Characterization of Five Purine Riboswitches in Cellular and Cell-Free Expression Systems.

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of the control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

The CRISPR toolbox for the gram-positive model bacterium Bacillus subtilis.

CRISPR has revolutionized the way we engineer genomes. Its simplicity and modularity have enabled the development of a great number of tools to edit genomes and to control gene expression. This powerful technology was first adapted to Bacillus subtilis in 2016 and has been intensely upgraded since then. Many tools have been successfully developed to build a CRISPR toolbox for this Gram-positive model and important industrial chassis. The toolbox includes tools, such as double-strand and single-strand cutting CRISPR for point mutation, gene insertion, and gene deletion up to 38 kb. Moreover, catalytic dead Cas proteins have been used for base editing, as well as for the control of gene expression (CRISPRi and CRISPRa). Many of these tools have been used for multiplex CRISPR with the most successful one targeting up to six loci simultaneously for point mutation. However, tools for efficient multiplex CRISPR for other functionalities are still missing in the toolbox. CRISPR engineering has already resulted in efficient protein and metabolite-producing strains, demonstrating its great potential. In this review, we cover all the important additions made to the B. subtilis CRISPR toolbox since 2016, and strain developments fomented by the technology.

Targeting riboswitches with synthetic small RNAs for metabolic engineering.

Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA tool to target bacterial riboswitches and activate gene expression (rtRNA). The synthetic rtRNA is suitable for regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used small RNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic rtRNAs to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the rtRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

Dataset for supporting a modular autoinduction device for control of gene expression in Bacillus subtilis.

Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1]. Here, we present raw and processed data regarding to B. subtilis growth measured as OD600, performed in three different scales: microcultivation on 96-well plates (200 µL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD600, maximum and minimum promoter activity, fold of induction, and the induction OD600.

A modular autoinduction device for control of gene expression in Bacillus subtilis.

Intense synthesis of proteins and chemicals in engineered microbes impose metabolic burden, frequently leading to reduced growth and heterogeneous cell population. Thus, the correct balance between growth and production is important. Such balance can be engineered through dynamic control of pathways, but few broadly applicable tools are available to achieve this. We present an autonomous control of gene expression mediated by quorum sensing in Bacillus subtilis, able to self-monitor and induce expression without human supervision. Two variations of the induction module and seven of the response module were engineered generating a range of induction folds and strengths for gene expression control. Our strongest response promoter is 2.5 and 3.2 times stronger than the well-characterized promoters PsrfA and Pveg, respectively. We applied our strongest autoinduction device for the production of the vitamin B2. This study presents a toolbox of autoinduction modules for B. subtilis that is modular and tunable.

Water-soluble fluorescent red colorant production by Talaromyces amestolkiae.

The replacement of synthetic colors in food products by natural alternatives has been boosted by consumers willing to pay more for healthier products. However, the success of microbial colorants depends not only on its acceptability on the market but also its production costs. Talaromyces species can produce water-soluble red colorants induced by glucose and monosodium glutamate (MSG). In this study, the influence of several conditions was evaluated to produce natural red colorants by submerged culture of Talaromyces amestolkiae. Under optimal conditions (g/L: glucose 10, MSG 25, MgSO4 0.012, FeSO4 0.01, CaCl2 0.015; and initial pH of 5.0), a 30-fold increase in the production was achieved, reaching a red colorant production of 13.44 UA500nm. Depending on the initial pH, colorants with different hues and chroma values were obtained. Deep yellow colorants were derived from neutral and basic pH, while deep red colors were derived from acidic pH. The fluorescence spectrum of culture broth obtained before and after complexation with salts presented red colorants with yellow fluorescence spectra. The information generated in this study would be useful for the formulation of industrial media for large-scale cultivation of T. amestolkiae, which have the potential to produce Talaromyces fermented colorants for use in health foods and pharmaceutics.

Improving the cost effectiveness of enhanced green fluorescent protein production using recombinant Escherichia coli BL21 (DE3): Decreasing the expression inducer concentration.

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-ß-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.

The copper-inducible copAB operon in Xanthomonas citri subsp. citri is regulated at transcriptional and translational levels.

Upstream open reading frames (ORFs) are frequently found in the 5'-flanking regions of genes and may have a regulatory role in gene expression. A small ORF (named cohL here) was identified upstream from the copAB copper operon in Xanthomonascitri subsp. citri (Xac). We previously demonstrated that copAB expression was induced by copper and that gene inactivation produced a mutant strain that was unable to grow in the presence of copper. Here, we address the role of cohL in copAB expression control. We demonstrate that cohL expression is induced by copper in a copAB-independent manner. Although cohL is transcribed, the CohL protein is either not expressed in vivo or is synthesized at undetectable levels. Inactivation of cohL (X. citri cohL polar mutant strain) leads to an inability to synthesize cohL and copAB transcripts and consequently the inability to grow in the presence of copper. Bioinformatic tools predicted a stem-loop structure for the cohL-copAB intergenic region and revealed that this region may arrange itself in a secondary structure. Using in vitro gene expression, we found out that the structured 5'-UTR mRNA of copAB is responsible for sequestering the ribosome-binding site that drives the translation of copA. However, copper alone was not able to release the sequence. Based on the results, we speculate that cohL plays a role as a regulatory RNA rather than as a protein-coding gene.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus

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.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus.

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.

A dual control mechanism synchronizes riboflavin and sulphur metabolism in Bacillus subtilis.

Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for biosynthesis and/or transport of riboflavin (rib genes). Cytoplasmic riboflavin is rapidly and almost completely converted to FMN by flavokinases. When cytoplasmic levels of FMN are sufficient ("high levels"), FMN binding to FMN riboswitches leads to a reduction of rib gene expression. We report here that the protein RibR counteracts the FMN-induced "turn-off" activities of both FMN riboswitches in Bacillus subtilis, allowing rib gene expression even in the presence of high levels of FMN. The reason for this secondary metabolic control by RibR is to couple sulfur metabolism with riboflavin metabolism.

Natural riboflavin analogs.

Riboflavin analogs have a good potential to serve as basic structures for the development of novel anti-infectives. Riboflavin analogs have multiple cellular targets, since riboflavin (as a precursor to flavin cofactors) is active at more than one site in the cell. As a result, the frequency of developing resistance to antimicrobials based on riboflavin analogs is expected to be significantly lower. The only known natural riboflavin analog with antibiotic function is roseoflavin from the bacterium Streptomyces davawensis. This antibiotic negatively affects flavoenzymes and FMN riboswitches. Another roseoflavin producer, Streptomyces cinnabarinus, was recently identified. Possibly, flavin analogs with antibiotic activity are more widespread than anticipated. The same could be true for flavin analogs yet to be discovered, which could constitute tools for cellular chemistry, thus allowing a further extension of the catalytic spectrum of flavoenzymes.

Bacterial flavin mononucleotide riboswitches as targets for flavin analogs.

Roseoflavin is a toxic riboflavin (vitamin B2) analog and naturally is produced by Streptomyces davawensis. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) by promiscuous flavokinases (EC 2.7.1.26). Flavin mononucleotide (FMN) riboswitches control the expression of genes involved in riboflavin biosynthesis and/or transport. RoFMN triggers FMN riboswitches and negatively (or positively) affects expression of the downstream genes. RoFMN binding to the aptamer portion of FMN riboswitch RNAs occurs in the course of transcription by cellular RNA polymerases. We developed an in vitro test system to functionally characterize the interaction between riboflavin/FMN analogs such as roseoflavin/RoFMN and FMN riboswitches in the context of an actively transcribing RNA polymerase.

Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation.

A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

Riboflavin analogs as antiinfectives: occurrence, mode of action, metabolism and resistance.

Antimetabolites are molecules, which are structurally similar to molecules needed to carry out primary metabolic reactions.The inhibitory activity of an antimetabolite depends on its successful competition with the natural substrate, ligand, modulator or cofactor of a given biomolecule. Antimetabolites are indispensable as molecular tools in order to understand biological processes. Beyond that,antimetabolites have a large variety of applications in the pharmaceutical and food industries. The identification of the structural riboflavin(vitamin B2) analog roseoflavin in Streptomyces davawensis demonstrates that anti-vitamins/cofactor analogs may serve as lead structures for the development of novel antibiotics. The latter is supported by the recent finding that roseoflavin had a profound inhibiting effect on the growth and infectivity of the human bacterial pathogen Listeria monocytogenes at very low concentrations. Roseoflavin is studied in our laboratory as a model compound. We investigate the biosynthesis, the possible large-scale production, the metabolization,the mechanism of action and the resistance mechanism of the producer organism in order to pave the way for the structured analysis of other vitamin analogs yet to be discovered. These compounds hopefully will help to replenish the arsenal of antimicrobials urgently needed to fight multiresistant bacterial pathogens.

A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis.

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B2) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures.

Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications, such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited maximal activity at pH 6.0 and 55-60 degrees C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min at 40, 50 and 55 degrees C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was enhanced in the presence of beta-mercaptoethanol, dithiothreitol, Co(2+), Mn(2+), Mg(2+), NH(4) (+), and Na(+) and was resistant to inhibition by Pb(2+).

Studies on productivity and characterization of polygalacturonase from Aspergillus giganteus submerged culture using citrus pectin and orange waste.

Polygalacturonases are part of the group of enzymes involved in pectin degradation. The aim of this work was to investigate some of the factors affecting polygalacturonase production by an Aspergillus giganteus strain and to characterize this pectinolytic activity. Several carbon sources, both pure substances and natural substrates, were tested in standing cultures, and the best results were obtained with orange bagasse and purified citrus pectin. On citrus pectin as sole carbon source, the highest extracellular activity (9.5 U/ml and 40.6 U/mg protein) was obtained in 4.5-day-old cultures shaken at 120 rpm, pH 3.5 and 30 degrees C, while on orange bagasse, the highest extracellular activity (48.5 U/ml and 78.3 U/mg protein) was obtained in 3.5-day-old cultures shaken at 120 rpm, pH 6.0 and 30 degrees C. Optimal polygalacturonase activity was observed in assays conducted at pH 5.5-6.5 and 55-60 degrees C. The activity showed good thermal stability, with half-lives of 90 and 30 min when incubated at 55 and 60 degrees C, respectively. High stability was observed from pH 4.5 to 8.5; more than 90% of the activity remained after 24 h in this pH range.