Minimal Medium Research Articles

Genomically recoded Escherichia coli with optimized functional phenotypes.

Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.

Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor

BackgroundSeveral two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor.ResultsThe ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869.ConclusionThis bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

The impact of heat treatment on E. coli cell physiology in rich and minimal media considering oxidative secondary stress.

This study investigates the cell physiology of thermally injured bacterial cells, with a specific focus on oxidative stress and the repair mechanisms associated with oxidative secondary stress. We explored the effect of heat treatment on the activity of two protective enzymes, levels of intracellular reactive oxygen species, and redox potential. The findings reveal that enzyme activity slightly increased after heat treatment, gradually returning to baseline levels during subculture. The response of Escherichia coli cells to heat treatment, as assessed by the level of superoxide radicals generated and redox potential, varied based on growth conditions, namely minimal and rich media. Notably, the viability of injured cells improved when antioxidants were added to agar media, even in the presence of metabolic inhibitors. These results suggest a complex system involved in repairing damage in heat-treated cells, particularly in rich media. While repairing membrane damage is crucial for cell regrowth and the electron transport system plays a critical role in the recovery process of injured cells under both tested conditions.

Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

Willingness and Attitude Towards Human Papilloma Virus (HPV) Vaccination Among Early-Adolescent Female School Students, in Harar, Ethiopia, 2024: A Cross-Sectional Study

Human papillomavirus (HPV) is a sexually transmitted disease that causes various cancers, particularly in women with early sexual activity, multiple partners, and unprotected sex. However, the willingness of adolescents to receive the vaccine is influenced by community, organization, policy, and parental conditions. A study was conducted in Harar, Ethiopia, from January to February 2024. The study used a questionnaire adapted from previous studies and data collection involved face-to-face interviews. Descriptive statistics were calculated and binary logistic was used to assess associated factors. The overall favorable attitude and willingness to take HPV vaccination in this study was 66.67% (95% CI: 61.9%, 71.2%) and 50.6% (95% CI: 45.7%, 55.5%), respectively. The factor associated with the willingness to get HPV vaccination was living with parents (AOR =2.06 95% CI 1.07, 3.95), having a father's education (AOR=1.63 95% CI 1.01, 2.66), vaccine hesitancy (AOR=0.63, 95% CI 0.41, 0.98), hesitancy and complacency (AOR=1.7, 95% CI 1.07, 2.69), (AOR=1.86, 95% CI1.18, 2.93) respectively. The factor associated with the attitude towards HPV vaccination was access to the minimal media, the occupation of the parents was non-governmental, vaccine hesitancy. The study indicates a moderate willingness and attitude towards HPV vaccination among early adolescent female students.

Iron starvation results in up-regulation of a probable Haloferax volcanii siderophore transporter.

The response of the haloarchaeal model organism Haloferax volcanii to iron starvation was analyzed at the proteome level by data-independent acquisition mass spectrometry. Cells grown in minimal medium with normal iron levels were compared to those grown under low iron conditions, with samples being separated into membrane and cytoplasmic fractions in order to focus on import/export processes which are frequently associated with metal homeostasis. Iron starvation not only caused a severe retardation of growth but also altered the levels of many proteins. Using a comprehensive annotated spectral library and data-independent acquisition mass spectrometry (DIA-MS), we found that iron starvation resulted in significant changes to both the membrane and the soluble proteomes of Hfx. volcanii. The most affected protein is the RND family permease HVO_A0467, which is 44-fold enriched in cells grown under iron starvation. The gene HVO_A0467 can be deleted suggesting that it is not essential under standard conditions. Compared to wild type cells the deletion strain shows only slight changes in growth and cell morphologies show no differences. Molecular docking predictions indicated that HVO_A0467 may be an exporter of the siderophore schizokinen for which a potential biosynthesis cluster is encoded in the Hfx. volcanii genome. Together, these findings confirm the importance of iron for archaeal cells and suggest HVO_0467 as a siderophore exporter.

Dialysis rolled scaffold bioreactor allows extended production of monoclonal antibody with reduced media use.

Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production.

Development of Broad-Range Microbial Minimal Culture Medium for Lanthanide Studies.

Rare Earth Elements (REE), also known as Lanthanides (Ln3+), are a group of 17 elements showing peculiar physical and chemical properties. Unlike technological applications, very little is known about the physiological role and toxicity of Ln3+ on biological systems, in particular on microorganisms (e.g., bacteria), which represent the most abundant domains on our planet. Up to now, very limited studies have been conducted due to Ln3+ precipitation with some anions commonly present in the culture media. Therefore, the development of a minimal medium is essential to allow the study of Ln3+-microbial interactions, limiting considerably the precipitation of insoluble salts. In this regard, a new minimal culture medium capable of solubilizing large amounts of Ln3+ and allowing the growth of different microbial taxa was successfully developed. Assays have shown that the medium is capable of solubilizing Ln3+ up to 100 times more than other common culture media and allowing the growth of 63 bacteria and 5 fungi. The kinetic growth of one yeast and one Gram-positive bacterium has been defined, proving to support superior growth and biomass compared to other commonly used minimal media. Moreover, the sensitivity and uptake/absorption of a Bacillus stratosphericus strain were tested, highlighting its capability to tolerate concentrations up to 10 mM of either Cerium, Gadolinium or Lanthanum and accumulate different quantities of the three.

Metabolic engineering of fast growing cyanobacteria for phototrophic production of 2,3-butanediol

Metabolic engineering of cyanobacteria holds great potential for sustainable photosynthetic production of platform chemicals from CO2. However, product titers have been significantly low due to the slow growth rates of available model strains and lack of adequate synthetic biology tools. Here, we engineered three newly isolated fast growing Synechococcus elongatus strains PCC 11801, PCC 11802, and IITB6 for production of the platform chemical 2,3-butanediol. Importantly, we used native cyanobacterial promoters to enable inducer-free gene expression and 2,3-butanediol production. Different combinations of these native promoters were employed to optimize expression of the three-gene 2,3-butanediol synthesis pathway. Among the strains tested in this study, the highest 2,3-butanediol titer of 1.62 g L−1 (130 mg L−1 Day−1) was obtained in IITB6 (PcpcB300:alsS::PpsbAIII:alsD::PrbcL:adh), with the highest reported photosynthetic productivity in cyanobacteria cultivated on minimal media. The findings from our study highlight the potential of using fast growing S. elongatus isolates with native cyanobacterial promoters for metabolic engineering applications.

CRP improves the survival and competitive fitness of Salmonella Typhimurium under starvation by controlling the cellular maintenance rate.

Catabolite repression is a mechanism of selectively utilizing preferred nutrient sources by redirecting the metabolic pathways. Therefore, it prevents non-essential energy expenditure by repressing the genes and proteins involved in the metabolism of other less favored nutrient sources. Catabolite repressor protein (CRP) is a chief mediator of catabolite repression in microorganisms. In this context, we investigated the role of CRP in starvation tolerance, at both cell physiology and molecular level, by comparing the growth, survival, competitive fitness, maintenance rate, and gene and protein expression of wild type (WT) and ∆crp of Salmonella Typhimurium, under nutrient-rich and minimal medium condition. The ∆crp shows slow growth upon the arrival of nutrient-limiting conditions, poor survival under prolong-starvation, and inability to compete with its counterpart WT strain in nutrient-rich [Luria broth (LB)] and glucose-supplemented M9 minimal medium. Surprisingly, we observed that the survival and competitive fitness of ∆crp are influenced by the composition of the growth medium. Consequently, compared to the glucose-supplemented M9 medium, ∆crp shows faster death and a higher maintenance rate in the LB medium. The comparative gene and protein expression studies of WT and ∆crp in LB medium show that ∆crp has partial or complete loss of repression from CRP-controlled genes, resulting in a high abundance of hundreds of proteins in ∆crp compared to WT. Subsequently, the addition of metabolizable sugar or fresh nutrients to the competing culture showed extended survival of ∆crp. Therefore, our results suggest that CRP-mediated gene repression improves starvation tolerance and competitive fitness of Salmonella Typhimurium by adapting its cellular maintenance rate to environmental conditions.IMPORTANCESalmonella Typhimurium is a master at adapting to chronic starvation conditions. However, the molecular mechanisms to adapt to such conditions are still unknown. In this context, we have evaluated the role of catabolite repressor protein (CRP), a dual transcriptional regulator, in providing survival and competitive fitness under starvation conditions. Also, it showed an association between CRP and nutrient composition. We observed that Δcrp growing on alternate carbon sources has lower survival and competitive fitness than Δcrp growing on glucose as a carbon source. We observed that this is due to the loss of repression from the glucose and CRP-controlled genes, resulting in elevated cellular metabolism (a high maintenance rate) of the Δcrp during growth in a medium having a carbon source other than glucose (e.g., Luria broth medium).

Isolation and Identification of Isolate, S1 with High Biotransformation Potential of Ferulic Acid to Vanillin

Nine actinobacterial isolates were purified from the sediment sample of Kogilvai village, Warangal, Telangana, based on their capability to grow on the minimal medium with Ferulic acid (FA) as only Carbon (C) source. FA to Vanillin conversion capacity of these isolates was identified by Thin-layer chromatography (TLC). Biotransformation of FA to Vanillin was high by four isolates, S1, S3, O3 and O4 when compared to other five isolates (O1, O2, S2, S4 and S5) with initial pH 7 in basal medium. Among these four isolates, optimal and rapid FA to Vanillin bioconversion of 140 mg/L was shown by isolate S1 with UV-spectrophotometry. Its conversion was confirmed by High Performance Liquid Chromatography (HPLC) analysis with retention time of 2.9 min after 28hrs of incubation at 37°C with 1g/L ferulic acid in the medium with 150 rpm. Isolate S1 could utilize Lactose, Maltose, Glycerol, Fructose, Galactose, Sucrose, Dextrose, L-Arabinose, ONPG, Esculin and not other carbohydrates present in the Himedia Hicarbo kit. Molecular characterization showed that 16S rDNA gene sequence of isolate S1 was 98.27% similar to Limosilactobacillus fermentum CECT 562 with completeness of 96.7% and was identified as Limosilactobacillus sp. 16S rDNA gene sequence of isolate S1 was submitted to NCBI GenBank and its accession number was OR136396. As this isolate has high potential of FA to Vanillin biotransformation capacity, it can be further explored to be used for industrial setups for commercial exploitation.

Effect of CUP1 copy number and pH on copper resistance of Saccharomyces cerevisiae enological strains

The widespread use of copper-based pesticides in winemaking can affect wine fermentation. Therefore, it is crucial to assess the resistance levels of Saccharomyces cerevisiae wine strains in enological growth conditions. In the context of winemaking, grape juice is a complex environment capable of chelating copper and is characterized by a distinctly acidic pH. In this work, the effects of copper concentration on the growth of 10 S. cerevisiae strains, isolated from an enological environment, and one commercial starter were tested in YNB minimal medium and synthetic must, mimicking enological conditions.In minimal medium, resistance to copper varied among yeasts (50–600 μM), revealing the presence of three resistance levels (high, intermediate, and low). Representative strains of the three groups were tested at a pH range from 5.2 to 3.0 at the copper concentration that showed a 20–25 % growth reduction. At pH range 5.2–4.5, a growth reduction was observed, while, conversely, a strain-specific recovery was observed at pH range 3.2–3.0.In synthetic must, the strains showed higher copper resistance levels than in minimal medium (50–4000 μM). In both synthetic must and minimal medium, a significant logarithmic correlation was found between copper resistance and CUP1 gene copy number. The copy number tended to better explain resistance in minimal medium compared to synthetic must. The results shed light on the role of CUP1 copy number within an enological environment.

Expression of an engineered salt-inducible proline biosynthetic operon in a Glutamic acid Over-Producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata GOP, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

Indigo carmine biodegradation by endophytic Microbacterium zeae K5: Enzymatic insights, degradation mechanism, and ecotoxicity analysis

The enormous amount of colored effluent release from dying industries into fresh and marine reservoirs, causing ecotoxicity and serious health problems. Pertaining to this, the current research emphasis on the decolorization and degradation treatment of Indigo carmine (IC). The decolorization of IC with endophytic Microbacteium zeae K5, isolated from the root of the Salix purpurea plant was studied. In a minimal salt medium with shaking, full dye decolorization (400 mg/L) was achieved within a 24 h incubation period. During dye degradation, the activities of enzymes such as laccase (12.02 U/g), manganese peroxidase (4.23 U/g), and quinone dehydrogenase (0.09 U/g) were also observed. The biodegradation of IC into isatin sulfonic acid and isatin was confirmed by UV–VIS spectrophotometry and GC-MS analysis of dye samples extracted with ethyl acetate. Finally, phytotoxicity studies revealed that the IC degraded metabolites toxicity was lower than that of the parent dye compound. The current study demonstrated isolate M. zeae K5 has ability to efficiently break down IC, indicating its potential for future bioremediation uses.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

Non-invasive VOCs detection to monitor the gut microbiota metabolism in-vitro

This work implemented a non-invasive volatile organic compounds (VOCs) monitoring approach to study how food components are metabolised by the gut microbiota in-vitro. The fermentability of a model food matrix rich in dietary fibre (oat bran), and a pure prebiotic (inulin), added to a minimal gut medium was compared by looking at global changes in the volatilome. The substrates were incubated with a stabilised human faecal inoculum over a 24-h period, and VOCs were monitored without interfering with biological processes. The fermentation was performed in nitrogen-filled vials, with controlled temperature, and tracked by automated headspace-solid-phase microextraction coupled with gas chromatography–mass spectrometry. To understand the molecular patterns over time, we applied a multivariate longitudinal statistical framework: repeated measurements—ANOVA simultaneous component analysis. The methodology was able to discriminate the studied groups by looking at VOCs temporal profiles. The volatilome showed a time-dependency that was more distinct after 12 h. Short to medium-chain fatty acids showed increased peak intensities, mainly for oat bran and for inulin, but with different kinetics. At the same time, alcohols, aldehydes, and esters showed distinct trends with discriminatory power. The proposed approach can be applied to study the intertwined pathways of gut microbiota food components interaction in-vitro.

Sexual Education for Children with Special Needs through Learning Media based on Universal Design for Learning

Sexual education is very important to be taught to young children, especially children with special needs. Sexual violence can intentionally occur to someone in a sexual manner. This can even extend to attempted rape, sadism in sexual relationships, other unwanted sexual activities, demeaning and hurting or injuring the victim. One effort to prevent sexual violence is to equip young children with knowledge, skills, and abilities to protect themselves from the threat of sexual violence. The method used is descriptive qualitative research. The subjects in this study are teachers in one Special School. Data collection in this study uses observation, interviews, and documentation techniques. Data analysis techniques use qualitative descriptive analysis techniques. The results of the study show that special teachers need learning media for more structured and systematic inclusive education according to the learning stage process. Until now, there is still minimal learning media used for sexual education, especially for children with special needs. Good learning media must have clear, communicative, concise, and systematic language use. The scope of the content in the learning media must also adjust to the conditions of students who will be given learning.

Growth on Douglas fir media facilitates Cryptococcus virulence factor production and enhances fungal survival against environmental and immune stressors.

The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.

Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

BackgroundAn important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation.ResultsThis study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%.ConclusionThe identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production.

Deciphering the Role of AMPK in Regulating Integrin Profile of Tumor-Derived Exosomes as a Potential Antimetastatic Strategy

Introduction: Cancer cell adapt their metabolic activity to survive in stressful environments with limited nutrients and oxygen. To sense and respond to nutrient cues, cells rely on a “fuel gauge” protein known as AMP-activated protein kinase (AMPK). While previous work has largely focused on AMPK’s role in cell metabolism, its role in metastasis is poorly defined. Interestingly, AMPK also regulates the membrane trafficking of integrins – key proteins in cell adhesion and migration. Recent studies have also shown that circulating integrins in exosomes are predictive of metastasis. Therefore, understanding the biology of AMPK signaling on the exosomal integrin profile not only allow for better cancer treatments but also help developing a potential predictive biomarker. I hypothesize that AMPK activation alters the integrin profile of exosomes in breast and endometrial cancer. Methods: Breast and endometrial cancer cells, MDA-MB-231 and HEC-1 respectively, will be cultured in full nutrient media conditions. 48 hours before exosome harvest, the media will be changed to minimal specialized media for extracellular vesicles (EV) collection. To induce AMPK activation, cells will be treated with limiting concentrations of glucose and glutamine, or with metabolic poisons 2-deoxyglucose (2-DG) and oligomycin. Total Exosome IsolationTM (TEI) solution will be used to enrich released exosomes that will be subjected to mass spectrometry to analyze the exosomal integrin profiles. Anticipated Results: Exosomes secreted from MDA-MB-231 and HEC-1 cells are expected to contain different exosomal integrin profile in both basal and induced AMPK activation conditions. It is anticipated that integrin subunits that promote metastatic and survival signaling such as αvβ5, α6β4, α6β1, will be upregulated in exosomes following AMPK activation. This could suggest tumor cells adapting to nutrient insufficiency by AMPK activation may present a more aggressive exosomal integrin profile to support cancer progression. Discussion: Understanding how metabolic cues such as nutrient insufficiency impact integrin content of tumour-derived exosomes can help identify novel metabolic drug targets to limit pro-survival and pro-metastatic signaling in primary breast tumors. Conclusion: This study investigates how AMPK activation modulates the exosomal integrin profile in breast and endometrial cancer cells, potentially uncovering predictive biomarkers and therapeutic targets for metastatic cancers.