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Background: Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb’s vulnerability and metabolic adaptation have been carried out. Results: A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. Conclusions: This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus.

The clinical and industrial importance of clavulanic acid (CA) promotes the search for novel strategies to boost productivity at lower costs. In this contribution, the production of CA was evaluated in two reference strains of Streptomyces clavuligerus (Sc), ATCC27064 and DSM41826; the strains were tested in different culture media, using product yields and culture media costs as selection criteria. On average, ScATCC reached a concentration of CA 1.6-fold higher than ScDSM, using the isolated soybean protein (ISP) culture medium. A central composite experimental design was used to optimize the medium, by studying the influence of ISP and K2HPO4 concentration on the production of CA. The maximum CA concentration obtained in this optimized medium was 56 mg·L-1, close to the theoretical optimum. Our results confirm that a culture medium containing a rich carbon source such as glycerol and/or starch provides the best conditions for CA production.

In this study the metabolic responses of Botryococcus braunii were analyzed upon different inorganic carbon dosages and nutrient limitation conditions in terms of lipid and biomass productivity, as well as photosynthetic performance. The nutritional schemes evaluated included different levels of sodium bicarbonate and nitrogen and phosphorus starvation, which were contrasted against standard cultures fed with CO2. Bicarbonate was found to be an advantageous carbon source since high dosages caused a significant increase in biomass and lipid productivity, in addition to an enhanced photosynthetic quantum yield and neutral lipids abundance. This contrasts to the commonly used approach of microalgae nutrient limitation, which leads to high lipid accumulation at the expense of impaired cellular growth, causing a decline in overall lipid productivity. The lipidome analysis served to hypothesize about the influence of the nutritional context on B. braunii structural and storage lipid metabolism, besides the adaptive responses exhibited by cells that underwent nutrient stress.

Background/Objectives: Clavulanic acid (CA) is produced by cell suspension cultures of Streptomyces clavuligerus ATCC 27064, and is widely used as a beta-lactamase inhibitor to combat antibiotic resistance. CA titers are moderate due to bioprocess complexity, prompting ongoing efforts to overcome these limitations. Methods: In this study, we aimed to evaluate the effect of live and inactivated Bacillus velezensis FZB42 cells on CA production in S. clavuligerus, and to explore the transcriptional response underlying this interaction using RNA-seq technology. Results: The addition of dead and live cells of B. velezensis improved CA production by 1.4 and 2.0-fold, respectively. Furthermore, the transcriptome of S. clavuligerus, obtained with live cells of B. velezensis FZB42 at the peak of maximum CA production, revealed that 410 genes were up-regulated and 594 were down-regulated under these conditions, with a padj < 0.05. Most of the genes from the cephamycin C and CA clusters were up-regulated, which correlates well with the increase in CA production. Likewise, S. clavuligerus ATCC 27064 enhanced the expression of genes encoding enzymes that scavenge endogenous H2O2, as well as other genes related to oxidative stress defense. Regarding downregulated genes, we found that S. clavuligerus decreased the expression of genes involved in the biosynthesis of terpenoids, polyketides, and lantibiotics, as well as the expression of the operon involved in the synthesis of the pyrroloquinoline quinone (PQQ) cofactor. Conclusions: These findings contribute to the understanding of S. clavuligerus metabolism and pave the way for future metabolic engineering efforts aimed at obtaining CA-overproducing strains.

Chitosan has gained agro-industrial interest due to its potential applications in food preservation. In this work, chitosan applications for exotic fruit coating, using feijoa as a case of study, were evaluated. For this, we synthetized and characterized chitosan from shrimp shells and tested its performance. Chemical formulations for coating preparation using chitosan were proposed and tested. Mechanical properties, porosity, permeability, and fungal and bactericidal characteristics were used to verify the potential application of the film in the protection of fruits. The results indicated that synthetized chitosan has comparable properties to commercial chitosan (deacetylation degree > 82%), and, for the case of feijoa, the chitosan coating achieved significant reduction of microorganisms and fungal growth (0 UFC/mL for sample 3). Further, membrane permeability allowed oxygen exchange suitable for fruit freshness and natural physiological weight loss, thus delaying oxidative degradation and prolonging shelf-life. Chitosan’s characteristic of a permeable film proved to be a promising alternative for the protection and extension of the freshness of post-harvest exotic fruits.

Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the β-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the Streptomyces genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of Streptomyces clavuligerus was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress. A flux balance analysis based on experimental evidence revealed that clavulanic acid biosynthetic reaction fluxes are favored in conditions of phosphate limitation, and this is correlated with enhanced activity of central and amino acid metabolism, as well as with enhanced oxygen uptake. In silico and experimental results show a possible slowing down of tricarboxylic acid (TCA) due to reduced oxygen availability in low shear stress conditions. In contrast, high shear stress conditions are connected with high intracellular oxygen availability favoring TCA activity, precursors availability and clavulanic acid (CA) production.

Microalgae biomass exploitation as a carbon–neutral energy source is currently limited by several factors, productivity being one of the most relevant. Due to the high absorption properties of light-harvesting antenna, photosynthetic cells tend to capture an excessive amount of energy that cannot be entirely channeled through the electron transfer chain that ends up dissipated as heat and fluorescence, reducing the overall light use efficiency. Aiming to minimize this hurdle, in this work we studied the effect of decreasing concentrations of Magnesium (Mg2+) on the chlorophyll a content, photosynthetic performance, biomass and lipid production of autotrophic cultures of Botryococcus braunii LB 572. We also performed, for the first time, a comparative lipidomic analysis to identify the influence of limited Mg2+ supply on the lipid profile of this algae. The results indicated that a level of 0.0037 g L−1 MgSO4 caused a significant decline on chlorophyll a content with a concomitant 2.3-fold reduction in the biomass absorption coefficient. In addition, the Mg2+ limitation caused a decrease in the total carbohydrate content and triggered lipid accumulation, achieving levels of up to 53% DCW, whereas the biomass productivity remained similar for all tested conditions. The lipidome analysis revealed that the lowest Mg2+ concentrations also caused a differential lipid profile distribution, with an enrichment of neutral lipids and an increase of structural lipids. In that sense, we showed that Mg2+ limitation represents an alternative optimization approach that not only enhances accumulation of neutral lipids in B. braunii cells but also may potentially lead to a better areal biomass productivity due to the reduction in the cellular light absorption properties of the cells.

Chitin is an aminopolysaccharide of industrial interest commonly obtained from shrimp processing waste through chemical or biotechnological means. Current environmental concerns offer a stimulating perspective for chitin bioextraction with lactic acid bacteria since a considerable reduction in the use of corrosive and pollutant products is possible. Nevertheless, the efficiency of this bioprocess is still a matter of discussion. In this work, the experimental studies of chitin bioextraction from Pacific white shrimp (Litopenaeus vannamei) waste with a mixed culture of Lactobacillus plantarum, Lactobacillus bulgaricus and Streptococcus thermophilus are used in process simulation using Aspen Plus software for the analysis of the potential application of a bioprocess on plant scale. The experimental results of characterization in shake flasks and 1-litre bioreactor indicated that 50 h of fermentation with the mixed culture of lactic acid bacteria was enough to extract more than 90 % of minerals and proteins from the shrimp waste. The use of experimental parameters in the simulation allowed a reliable representation of the bioprocess yielding normalized root mean square values below 10 %. Simulation was used for the assessment of the impact of the raw material variability on the production costs and gross margin. In this regard, the gross margin of the operation ranged from 42 to 52 % depending on the raw material composition and product yield

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.