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It is essential to evaluate the effects of operating conditions in submerged cultures of filamentous microorganisms. In particular, the impeller type influences the flow pattern, power consumption, and energy dissipation, leading to differences in the hydrodynamic environment that affect the morphology of the microorganism. This work investigated the effect of different impeller types, namely the Rushton turbine (RT-RT) and Elephant Ear impellers in up-pumping (EEUP) and down-pumping (EEDP) modes, on cellular morphology and clavulanic acid (CA) production by Streptomyces clavuligerus in a stirred-tank bioreactor. At 800 rpm and 0.5 vvm, the cultivations performed using RT-RT and EEUP impellers provided higher shear conditions and oxygen transfer rates than those observed with EEDP. These conditions resulted in higher clavulanic acid production using RT-RT (380.7 mg/L) and EEUP (453.3 mg/L) impellers, compared to EEDP (196.6 mg/L). Although the maximum CA concentration exhibited the same order of magnitude for RT-RT and EEUP impellers, the latter presented 40% of the specific power consumption (4.9 kW/m3) compared to the classical RT-RT (12.0 kW/m3). The specific energy for CA production ( E CA ), defined as the energy cost to produce 1 mg of CA, was 3.5 times lower using the EEUP impeller (1.91 kJ/mgCA) when compared to RT-RT (5.91 kJ/mgCA). Besides, the specific energy for O2 transfer ( E O 2 ), the energy required to transfer 1 mmol of O2, was 2.3 times lower comparing the EEUP impeller (3.28 kJ/mmolO2) to RT-RT (7.65 kJ/mmolO2). The results demonstrated the importance of choosing the most suitable impeller configuration in conventional bioreactors to manufacture bioproducts.

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Sigma factors and sigma factor-related mechanisms control antibiotic production in Streptomyces. In this contribution, the orf21 gene was overexpressed in the wild-type strain of Streptomyces clavuligerus ATCC2764, yielding S. clavuligerus/pIORF21, to further evaluate its regulatory effect on clavulanic acid (CA) biosynthesis under different culture medium conditions. The orf21 overexpression, regulated under the constitutive promoter ermE*, led to 2.6-fold increase in CA production in GSPG medium, and a 1.8-fold decrease using ISP medium. As for GYM and MYM media, S. clavuligerus/pIORF21 strain showed higher aerial mycelium production compared to control. Glycerol uptake rate profile was affected by orf21 overexpression. Furthermore, in GSPG, S. clavuligerus/pIORF21 slightly increased the expression of adpA and gcas genes, whilst, in ISP, the claR gene expression was drastically reduced, which is consistent with a decreased CA production, observed in this medium. These findings suggest the protein encoded by the orf21 gene plays a role in the regulation of CA biosynthesis as a response to the nutritional composition of the medium.

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Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

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Clavulanic acid (CA) is an irreversible ß-lactamase enzyme inhibitor with a weak antibacterial activity produced by Streptomyces clavuligerus (S. clavuligerus). CA is typically co-formulated with broad-spectrum ß­lactam antibiotics such as amoxicillin, conferring them high potential to treat diseases caused by bacteria that possess ß­lactam resistance. The clinical importance of CA and the complexity of the production process motivate improvements from an interdisciplinary standpoint by integrating metabolic engineering strategies and knowledge on metabolic and regulatory events through systems biology and multi-omics approaches. In the large-scale bioprocessing, optimization of culture conditions, bioreactor design, agitation regime, as well as advances in CA separation and purification are required to improve the cost structure associated to CA production. This review presents the recent insights in CA production by S. clavuligerus, emphasizing on systems biology approaches, strain engineering, and downstream processing.

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Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the ß-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.

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lysA gene encoding meso-diaminopimelic acid (DAP) decarboxylase enzyme that catalyzes L-lysine biosynthesis in the aspartate pathway in Streptomyces clavuligerus was overexpressed, and its effects on cephamycin C (CephC), clavulanic acid (CA), and tunicamycin productions were investigated. Multicopy expression of lysA gene under the control of glpF promoter (glpFp) in S. clavuligerus pCOlysA led to higher expression levels ranging from 2- to 6-fold increase at both lysA gene and CephC biosynthetic gene cluster at T36 and T48 of TSBG fermentation. These results accorded well with CephC production. Thus, 1.86- and 3.14-fold higher volumetric as well as 1.26- and 1.71-fold increased specific CephC yields were recorded in S. clavuligerus pCOlysA in comparison with the wild-type and its control strain, respectively, at 48th h. Increasing the expression of lysA provided 4.3 times more tunicamycin yields in the recombinant strain. These findings suggested that lysA overexpression in S. clavuligerus made the strain more productive for CephC and tunicamycin. The results also supported the presence of complex interactions among antibiotic biosynthesis pathways in S. clavuligerus.

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The performance of software tools for de novo transcriptome assembly greatly depends on the selection of software parameters. Up to now, the development of de novo transcriptome assembly for prokaryotes has not been as remarkable as that for eukaryotes. In this contribution, Rockhopper2 was used to perform a comparative transcriptome analysis of Streptomyces clavuligerus exposed to diverse environmental conditions. The study focused on assessing the incidence of software parameters on software performance for the identification of differentially expressed genes as a final goal. For this, a statistical optimization was performed using the Transrate Assembly Score (TAS). TAS was also used for evaluating the software performance and for comparing it with related tools, e.g., Trinity. Transcriptome redundancy and completeness were also considered for this analysis. Rockhopper2 and Trinity reached a TAS value of 0.55092 and 0.58337, respectively. Trinity assembles transcriptomes with high redundancy, with 55.6% of transcripts having some duplicates. Additionally, we observed that the total number of differentially expressed genes (DEG) and their annotation greatly depends on the method used for removing redundancy and the tools used for transcript quantification. To our knowledge, this is the first work aimed at assessing de novo assembly software for prokaryotic organisms.

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Background: Clavulanic acid (CA), a ß-lactamase inhibitor, is industrially produced by the fermentation of Streptomyces clavuligerus. The efficiency of CA production is associated with media composition, culture conditions and physiological and genetic strain characteristics. However, the molecular pathways that govern CA regulation in S. clavuligerus remain unknown. Methods and Results: Here we used RNA-seq to perform a comparative transcriptome analysis of S. clavuligerus ATCC 27064 wild-type strain grown in both a favorable soybean-based medium and in limited media conditions to further contribute to the understanding of S. clavuligerus metabolism and its regulation. A total of 350 genes were found to be differentially expressed between conditions; 245 genes were up-regulated in favorable conditions compared to unfavorable. Conclusion: The up-regulated expression of many regulatory and biosynthetic CA genes was positively associated with the favorable complex media condition along with pleiotropic regulators, including proteases and some genes whose biological function have not been previously reported. Knowledge from differences between transcriptomes from complex/defined media represents an advance in the understanding of regulatory paths involved in S. clavuligerus' metabolic response, enabling the rational design of future experiments.

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Clavulanic acid (CA) is frequently prescribed for treatment of bacterial infections. Despite the large number of studies concerning CA production, there is still a need to search for more effective and productive processes because it is mainly produced by biochemical route and is chemically unstable. This paper evaluates the influence of acid and cold stresses on CA production by Streptomyces clavuligerus in bench scale stirred tank bioreactor. Four batch cultures were conducted at constant pH (6.8 or 6.3) and temperature (30, 25, or 20 °C) and five batch cultures were performed with application of acid stress (pH reduction from 6.8 to 6.3), cold stress (reduction from 30 to 20 °C), or both. The highest maximum CA concentration (684.4 mg L-1) was obtained in the culture conducted at constant temperature of 20 °C. However, the culture under acid stress, in which the pH was reduced from 6.8 to 6.3 at a rate of 0.1 pH unit every 6 h, provided the most promising result, exhibiting a global yield coefficient of CA relative to cell formation (YCA/X) of 851.1 mgCA gX-1. High YCA/X values indicate that a small number of cells are able to produce a large amount of antibiotic with formation of smaller amounts of side byproducts. This could be especially attractive for decreasing the complexity and cost of the downstream processing, enhancing CA production.

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ABSTRACT Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20 g L-1), threonine (0.0-1.44 g L-1), ornithine (0.0-4.08 g L-1), and glutamate (0.0-8.16 g L-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L-1, while a formulation without this salt produced only 41 mg L-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

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Clavulanic acid is a beta-lactam compound with potent inhibitory activity against beta-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.03.20 g L-1), threonine (0.01.44 g L-1), ornithine (0.04.08 g L-1), and glutamate (0.08.16 g L-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L-1, while a formulation without this salt produced only 41 mg L-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.(AU)

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Clavulanic acid (CA), a potent inhibitor of the ß-lactam, ase enzyme, is frequently co-formulated with a broad spectrum of antibiotics to treat infections caused by ß-lactamase-producing pathogens. In order to evaluate the impact and the progress of CA studies in the last four decades, a bibliometric analysis of the global scientific production of CA was carried out. A total of 39,758 records in the field of CA were indexed in the Scopus database for a 43-year period of study (1975⁻2017). The results indicated that CA studies have grown, showing three phases (1975⁻1999, 2000⁻2003 and 2004⁻2017) based on records of publications; the results showed a sigmoidal profile. Medicine was the main subject area for CA studies, whereas biochemistry, genetics and molecular biology were areas of research for CA production by Streptomyces clavuligerus (S. clavuligerus). Nevertheless, chemical engineering (as a subject area) had the highest increase in the percentage of publications related to CA production by S. clavuligerus. The United States, France, the United Kingdom, Spain and Brazil were the leading countries in the scientific production of studies on both CA and CA related to S. clavuligerus. This analysis allowed the identification of the area of knowledge with the highest impact on CA studies, the top researchers and their geographic distribution, and also helped to highlight the existence of antibiotic-resistant bacteria as an emergent area in CA research.

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Clavulanic acid is a ß-lactam compound with potent inhibitory activity against ß-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20gL-1), threonine (0.0-1.44gL-1), ornithine (0.0-4.08gL-1), and glutamate (0.0-8.16gL-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437mgL-1, while a formulation without this salt produced only 41mgL-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

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Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.

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In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

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This work evaluated a wild-type Streptomyces clavuligerus strain as a whole-cell lipase (Sc-WCL) producer by submerged fermentation. In an orbital shaker, lipase hydrolytic activity of 3000 U L-1, measured at pH 9.0 and 37 °C by using p-nitrophenyl palmitate as substrate, was achieved after 36 h fermentation using glycerol-free production medium in a baffled Erlenmeyer flask at 28 °C and pH 6.8. Maximum productivity of 52.5 U L-1 h-1 was achieved after 24 h in bioreactor using glycerol-free production medium at pH 6.8 and 28 °C, with agitation at 400 rpm and aeration at 1 vvm. Sc-WCL was shown to be more active at 60 °C and pH 10.7, while higher activity retention was observed at 30-40 °C after 1 h incubation at pH 10. Sc-WCL showed to have potential to be used as biocatalyst in hydrolysis and esterification reactions. In the hydrolysis of p-nitrophenyl palmitate, lyophilized Sc-WCL expressed a hydrolytic activity (330 units g-1 solid, measured at 37 °C and pH 9.0) around 100-fold higher than the ones declared by a supplier of lyophilized powders of mixtures of intracellular lipases from Thermus thermophiles and Thermus flavus (≥3.0 units g-1 solid, measured at 65 °C and pH 8.0). In the synthesis of butyl butyrate in anhydrous medium, 85% ester conversion was achieved at 37 °C after 8 h reaction. Thus, Sc-WCL showed to be a promising biocatalyst because it is cheaper than the isolated and purified lipases.

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Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial ß-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.

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Streptomyces clavuligerus produces simultaneously cephamycin C (CephC) and clavulanic acid (CA). Adding 1,3-diaminopropane to culture medium stimulates production of beta-lactam antibiotics. However, there are no studies on the influence of this diamine on coordinated production of CephC and CA. This study indicates that 1,3-diaminopropane can dissociate CephC and CA productions. Results indicated that low diamine concentrations (below 1.25 g l(-1)) in culture medium increased CA production by 200%, but not that of CephC. Conversely, CephC production increased by 300% when 10 g l(-1) 1,3-diaminopropane was added to culture medium. Addition of just L-lysine (18.3 g l(-1)) to culture medium increased both biocompounds. On the other hand, while L-lysine plus 7.5 g l(-1) 1,3-diaminopropane increased volumetric production of CephC by 1100%, its impact on CA production was insignificant. The combined results suggest that extracellular concentration of 1,3-diaminopropane may trigger the dissociation of CephC and CA biosynthesis in S. clavuligerus.

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In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α-ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production.

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Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

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