Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites.

In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

Microwave-Assisted Extraction of Polyphenols from Bitter Orange Industrial Waste and Identification of the Main Compounds.

In this work, the extraction of phenolic compounds from orange waste (OW) obtained after the industrial extraction of neohesperidin from bitter oranges (Seville oranges) was assayed by microwave-assisted extraction (MAE) and Soxhlet extraction (SE). The extraction agents were ethanol and acetone. For SE, aqueous solutions of both extraction agents were used at 50%, 75%, and 100% (v/v). For MAE, a design of experiments was applied to determine the conditions that maximize the extraction yield. The independent variables were temperature (from 20 to 75 °C), process time (between 10 and 20 min), and percentage of extraction agent (v/v) in the extraction solution (50%, 75%, and 100%). Following that, the extracts were analyzed by ultra-high-performance liquid chromatography to identify the main phenolic compounds extracted. Results showed that 50% (v/v) ethanol or acetone was the extraction agent concentration that maximized the extraction yield for both SE and MAE, with the yields of MAE being higher than those of SE. Thus, the highest extraction yields on a dry basis achieved for MAE were 16.7 g/100 OW for 50% acetone, 75 °C, and 15 min, and 20.2 g/100 OW for 50% ethanol, 75 °C, and 10.8 min, respectively. Finally, the main phenolic compounds found in the orange waste were naringin, hesperidin, neohesperidin, and naringenin (i.e., flavonoids).

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.

Vacuolal and Peroxisomal Calcium Ion Transporters in Yeasts and Fungi: Key Role in the Translocation of Intermediates in the Biosynthesis of Fungal Metabolites.

The intracellular calcium content in fungal cells is influenced by a large number of environmental and nutritional factors. Sharp changes in the cytosolic calcium level act as signals that are decoded by the cell gene expression machinery, resulting in several physiological responses, including differentiation and secondary metabolites biosynthesis. Expression of the three penicillin biosynthetic genes is regulated by calcium ions, but there is still little information on the role of this ion in the translocation of penicillin intermediates between different subcellular compartments. Using advanced information on the transport of calcium in organelles in yeast as a model, this article reviews the recent progress on the transport of calcium in vacuoles and peroxisomes and its relation to the translocation of biosynthetic intermediates in filamentous fungi. The Penicillium chrysogenum PenV vacuole transporter and the Acremonium chrysogenum CefP peroxisomal transporter belong to the transient receptor potential (TRP) class CSC of calcium ion channels. The PenV transporter plays an important role in providing precursors for the biosynthesis of the tripeptide δ-(-α-aminoadipyl-L-cysteinyl-D-valine), the first intermediate of penicillin biosynthesis in P. chrysogenum. Similarly, CefP exerts a key function in the conversion of isopenicillin N to penicillin N in peroxisomes of A. chrysogenum. These TRP transporters are different from other TRP ion channels of Giberella zeae that belong to the Yvc1 class of yeast TRPs. Recent advances in filamentous fungi indicate that the cytosolic calcium concentration signal is connected to the calcitonin/calcineurin signal transduction cascade that controls the expression of genes involved in the subcellular translocation of intermediates during fungal metabolite biosynthesis. These advances open new possibilities to enhance the expression of important biosynthetic genes in fungi.

Penicillin-Binding Proteins, ß-Lactamases, and ß-Lactamase Inhibitors in ß-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the ß-lactam antibiotic cephamycin C, a class A ß-lactamase, and the ß lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the ß-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their ß-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel ß-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the ß-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the ß-lactamase in S. clavuligerus may be modulated by the ß-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the ß-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between ß-lactam biosynthesis and ß-lactam resistance mechanisms in the producer actinobacteria.

Comparative Molecular Mechanisms of Biosynthesis of Naringenin and Related Chalcones in Actinobacteria and Plants: Relevance for the Obtention of Potent Bioactive Metabolites.

Naringenin and its glycosylated derivative naringin are flavonoids that are synthesized by the phenylpropanoid pathway in plants. We found that naringenin is also formed by the actinobacterium Streptomyces clavuligerus, a well-known microorganism used to industrially produce clavulanic acid. The production of naringenin in S. clavuligerus involves a chalcone synthase that uses p-coumaric as a starter unit and a P450 monoxygenase, encoded by two adjacent genes (ncs-ncyP). The p-coumaric acid starter unit is formed by a tyrosine ammonia lyase encoded by an unlinked, tal, gene. Deletion and complementation studies demonstrate that these three genes are required for biosynthesis of naringenin in S. clavuligerus. Other actinobacteria chalcone synthases use caffeic acid, ferulic acid, sinapic acid or benzoic acid as starter units in the formation of different antibiotics and antitumor agents. The biosynthesis of naringenin is restricted to a few Streptomycess species and the encoding gene cluster is present also in some Saccharotrix and Kitasatospora species. Phylogenetic comparison of S. clavuligerus naringenin chalcone synthase with homologous proteins of other actinobacteria reveal that this protein is closely related to chalcone synthases that use malonyl-CoA as a starter unit for the formation of red-brown pigment. The function of the core enzymes in the pathway, such as the chalcone synthase and the tyrosine ammonia lyase, is conserved in plants and actinobacteria. However, S. clavuligerus use a P450 monooxygenase proposed to complete the cyclization step of the naringenin chalcone, whereas this reaction in plants is performed by a chalcone isomerase. Comparison of the plant and S. clavuligerus chalcone synthases indicates that they have not been transmitted between these organisms by a recent horizontal gene transfer phenomenon. We provide a comprehensive view of the molecular genetics and biochemistry of chalcone synthases and their impact on the development of antibacterial and antitumor compounds. These advances allow new bioactive compounds to be obtained using combinatorial strategies. In addition, processes of heterologous expression and bioconversion for the production of naringenin and naringenin-derived compounds in yeasts are described.

Streptomyces clavuligerus: The Omics Era.

The Streptomyces clavuligerus genome consists in a linear chromosome of about 6.7 Mb and four plasmids (pSCL1 to pSCL4), the latter one of 1.8 Mb. Deletion of pSCL4, results in viable mutants with high instability in the chromosome arms, which may lead to chromosome circularisation. Transcriptomic and proteomic studies comparing different mutants with the wild-type strain improved our knowledge on the biosynthesis and regulation of clavulanic acid, cephamycin C and holomycin. Additional knowledge has been obtained on the SARP-type CcaR activator and the network of connections with other regulators (Brp, AreB, AdpA, BldG, RelA) controlling ccaR expression. The transcriptional pattern of the cephamycin and clavulanic acid clusters is supported by the binding of CcaR to different promoters and confirmed that ClaR is a CcaR-dependent activator that controls the late steps of clavulanic biosynthesis. Metabolomic studies allowed the detection of new metabolites produced by S. clavuligerus such as naringenin, desferroxamines, several N-acyl tunicamycins, the terpenes carveol and cuminyl alcohol or bafilomycin J. Heterologous expression of S. clavuligerus terpene synthases resulted in the formation of no less than 15 different terpenes, although none of them was detected in S. clavuligerus culture broth. In summary, application of the Omic tools results in a better understanding of the molecular biology of S. clavuligerus, that allows the use of this strain as an industrial actinobacterial platform and helps to improve CA production.

Modulation of Gene Expression in Actinobacteria by Translational Modification of Transcriptional Factors and Secondary Metabolite Biosynthetic Enzymes.

Different types of post-translational modifications are present in bacteria that play essential roles in bacterial metabolism modulation. Nevertheless, limited information is available on these types of modifications in actinobacteria, particularly on their effects on secondary metabolite biosynthesis. Recently, phosphorylation, acetylation, or phosphopantetheneylation of transcriptional factors and key enzymes involved in secondary metabolite biosynthesis have been reported. There are two types of phosphorylations involved in the control of transcriptional factors: (1) phosphorylation of sensor kinases and transfer of the phosphate group to the receiver domain of response regulators, which alters the expression of regulator target genes. (2) Phosphorylation systems involving promiscuous serine/threonine/tyrosine kinases that modify proteins at several amino acid residues, e.g., the phosphorylation of the global nitrogen regulator GlnR. Another post-translational modification is the acetylation at the epsilon amino group of lysine residues. The protein acetylation/deacetylation controls the activity of many short and long-chain acyl-CoA synthetases, transcriptional factors, key proteins of bacterial metabolism, and enzymes for the biosynthesis of non-ribosomal peptides, desferrioxamine, streptomycin, or phosphinic acid-derived antibiotics. Acetyltransferases catalyze acetylation reactions showing different specificity for the acyl-CoA donor. Although it functions as acetyltransferase, there are examples of malonylation, crotonylation, succinylation, or in a few cases acylation activities using bulky acyl-CoA derivatives. Substrates activation by nucleoside triphosphates is one of the central reactions inhibited by lysine acetyltransferases. Phosphorylation/dephosphorylation or acylation/deacylation reactions on global regulators like PhoP, GlnR, AfsR, and the carbon catabolite regulator glucokinase strongly affects the expression of genes controlled by these regulators. Finally, a different type of post-translational protein modification is the phosphopantetheinylation, catalized by phosphopantetheinyl transferases (PPTases). This reaction is essential to modify those enzymes requiring phosphopantetheine groups like non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. Up to five PPTases are present in S. tsukubaensis and S. avermitilis. Different PPTases modify substrate proteins in the PCP or ACP domains of tacrolimus biosynthetic enzymes. Directed mutations of genes encoding enzymes involved in the post-translational modification is a promising tool to enhance the production of bioactive metabolites.

Purification and Chemical Characterization of a Potent Acaricide and a Closely Related Inactive Metabolite Produced by Eurotium rubrum C47.

Mites are arthropods and some of them infest dry meat cured products and produce allergic reactions. Some mites, such as Tyrolichus casei, Tyrophagus putrescentiae, or Tyrophagus longior feed on filamentous fungi that grow during the meat curing process. Removal of mite infestation of meat products is extremely difficult and there are no adequate miticidal compounds. The filamentous fungus Eurotium rubrum growing on the surface of ham is able to exert a biocontrol of the population of mites due to the production of miticidal compound(s). We have purified two compounds by silica gel chromatography, gel filtration, semipreparative and analytical HPLC and determined their miticidal activity against T. casei using a mite feeding assay. Mass spectrometry and NMR analysis showed that these two compounds are prenylated salicilyl aldehydes with a C-7 alkyl chain differing in a double bond in the C-7 alkyl chain. Structures correspond to those of flavoglaucin and aspergin. Pure flavoglaucin has a miticidal activity resulting in more than 90% mite mortality whereas aspergin does not affect the mites. Both compounds were formed simultaneously by E. rubrum C47 cultures in different media suggesting that they are synthesized by the same pathway. Production of both compounds was higher in solid culture media and the products were associated with abundant formation of cleistothecia. In liquid cultures both compounds remained mainly cell-associated and only about 10% of the total compounds was released to the culture broth. This miticidal compound may be used to combat efficiently mite infestation in different habitats. These results, will promote further advances on the utilization of flavoglaucin in food preservation and in human health since this compound has antitumor activity.

Hyperspectral Imaging Coupled with Multivariate Analysis and Image Processing for Detection and Visualisation of Colour in Cooked Sausages Stuffed in Different Modified Casings.

A hyperspectral imaging system was for the first time exploited to estimate the core colour of sausages stuffed in natural hog casings or in two hog casings treated with solutions containing surfactants and lactic acid in slush salt. Yellowness of sausages stuffed in natural hog casings (control group, 20.26 ± 4.81) was significantly higher than that of sausages stuffed in casings modified by submersion for 90 min in a solution containing 1:30 (w/w) soy lecithin:distilled water, 2.5% wt. soy oil, and 21 mL lactic acid per kg NaCl (17.66 ± 2.89) (p < 0.05). When predicting the lightness and redness of the sausage core, a partial least squares regression model developed from spectra pre-treated with a second derivative showed calibration coefficients of determination (Rc2) of 0.73 and 0.76, respectively. Ten, ten, and seven wavelengths were selected as the important optimal wavelengths for lightness, redness, and yellowness, respectively. Those wavelengths provide meaningful information for developing a simple, cost-effective multispectral system to rapidly differentiate sausages based on their core colour. According to the canonical discriminant analysis, lightness possessed the highest discriminant power with which to differentiate sausages stuffed in different casings.

Omics Approaches Applied to Penicillium chrysogenum and Penicillin Production: Revealing the Secrets of Improved Productivity.

Penicillin biosynthesis by Penicillium chrysogenum is one of the best-characterized biological processes from the genetic, molecular, biochemical, and subcellular points of view. Several omics studies have been carried out in this filamentous fungus during the last decade, which have contributed to gathering a deep knowledge about the molecular mechanisms underlying improved productivity in industrial strains. The information provided by these studies is extremely useful for enhancing the production of penicillin or other bioactive secondary metabolites by means of Biotechnology or Synthetic Biology.

Insight into the Genome of Diverse Penicillium chrysogenum Strains: Specific Genes, Cluster Duplications and DNA Fragment Translocations.

BACKGROUND: There are eighteen species within the Penicillium genus section chrysogena, including the original penicillin producers Penicillium notatum (Fleming strain) and Penicillium chrysogenum NRRL 1951. Other wild type isolates of the Penicillium genus are relevant for the production of useful proteins and primary or secondary metabolites. The aim of this article is to characterize strain specific genes and those genes which are involved in secondary metabolite biosynthesis, particularly the mutations that have been introduced during the ß-lactams strain improvement programs. RESULTS: The available genomes of several classical and novel P. chrysogenum strains have been compared. The first genome sequenced was that of the reference strain P. chrysogenum Wis54-1255, which derives from the wild type P. chrysogenum NRRL 1951; its genome size is 32.19 Mb and it encodes 12,943 proteins. Four chromosomes were resolved in P. chrysogenum and P. notatum by pulse field gel electrophoresis. The genomes of three industrial strains have a similar size but contain gene duplications and truncations; the penicillin gene cluster copy number ranges from one in the wild type to twelve in the P. chrysogenum ASP-E1 industrial strain and is organized in head to tail tandem repeats. The genomes of two new strains, P. chrysogenum KF-25, a producer of antifungal proteins isolated from a soil sample, and P. chrysogenum HKF2, a strain with carbohydrate-converting activities isolated from a sludge treatment plant, showed strain specific genes. CONCLUSIONS: The overall comparison of all available P. chrysogenum genomes indicates that there are a significant number of strain-specific genes, mutations of structural and regulatory genes, gene cluster duplications and DNA fragment translocations. This information provides important leads to improve the biosynthesis of enzymes, antifungal agents, prebiotics or different types of secondary metabolites.

Transport systems, intracellular traffic of intermediates and secretion of ß-lactam antibiotics in fungi.

Fungal secondary metabolites are synthesized by complex biosynthetic pathways catalized by enzymes located in different subcellular compartments, thus requiring traffic of precursors and intermediates between them. The ß-lactam antibiotics penicillin and cephalosporin C serve as an excellent model to understand the molecular mechanisms that control the subcellular localization of secondary metabolites biosynthetic enzymes. Optimal functioning of the ß-lactam biosynthetic enzymes relies on a sophisticated temporal and spatial organization of the enzymes, the intermediates and the final products. The first and second enzymes of the penicillin pathway, ACV synthetase and IPN synthase, in Penicillium chrysogenum and Aspergillus nidulans are cytosolic. In contrast, the last two enzymes of the penicillin pathway, phenylacetyl-CoA ligase and isopenicillin N acyltransferase, are located in peroxisomes working as a tandem at their optimal pH that coincides with the peroxisomes pH. Two MFS transporters, PenM and PaaT have been found to be involved in the import of the intermediates isopenicillin N and phenylacetic acid, respectively, into peroxisomes. Similar compartmentalization of intermediates occurs in Acremonium chrysogenum; two enzymes isopenicillin N-CoA ligase and isopenicillin N-CoA epimerase, that catalyse the conversion of isopenicillin N in penicillin N, are located in peroxisomes. Two genes encoding MFS transporters, cefP and cefM, are located in the early cephalosporin gene cluster. These transporters have been localized in peroxisomes by confocal fluorescence microscopy. A third gene of A. chrysogenum, cefT, encodes an MFS protein, located in the cell membrane involved in the secretion of cephalosporin C, although cefT-disrupted mutants are still able to export cephalosporin by redundant transporters. The secretion of penicillin from peroxisomes to the extracellular medium is still unclear. Attempts have been made to identify a gene encoding the penicillin secretion protein among the 48 ABC-transporters of P. chrysogenum. The highly efficient secretion system that exports penicillin against a concentration gradient may involve active penicillin extrusion systems mediated by vesicles that fuse to the cell membrane. However, there is no correlation of pexophagy with penicillin or cephalosporin formation since inactivation of pexophagy leads to increased penicillin or cephalosporin biosynthesis due to preservation of peroxisomes. The penicillin biosynthesis finding shows that in order to increase biosynthesis of novel secondary metabolites it is essential to adequately target enzymes to organelles.

Regulation of Geldanamycin Biosynthesis by Cluster-Situated Transcription Factors and the Master Regulator PhoP.

Geldanamycin and the closely related herbimycins A, B, and C are benzoquinone-type ansamycins with antitumoral activity. They are produced by Streptomyces hygroscopicus var. geldanus, Streptomyces lydicus and Streptomyces autolyticus among other Streptomyces strains. Geldanamycins interact with the Hsp-90 chaperone, a protein that has a key role in tumorigenesis of human cells. Geldanamycin is a polyketide antibiotic and the polyketide synthase contain seven modules organized in three geldanamycin synthases genes named gdmAI, gdmAII, and gdmAIII. The loading domain of GdmI activates AHBA, and also related hydroxybenzoic acid derivatives, forming geldanamycin analogues. Three regulatory genes, gdmRI, gdmRII, and gdmRIII were found associated with the geldanamycin gene cluster in S. hygroscopicus strains. GdmRI and GdmRII are LAL-type (large ATP binding regulators of the LuxR family) transcriptional regulators, while GdmRIII belongs to the TetR-family. All three are positive regulators of geldanamycin biosynthesis and are strictly required for expression of the geldanamycin polyketide synthases. In S. autolyticus the gdmRIII regulates geldanamycin biosynthesis and also expression of genes in the elaiophylin gene cluster, an unrelated macrodiolide antibiotic. The biosynthesis of geldanamycin is very sensitive to the inorganic phosphate concentration in the medium. This regulation is exerted through the two components system PhoR-PhoP. The phoRP genes of S. hygroscopicus are linked to phoU encoding a transcriptional modulator. The phoP gene was deleted in S. hygroscopicus var geldanus and the mutant was unable to grow in SPG medium unless supplemented with 5 mM phosphate. Also, the S. hygroscopicus pstS gene involved in the high affinity phosphate transport was cloned, and PhoP binding sequences (PHO boxes), were found upstream of phoU, phoRP, and pstS; the phoRP-phoU sequences were confirmed by EMSA and nuclease footprinting protection assays. The PhoP binding sequence consists of 11 nucleotide direct repeat units that are similar to those found in S. coelicolor Streptomyces avermitilis and other Streptomyces species. The available genetic information provides interesting tools for modification of the biosynthetic and regulatory mechanisms in order to increase geldanamycin production and to obtain new geldanamycin analogues with better antitumor properties.

Harnessing microbiota interactions to produce bioactive metabolites: communication signals and receptor proteins.

Numerous microbial communities live in soil, aquatic habitats, plants, and animal bodies. Microbial genome sequences have revealed that thousands of biosynthetic gene clusters (BGCs) are present in different bacteria and filamentous fungi. Many of these BGCs are not expressed in pure cultures in the laboratory. However, a large part of these silent clusters is expressed in nature when complex microbial populations are studied. The encoding specialized metabolites are frequently produced at very low concentrations but still they serve as communication signals that produce important biochemical and differentiation effects on other microorganisms of the consortium. Many specialized metabolites acting as communication signals have been identified, including autoinducers, intergeneric, and interkingdom cues. These signals trigger expression of silent BGCs in other microorganisms, thus providing new compounds with interesting biological and pharmacological activities. Examples of interactions between different bacteria or between bacteria and fungi are described here. Finally, the relevance of the human microbiota and the production in vivo of specialized metabolites of medical interest is discussed.

The Balance Metabolism Safety Net: Integration of Stress Signals by Interacting Transcriptional Factors in Streptomyces and Related Actinobacteria.

Soil dwelling Streptomyces species are faced with large variations in carbon or nitrogen sources, phosphate, oxygen, iron, sulfur, and other nutrients. These drastic changes in key nutrients result in an unbalanced metabolism that have undesirable consequences for growth, cell differentiation, reproduction, and secondary metabolites biosynthesis. In the last decades evidence has accumulated indicating that mechanisms to correct metabolic unbalances in Streptomyces species take place at the transcriptional level, mediated by different transcriptional factors. For example, the master regulator PhoP and the large SARP-type regulator AfsR bind to overlapping sequences in the afsS promoter and, therefore, compete in the integration of signals of phosphate starvation and S-adenosylmethionine (SAM) concentrations. The cross-talk between phosphate control of metabolism, mediated by the PhoR-PhoP system, and the pleiotropic orphan nitrogen regulator GlnR, is very interesting; PhoP represses GlnR and other nitrogen metabolism genes. The mechanisms of control by GlnR of several promoters of ATP binding cassettes (ABC) sugar transporters and carbon metabolism are highly elaborated. Another important cross-talk that governs nitrogen metabolism involves the competition between GlnR and the transcriptional factor MtrA. GlnR and MtrA exert opposite effects on expression of nitrogen metabolism genes. MtrA, under nitrogen rich conditions, represses expression of nitrogen assimilation and regulatory genes, including GlnR, and competes with GlnR for the GlnR binding sites. Strikingly, these sites also bind to PhoP. Novel examples of interacting transcriptional factors, discovered recently, are discussed to provide a broad view of this interactions. Altogether, these findings indicate that cross-talks between the major transcriptional factors protect the cell metabolic balance. A detailed analysis of the transcriptional factors binding sequences suggests that the transcriptional factors interact with specific regions, either by overlapping the recognition sequence of other factors or by binding to adjacent sites in those regions. Additional interactions on the regulatory backbone are provided by sigma factors, highly phosphorylated nucleotides, cyclic dinucleotides, and small ligands that interact with cognate receptor proteins and with TetR-type transcriptional regulators. We propose to define the signal integration DNA regions (so called integrator sites) that assemble responses to different stress, nutritional or environmental signals. These integrator sites constitute nodes recognized by two, three, or more transcriptional factors to compensate the unbalances produced by metabolic stresses. This interplay mechanism acts as a safety net to prevent major damage to the metabolism under extreme nutritional and environmental conditions.

Genome-wide transcriptome response of Streptomyces tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis.

Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces tsukubaensis: differences with the model organism Streptomyces coelicolor.

Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. tsukubaensis and the model organism S. coelicolor.

Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces tsukubaensis through omic Approaches.

Streptomyces tsukubaensis stands out among actinomycetes by its ability to produce the immunosuppressant tacrolimus. Discovered about 30 years ago, this macrolide is widely used as immunosuppressant in current clinics. Other potential applications for the treatment of cancer and as neuroprotective agent have been proposed in the last years. In this review we introduce the discovery of S. tsukubaensis and tacrolimus, its biosynthetic pathway and gene cluster (fkb) regulation. We have focused this work on the omic studies performed in this species in order to understand tacrolimus production. Transcriptomics, proteomics and metabolomics have improved our knowledge about the fkb transcriptional regulation and have given important clues about nutritional regulation of tacrolimus production that can be applied to improve production yields. Finally, we address some points of S. tsukubaensis biology that deserve more attention.

Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases.

The clavine alkaloids produced by the fungi of the Aspergillaceae and Arthrodermatacea families differ from the ergot alkaloids produced by Claviceps and Neotyphodium. The clavine alkaloids lack the extensive peptide chain modifications that occur in lysergic acid derived ergot alkaloids. Both clavine and ergot alkaloids arise from the condensation of tryptophan and dimethylallylpyrophosphate by the action of the dimethylallyltryptophan synthase. The first five steps of the biosynthetic pathway that convert tryptophan and dimethylallyl-pyrophosphate (DMA-PP) in chanoclavine-1-aldehyde are common to both clavine and ergot alkaloids. The biosynthesis of ergot alkaloids has been extensively studied and is not considered in this article. We focus this review on recent advances in the gene clusters for clavine alkaloids in the species of Penicillium, Aspergillus (Neosartorya), Arthroderma and Trychophyton and the enzymes encoded by them. The final products of the clavine alkaloids pathways derive from the tetracyclic ergoline ring, which is modified by late enzymes, including a reverse type prenyltransferase, P450 monooxygenases and acetyltransferases. In Aspergillus japonicus, a α-ketoglutarate and Fe2+-dependent dioxygenase is involved in the cyclization of a festuclavine-like unknown type intermediate into cycloclavine. Related dioxygenases occur in the biosynthetic gene clusters of ergot alkaloids in Claviceps purpurea and also in the clavine clusters in Penicillium species. The final products of the clavine alkaloid pathway in these fungi differ from each other depending on the late biosynthetic enzymes involved. An important difference between clavine and ergot alkaloid pathways is that clavine producers lack the enzyme CloA, a P450 monooxygenase, involved in one of the steps of the conversion of chanoclavine-1-aldehyde into lysergic acid. Bioinformatic analysis of the sequenced genomes of the Aspergillaceae and Arthrodermataceae fungi showed the presence of clavine gene clusters in Arthroderma species, Penicillium roqueforti, Penicillium commune, Penicillium camemberti, Penicillium expansum, Penicillium steckii and Penicillium griseofulvum. Analysis of the gene clusters in several clavine alkaloid producers indicates that there are gene gains, gene losses and gene rearrangements. These findings may be explained by a divergent evolution of the gene clusters of ergot and clavine alkaloids from a common ancestral progenitor six genes cluster although horizontal gene transfer of some specific genes may have occurred more recently.