment induced G2/M arrest and polyploidy. Six out of the eighteen PDX models responded to single agent TAK-960 therapy (TGII< 20). The addition of TAK-960 to standard of care chemotherapy resulted in largely additive antitumor effects. CONCLUSION: TAK-960 is an active anti-proliferative agent against CRC cell lines and PDX models. Collectively, these data suggest that TAK-960 may be of therapeutic benefit alone or in combination with other agents, although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations.

Production of Antifungal p-Aminobenzoic Acid in Lysobacter antibioticus OH13.

Among Lysobacter species, Lysobacter antibioticus has been demonstrated to be an interesting source of antimicrobial metabolites for the biocontrol of plant diseases. Although the antibacterial activity was attributed to N-oxide phenazines, the active compounds involved in the antifungal function remained unknown. In this work, an antifungal compound was isolated and identified as p-aminobenzoic acid (pABA). Antifungal activity screening revealed that pABA shows activity against a number of plant pathogens. The genes involved in the synthetic route of this compound in OH13 were identified. Further, the production of pABA was optimized by modification of the carbon source using engineered L. antibioticus OH13 strains.

Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection.

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential 1 . Periodontitis, which is the sixth most prevalent infectious disease worldwide 2 , ensues from the action of dysbiotic polymicrobial communities 3 . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo 3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

A nutrient mediates intraspecific competition between rodent malaria parasites in vivo.

Hosts are often infected with multiple strains of a single parasite species. Within-host competition between parasite strains can be intense and has implications for the evolution of traits that impact patient health, such as drug resistance and virulence. Yet the mechanistic basis of within-host competition is poorly understood. Here, we demonstrate that a parasite nutrient, para-aminobenzoic acid (pABA), mediates competition between a drug resistant and drug susceptible strain of the malaria parasite, Plasmodium chabaudi We further show that increasing pABA supply to hosts infected with the resistant strain worsens disease and changes the relationship between parasite burden and pathology. Our experiments demonstrate that, even when there is profound top-down regulation (immunity), bottom-up regulation of pathogen populations can occur and that its importance may vary during an infection. The identification of resources that can be experimentally controlled opens up the opportunity to manipulate competitive interactions between parasites and hence their evolution.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

Myeloperoxidase Inhibition Increases Neurogenesis after Ischemic Stroke.

The relationship between inflammation and neurogenesis in stroke is currently not well understood. Focal ischemia enhances cell proliferation and neurogenesis in the neurogenic regions, including the subventricular zone (SVZ), dentate gyrus, as well as the non-neurogenic striatum, and cortex in the ischemic hemisphere. Myeloperoxidase (MPO) is a potent oxidizing enzyme secreted during inflammation by activated leukocytes, and its enzymatic activity is highly elevated after stroke. In this study, we investigated whether the inhibition of MPO activity by a specific irreversible inhibitor, 4-aminobenzoic acid hydrazide (ABAH) (MPO-/- mice) can increase neurogenesis after transient middle cerebral artery occlusion in mice. ABAH administration increased the number of proliferating bromodeoxyuridine (BrdU)-positive cells expressing markers for neural stems cells, astrocytes, neuroprogenitor cells (Nestin), and neuroblasts (doublecortin) in the ischemic SVZ, anterior SVZ, striatum, and cortex. MPO inhibition also increased levels of brain-derived neurotrophic factor, phosphorylation of cAMP response element-binding protein (Ser133), acetylated H3, and NeuN to promote neurogenesis in the ischemic SVZ. ABAH treatment also increased chemokine CXC receptor 4 expression in the ischemic SVZ. MPO-deficient mice treated with vehicle or ABAH both showed similar effects on the number of BrdU+ cells in the ischemic hemisphere, demonstrating that ABAH is specific to MPO. Taken together, our results underscore a detrimental role of MPO activity to postischemia neurogenesis and that a strategy to inhibit MPO activity can increase cell proliferation and improve neurogenesis after ischemic stroke.

Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

OBJECTIVES: To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. RESULTS: Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. CONCLUSIONS: A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.