Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Coenzyme biosynthesis in response to precursor availability reveals incorporation of ß-alanine from pantothenate in prototrophic bacteria.

Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon sources and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and ß-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for ß-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Dynamic flux balance analysis of high cell density fed-batch culture of Escherichia coli BL21 (DE3) with mass spectrometry-based spent media analysis.

Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism-specific genome-scale metabolic models (GSMM) and by assuming instantaneous pseudo-steady states for processes that are inherently dynamic. This technique is well-suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed-batch, high-cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri-carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process.

The metabolic landscape of the bacteria Rhodococcus erythropolis used in the biodesulfurization of petroleum products: an emphasis on 2-hydroxybiphenyl.

Microorganisms produce diverse classes of metabolites under various physiological conditions. Many bacterial strains have been reported to carry out the process of desulfurization in a cost-effective manner by converting dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) and then using the 2-HBP as a carbon source for growth and development. Key rate-limiting factors and an increased concentration of 2HBP (400 µM) affect the biodesulfurization activity of bacteria through the produced metabolites. Thus, this study was designed to explore the nature of the metabolites produced by Rhodococcus erythropolis in the presence of DBT and 2HBP supplemented with a culture medium. A total of 330 metabolites were detected, and the key metabolites identified were 11Z-eicosaenoyl-EA, 1-carboxyethylisoleucine, 1(3)-glyceryl-PGF2alpha, taurine, 2-hydroxynicotinic acid, 4,4-dimethyl-14alpha-hydroxymethyl-5alpha-cholest-8-en-3beta-ol, and 10-nitrooleic acid. The supplementation of DBT and DBT-2HBP resulted in the differential regulation of these metabolites, either through downregulation or overexpression. Furthermore, at high concentrations of 2-HBP, 1-carboxyethylisoleucine, taurine, 2-hydroxynicotinic acid, and nicotinic acid were upregulated. This work proposes that the identified metabolites may play a role in bacteria-mediated desulphurization and could be beneficial in developing a cost-effective method of desulphurization for refining petroleum.

Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4 g/L of glucose, 17.3 g/L of beef extract, and 0.28 mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303 g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95 µg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.

Functional and structural responses of a halophilic consortium to oily sludge during biodegradation.

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

Comparative Analysis of Effect of Culture Conditions on Growth and C-Phycocyanin Production in Helical and Linear Spirulina.

Spirulina (Arthrospira and Spirulina spp.) has always been characterized by the helical trichomes, despite the existence of linear forms. A great debate is now open on the morphological flexibility of Spirulina, but it seems that both trichome morphology and C-phycocyanin (C-PC) concentrations are influenced by the culture conditions.This work compared the effect of some key growth factors (medium pH as well as its carbon, potassium, and salt contents) on the growth and C-PC concentration of helical and linear Spirulina strains. Further, two-phase strategies, including light and nitrogen variation, were applied to increase the in vivo C-PC accumulation into the trichomes. Results showed that high pH induced trichomes elongation and improved growth but decreased C-PC content (+ 65 and + 43% vs. -83 and -49%, for helical and linear strains, respectively). Variations in carbon and salt concentrations negatively impacted growth and C-PC content, even if the linear strain was more robust against these fluctuations. It was also interesting to see that potassium increasing improved growth and C-PC content for both strains.The variation of light wavelength during the enrichment phase (in the two-phase strategy) improved by 50% C-PC accumulation in trichomes, especially after blue lighting for 96 h. Similar result was obtained after 48 h of nitrogen reduction, while its removal from the medium caused trichomes disintegration. The current work highlights the robustness of linear Spirulina strain and presents an efficient and scalable way to increase C-PC in vivo without affecting growth.

Heat resistance of five spoilage microorganisms in a carbonated broth.

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO2 on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO2 and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment. Five microorganisms were selected for this study: Alicyclobacillus acidoterrestris (spores), Aspergillus niger (spores), Byssochlamys fulva (spores), Saccharomyces cerevisiae (vegetative cells), and Zygosaccharomyces parabailii (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance. The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO2 concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for As. niger, for which the presence of dissolved CO2 reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO2.

Initial culture media pH influences the antibacterial activity and metabolic footprint of Lactobacillus acidophilus BIOTECH 1900.

This study investigated the impact of initial culture media pH on the antibacterial properties and metabolic profile of cell-free supernatants (CFSs) from Lactobacillus acidophilus BIOTECH 1900 (LAB1900). The CFSs harvested from LAB1900 grown in de Man, Rogosa, Sharpe broth with initial pH of 5.5 (CFS5.5) and 6.6 (CFS6.6) were tested. The two CFSs elicited varying degrees of activity against three gram-negative bacteria. In the agar-well diffusion against Pseudomonas aeruginosa, CFS5.5 and CFS6.6 recorded 14.36 ± 1.34 and 13.06 ± 1.29 mm inhibition, respectively. Interestingly, against Klebsiella pneumoniae, CFS5.5 showed 14.36 ± 1.56 mm inhibition which was significantly higher than the 12.22 ± 1.31 mm inhibition of CFS6.6 (p = 0.0464). While against Acinetobacter baumannii, significantly higher inhibition of 10.66 ± 0.51 mm was observed in CFS6.6 compared to the 7.58 ± 1.93 mm inhibition of CFS5.5 (p = 0.0087). Nonetheless, both CFSs were bactericidal, with a minimum inhibitory and bactericidal concentration range of 3.90625-7.8125 mg/mL. The varied antibacterial activities may be attributed to the metabolite compositions of CFSs. A total of 152 metabolites driving the separation between CFSs were noted, with the majority upregulated in CFS5.5. Furthermore, 15 were putatively identified belonging to acylcarnities, vitamins, gibberellins, glycerophospholipids, and peptides. In summary, initial culture media pH affects the production of microbial metabolites with antibacterial properties.

Systematic comparison of culture media uncovers phenotypic shift of primary human microglia defined by reduced reliance to CSF1R signaling.

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (colony stimulating factor 1 receptor [CSF1R] ligands and TGF-ß1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia (hMGL). Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of hMGL in culture, with observations that diverge from those previously made in rodent microglia.

Comparison of culture media reveals that non-essential amino acids strongly affect the phenotype of human monocyte-derived macrophages.

Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL-10, SIRPα, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.

Fibrinogen supports self-renewal of mesenchymal stem cells under serum-reduced condition through autophagy activation.

Mesenchymal stem cells (MSCs) are somatic stem cells used in cell transplantation therapy for tissue injuries and inflammatory diseases because of their ability to support tissue regeneration and to suppress inflammation. While their applications are expanding, needs for automation of culture procedures with reduction of animal-derived materials to meet stable quality and suppliability are also increasing. On the other hand, the development of molecules that safely support cell adherence and expansion on a variety of interfaces under the serum-reduced culture condition remains a challenge. We report here that fibrinogen enables MSC culture on various materials with low cell adhesion property even under serum-reduced culture conditions. Fibrinogen promoted MSC adhesion and proliferation by stabilizing basic fibroblast growth factor (bFGF), which was secreted in the culture medium by autocrine, and also activated autophagy to suppress cellar senescence. Fibrinogen coating allowed MSCs expansion even on the polyether sulfone membrane that represents very low cell adhesion, and the MSCs showed therapeutic effects in a pulmonary fibrosis model. This study demonstrates that fibrinogen is currently the safest and most widely available extracellular matrix and can be used as a versatile scaffold for cell culture in regenerative medicine.

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae.

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.

Optimization of glutathione production in Saccharomyces cerevisiae HBSD-W08 using Plackett-Burman and central composite rotatable designs.

BACKGROUND: Glutathione is an important bioactive tripeptide and is widely used in the food, medicine, and cosmetics industries. The aim of this study was to provide an efficient method for producing GSH and to explore its synthesis mechanism. Saccharomyces cerevisiae strain HBSD-W08 was screened for GSH production, and its fermentation medium was optimized using single-factor experiments of the Plackett-Burman and central composite rotatable designs. This method was used to analyze the effects of the presence and concentration of various carbon sources, organic and inorganic nitrogen sources, metal ions, and precursor amino acids on GSH production and catalase, superoxide dismutase, and γ-glutamylcysteine synthetase activity. RESULTS: The three most significant factors affecting GSH production were peptone (optimal concentration [OC]: 2.50 g L- 1), KH2PO4 (OC: 0.13 g L- 1), and glutamic acid (OC: 0.10 g L- 1). GSH productivity of HBSD-W08 was obtained at 3.70 g L- 1 in the optimized medium. The activity of γ-GCS, which is a marker for oxidative stress, was found to be highly positively correlated with GSH production. CONCLUSIONS: This finding revealed an underlying relationship between GSH synthesis and oxidative stress, providing useful information for developing effective GSH fermentation control strategies.

Single step method for high yield human platelet lysate production.

BACKGROUND: We aimed to develop a single step method for the production of human platelet lysate (hPL). The method must result in high hPL yields, be closed system and avoid heparin use. STUDY DESIGN AND METHODS: The method aimed at using glass beads and calcium. An optimal concentration of calcium and glass beads was determined by serial dilution. This was translated to a novel method and compared to known methods: freeze-thawing and high calcium. Quality outcome measures were transmittance, fibrinogen and growth factor content, and cell doubling time. RESULTS: An optimal concentration of 5 mM Ca2+ and 0.2 g/ml glass beads resulted in hPL with yields of 92% ± 1% (n = 50) independent of source material (apheresis or buffy coat-derived). The transmittance was highest (56% ± 9%) compared to known methods (<39%). The fibrinogen concentration (7.0 ± 1.1 µg/ml) was well below the threshold, avoiding the need for heparin. Growth factor content was similar across hPL production methods. The cell doubling time of adipose derived stem cells was 25 ± 1 h and not different across methods. Batch consistency was determined across six batches of hPL (each n = 25 constituting concentrates) and was <11% for all parameters including cell doubling time. Calcium precipitation formed after 4 days of culturing stem cells in media with hPL prepared by the high (15 mM) Ca2+ method, but not with hPL prepared by glass bead method. DISCUSSION: The novel method transforms platelet concentrates to hPL with little hands-on time. The method results in high yield, is closed system, without heparin and non-inferior to published methods.

Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification.

Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH4 )2 SO4 and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.

Genome mining reveals secondary metabolites of Antarctic bacterium Streptomyces albidoflavus related to antimicrobial and antiproliferative activities.

The urgent need for new antimicrobials arises from antimicrobial resistance. Actinobacteria, especially Streptomyces genus, are responsible for production of numerous clinical antibiotics and anticancer agents. Genome mining reveals the biosynthetic gene clusters (BGCs) related to secondary metabolites and the genetic potential of a strain to produce natural products. However, this potential may not be expressed under laboratory conditions. In the present study, the Antarctic bacterium was taxonomically affiliated as Streptomyces albidoflavus ANT_B131 (CBMAI 1855). The crude extracts showed antimicrobial activity against both fungi, Gram-positive and Gram-negative bacteria and antiproliferative activity against five human tumor cell lines. Whole-genome sequencing reveals a genome size of 6.96 Mb, and the genome mining identified 24 BGCs, representing 13.3% of the genome. The use of three culture media and three extraction methods reveals the expression and recovery of 20.8% of the BGCs. The natural products identified included compounds, such as surugamide A, surugamide D, desferrioxamine B + Al, desferrioxamine E, and ectoine. This study reveals the potential of S. albidoflavus ANT_B131 as a natural product producer. Yet, the diversity of culture media and extraction methods could enhance the BGCs expression and recovery of natural products, and could be a strategy to intensify the BGC expression of natural products.

Enhancing the expression of multi-antigen chimeric TGAGS/BST protein from Toxoplasma gondii in Escherichia coli BL 21 Star during batch cultivation.

Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promising alternative for the process of obtaining a vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented by T. gondii. In this context, the present study evaluates batch culture strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii. Several exploratory cultures were initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different medium compositions without the addition of IPTG (inducer). Cultures of E. coli B21 Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1, and 6 h) to evaluate the effects on cell growth, production of the protein of interest, culture pH, and acetic acid formation. The results showed that among the culture media evaluated, 2xTY and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05 g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of culture.