Regulation Of Secondary Metabolism Research Articles

The Aspergillus flavus fluP-associated metabolite promotes sclerotial production

Aspergillus flavus is able to synthesize a variety of polyketide-derived secondary metabolites including the hepatocarcinogen, aflatoxin B1. The fungus reproduces and disseminates predominantly by production of conidia. It also produces hardened mycelial aggregates called sclerotia that are used to cope with unfavourable growth environments. In the present study, we examined the role of A. flavus fluP, the backbone polyketide synthase gene of secondary metabolite gene cluster 41, on fungal development. The A. flavus CA14 fluP deletion mutant (AfΔfluP) grew and accumulated aflatoxin normally but produced a lower amount of sclerotia than the parental strain. This was also true for the Aspergillus parasiticus BN9 fluP deletion mutant (ApΔfluP). The A. flavus fluP gene was positively regulated by developmental regulators of VeA and VelB but not by the global regulator of secondary metabolism, LaeA. Overexpression of fluP in AfΔfluP (OEfluP) elevated its ability to produce sclerotia compared to that of the parental strain. Coculture of OEfluP with CA14, AfΔfluP, ApΔfluP, or an A. flavus pptA deletion mutant incapable of producing functional polyketide synthases also allowed increased sclerotial production of the respective strains at edges where colonies made contact. Acetone extracts of OEfluP but not of AfΔfluP exhibited the same effect in promoting sclerotial production of AfΔfluP. These results suggest that FluP polyketide synthase is involved in the synthesis of a diffusible metabolite that could serve as a signal molecule to regulate sclerotiogenesis.

Comparative Transcriptional Profiling of Primed and Non-primed Rice Seedlings under Submergence Stress.

Submergence stress is a limiting factor for direct-seeded rice systems in rainfed lowlands and flood-prone areas of South and Southeast Asia. The present study demonstrated that submergence stress severely hampered the germination and seedling growth of rice, however, seed priming alleviated the detrimental effects of submergence stress. To elucidate the molecular basis of seed priming-induced submergence tolerance, transcriptome analyses were performed using 4-day-old primed (selenium-Se and salicylic acid-SA priming) and non-primed rice seedlings under submergence stress. Genomewide transcriptomic profiling identified 2371 and 2405 transcripts with Se- and SA-priming, respectively, that were differentially expressed in rice compared with non-priming treatment under submergence. Pathway and gene ontology term enrichment analyses revealed that genes involved in regulation of secondary metabolism, development, cell, transport, protein, and metal handling were over-represented after Se- or SA-priming. These coordinated factors might have enhanced the submergence tolerance and maintained the better germination and vigorous seedling growth of primed rice seedlings. It was also found that many genes involved in cellular and metabolic processes such as carbohydrate metabolism, cellular, and metabolic biosynthesis, nitrogen compound metabolic process, transcription, and response to oxidative stress were induced and overlapped in seed priming treatments, a finding which reveals the common mechanism of seed priming-induced submergence tolerance. Taken together, these results may provide new avenues for understanding and advancing priming-induced responses to submergence tolerance in crop plants.

Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica.

MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica.

Mitochondrial Complex I Is a Global Regulator of Secondary Metabolism, Virulence and Azole Sensitivity in Fungi.

Recent estimates of the global burden of fungal disease suggest that that their incidence has been drastically underestimated and that mortality may rival that of malaria or tuberculosis. Azoles are the principal class of antifungal drug and the only available oral treatment for fungal disease. Recent occurrence and increase in azole resistance is a major concern worldwide. Known azole resistance mechanisms include over—expression of efflux pumps and mutation of the gene encoding the target protein cyp51a, however, for one of the most important fungal pathogens of humans, Aspergillus fumigatus, much of the observed azole resistance does not appear to involve such mechanisms. Here we present evidence that azole resistance in A. fumigatus can arise through mutation of components of mitochondrial complex I. Gene deletions of the 29.9KD subunit of this complex are azole resistant, less virulent and exhibit dysregulation of secondary metabolite gene clusters in a manner analogous to deletion mutants of the secondary metabolism regulator, LaeA. Additionally we observe that a mutation leading to an E180D amino acid change in the 29.9 KD subunit is strongly associated with clinical azole resistant A. fumigatus isolates. Evidence presented in this paper suggests that complex I may play a role in the hypoxic response and that one possible mechanism for cell death during azole treatment is a dysfunctional hypoxic response that may be restored by dysregulation of complex I. Both deletion of the 29.9 KD subunit of complex I and azole treatment alone profoundly change expression of gene clusters involved in secondary metabolism and immunotoxin production raising potential concerns about long term azole therapy.

Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development.

Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P.chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P.chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P.chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P.chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P.chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA.

Decoding options and accuracy of translation of developmentally regulated UUA codon in Streptomyces: bioinformatic analysis

BackgroundThe gene bldA for leucyl {text{tRNA}}^{text{Leu}}{}_{text{UAA}} is known for almost 30 years as a key regulator of morphogenesis and secondary metabolism in genus Streptomyces. Codon UUA is the rarest one in Streptomyces genomes and is present exclusively in genes with auxiliary functions. Delayed accumulation of translation-competent {text{tRNA}}^{text{Leu}}{}_{text{UAA}} is believed to confine the expression of UUA-containing transcripts to stationary phase. Implicit to the regulatory function of UUA codon is the assumption about high accuracy of its translation, e.g. the latter should not occur in the absence of cognate {text{tRNA}}^{text{Leu}}{}_{text{UAA}}. However, a growing body of facts points to the possibility of mistranslation of UUA-containing transcripts in the bldA-deficient mutants. It is not known what type of near-cognate tRNA(s) may decode UUA in the absence of cognate tRNA in Streptomyces, and whether UUA possesses certain inherent properties (such as increased/decreased accuracy of decoding) that would favor its use for regulatory purposes.FindingsHere we took bioinformatic approach to address these questions. We catalogued the entire complement of tRNA genes from several relevant Streptomyces and identified genes for posttranscriptional modifications of tRNA that might be involved in UUA decoding by cognate and near-cognate tRNAs.ConclusionsBased on tRNA gene content in Streptomyces genomes, we propose possible scenarios of UUA codon mistranslation. UUA is not associated with an increased rate of missense errors as compared to other leucyl codons, contrasting general belief that low-abundant codons are more error-prone than the high-abundant ones.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2683-6) contains supplementary material, which is available to authorized users.

Physiological and transcriptomic analyses reveal mechanistic insight into the adaption of marine Bacillus subtilis C01 to alumina nanoparticles.

An increasing number of studies have investigated the effects of nanoparticles (NPs) on microbial systems; however, few existing reports have focused on the defense mechanisms of bacteria against NPs. Whether secondary metabolism biosynthesis is a response to NP stress and contributes to the adaption of bacteria to NPs is unclear. Here, a significant induction in the surfactin production and biofilm formation were detected by adding Al2O3 NPs to the B. subtilis fermentation broth. Physiological analysis showed that Al2O3 NP stress could also affect the cell and colony morphogenesis and inhibit the motility and sporulation. Exogenously adding commercial surfactin restored the swarming motility. Additionally, a suite of toxicity assays analyzing membrane damage, cellular ROS generation, electron transport activity and membrane potential was used to determine the molecular mechanisms of toxicity of Al2O3 NPs. Furthermore, whole transcriptomic analysis was used to elucidate the mechanisms of B. subtilis adaption to Al2O3 NPs. These results revealed several mechanisms by which marine B. subtilis C01 adapt to Al2O3 NPs. Additionally, this study broadens the applications of nanomaterials and describes the important effects on secondary metabolism and multicellularity regulation by using Al2O3 NPs or other nano-products.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors.

Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio’s silent genome for generating new modulators of fungal secondary metabolism.

On the Developmental and Environmental Regulation of Secondary Metabolism in Vaccinium spp. Berries

Secondary metabolites have important defense and signaling roles, and they contribute to the overall quality of developing and ripening fruits. Blueberries, bilberries, cranberries, and other Vaccinium berries are fleshy berry fruits recognized for the high levels of bioactive compounds, especially anthocyanin pigments. Besides anthocyanins and other products of the phenylpropanoid and flavonoid pathways, these berries also contain other metabolites of interest, such as carotenoid derivatives, vitamins and flavor compounds. Recently, new information has been achieved on the mechanisms related with developmental, environmental, and genetic factors involved in the regulation of secondary metabolism in Vaccinium fruits. Especially light conditions and temperature are demonstrated to have a prominent role on the composition of phenolic compounds. The present review focuses on the studies on mechanisms associated with the regulation of key secondary metabolites, mainly phenolic compounds, in Vaccinium berries. The advances in the research concerning biosynthesis of phenolic compounds in Vaccinium species, including specific studies with mutant genotypes in addition to controlled and field experiments on the genotype × environment (G×E) interaction, are discussed. The recently published Vaccinium transcriptome and genome databases provide new tools for the studies on the metabolic routes.

Influence of Niche-Specific Nutrients on Secondary Metabolism in Vibrionaceae.

Many factors, such as the substrate and the growth phase, influence biosynthesis of secondary metabolites in microorganisms. Therefore, it is crucial to consider these factors when establishing a bioprospecting strategy. Mimicking the conditions of the natural environment has been suggested as a means of inducing or influencing microbial secondary metabolite production. The purpose of the present study was to determine how the bioactivity of Vibrionaceae was influenced by carbon sources typical of their natural environment. We determined how mannose and chitin, compared to glucose, influenced the antibacterial activity of a collection of Vibrionaceae strains isolated because of their ability to produce antibacterial compounds but that in subsequent screenings seemed to have lost this ability. The numbers of bioactive isolates were 2- and 3.5-fold higher when strains were grown on mannose and chitin, respectively, than on glucose. As secondary metabolites are typically produced during late growth, potential producers were also allowed 1 to 2 days of growth before exposure to the pathogen. This strategy led to a 3-fold increase in the number of bioactive strains on glucose and an 8-fold increase on both chitin and mannose. We selected two bioactive strains belonging to species for which antibacterial activity had not previously been identified. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry and bioassay-guided fractionation, we found that the siderophore fluvibactin was responsible for the antibacterial activity of Vibrio furnissii and Vibrio fluvialis These results suggest a role of chitin in the regulation of secondary metabolism in vibrios and demonstrate that considering bacterial ecophysiology during development of screening strategies will facilitate bioprospecting. A challenge in microbial natural product discovery is the elicitation of the biosynthetic gene clusters that are silent when microorganisms are grown under standard laboratory conditions. We hypothesized that, since the clusters are not lost during proliferation in the natural niche of the microorganisms, they must, under such conditions, be functional. Here, we demonstrate that an ecology-based approach in which the producer organism is allowed a temporal advantage and where growth conditions are mimicking the natural niche remarkably increases the number of Vibrionaceae strains producing antibacterial compounds.

Characterization of salA, syrF, and syrG Genes and Attendant Regulatory Networks Involved in Plant Pathogenesis by Pseudomonas syringae pv. syringae B728a.

Pseudomonas syringae pv. syringae B728a, causal agent of brown spot on bean, is an economically important plant pathogen that utilizes extracellular signaling to initiate a lifestyle change from an epiphyte to a pathogen. LuxR regulatory proteins play an important role in the transcriptional regulation of a variety of biological processes involving two-component signaling, quorum sensing, and secondary metabolism. Analysis of the B728a genome identified 24 LuxR-like proteins, three of which are encoded by salA, syrF, and syrG located adjacent to the syringomycin gene cluster. The LuxR-like proteins encoded by these three genes exhibit a domain architecture that places them in a subfamily of LuxR-like proteins associated with regulation of secondary metabolism in B728a. Deletion mutants of salA, syrF, and syrG failed to produce syringomycin and displayed reduction of virulence on bean. The transcriptional start sites of salA, syrG, and syrF were located 63, 235, and 498 bp upstream of the start codons, respectively, using primer extension analysis. The predicted -10/-35 promoter regions of syrF and syrG were confirmed using site-directed mutagenesis and GFP reporters that showed conserved promoter sequences around the -35 promoter region. Overexpression analysis and GFP reporters identified SyrG as an upstream transcriptional activator of syrF, where both SyrG and SyrF activate promoters of syringomycin biosynthesis genes. This study shows that syrG and syrF encode important transcriptional regulators of syringomycin biosynthesis genes.

Light regulation of mycotoxin biosynthesis: new perspectives for food safety

Mycotoxins are secondary metabolites produced by toxigenic fungi contaminating foods and feeds in pre-, post-harvest and processing, and represent a great concern worldwide, both for the economic implications and for the health of the consumers. Many environmental conditions are involved in the regulation of mycotoxin biosynthesis. Among these, light represents one of the most important signals for fungi, influencing several physiological responses such as pigmentation, sexual development and asexual conidiation, primary and secondary metabolism, including mycotoxin biosynthesis. In this review we summarise some recent findings on the effect of specific light wavelength and intensity on mycotoxin biosynthesis in the main toxigenic fungal genera. We describe the molecular mechanism underlying light perception and its involvement in the regulation of secondary metabolism, focusing on VeA, global regulator inAspergillus nidulans, and the White-Collar proteins, key components of light response inNeurospora crassa. Light of specific wavelength and intensity exerts different effects both on growth and on toxin production depending on the fungal genus. InPenicilliumspp. red (627 nm) and blue wavelengths (455-470 nm) reduce ochratoxin A (OTA) biosynthesis by modulating the level of expression of the ochratoxin polyketide synthase. Furthermore a mutual regulation between citrinin and OTA production is reported inPenicilliumtoxigenic species. InAspergillusspp. the effect of light treatment is strongly dependent on the species and culture conditions. Royal blue wavelength (455 nm) of high intensity (1,700 Lux) is capable of completely inhibit fungal growth and OTA production inAspergillus stenyiiandPenicillum verrucosum. InFusariumspp. the effect of light exposure is less effective; mycotoxin-producing species, such asFusarium verticillioidesandFusarium proliferatum, grow better under light conditions, and fumonisin production increased. This review provides a comprehensive picture on light regulation of mycotoxin biosynthesis and discusses possible new applications of this resource in food safety.

A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb.

Dendrobium officinale is an important traditional Chinese herb. Here, we did a transcriptome-wide, organ-specific study on this valuable plant by combining RNA, small RNA (sRNA) and degradome sequencing. RNA sequencing of four organs (flower, root, leaf and stem) of Dendrobium officinale enabled us to obtain 536,558 assembled transcripts, from which 2,645, 256, 42 and 54 were identified to be highly expressed in the four organs respectively. Based on sRNA sequencing, 2,038, 2, 21 and 24 sRNAs were identified to be specifically accumulated in the four organs respectively. A total of 1,047 mature microRNA (miRNA) candidates were detected. Based on secondary structure predictions and sequencing, tens of potential miRNA precursors were identified from the assembled transcripts. Interestingly, phase-distributed sRNAs with degradome-based processing evidences were discovered on the long-stem structures of two precursors. Target identification was performed for the 1,047 miRNA candidates, resulting in the discovery of 1,257 miRNA--target pairs. Finally, some biological meaningful subnetworks involving hormone signaling, development, secondary metabolism and Argonaute 1-related regulation were established. All of the sequencing data sets are available at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/). Summarily, our study provides a valuable resource for the in-depth molecular and functional studies on this important Chinese orchid herb.

Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi.

Filamentous fungi are historically known as rich sources for production of biologically active natural products, so-called secondary metabolites. One particularly pharmaceutically relevant chemical group of secondary metabolites is the nonribosomal peptides synthesized by nonribosomal peptide synthetases (NRPSs). As most of the fungal NRPS gene clusters leading to production of the desired molecules are not expressed under laboratory conditions, efforts to overcome this impediment are crucial to unlock the full chemical potential of each fungal species. One way to activate these silent clusters is by overexpressing and deleting global regulators of secondary metabolism. The conserved fungal-specific regulator of secondary metabolism, LaeA, was shown to be a valuable target for sleuthing of novel gene clusters and metabolites. Additionally, modulation of chromatin structures by either chemical or genetic manipulation has been shown to activate cryptic metabolites. Furthermore, NRPS-derived molecules seem to be affected by cross talk between the specific gene clusters and some of these metabolites have a tissue- or developmental-specific regulation. This chapter summarizes how this knowledge of different tiers of regulation can be combined to increase production of NRPS-derived metabolites in fungal species.

The Regulation of Secondary Metabolism in Photorhabdus.

A general definition of secondary metabolism is that it consists of the metabolic pathways and the products of metabolism that are not absolutely required for the survival of the organism. Using this definition, it is now well established that Photorhabdus elaborate an extensive secondary metabolism during the post-exponential phase of bacterial growth. This secondary metabolism includes, but is not limited to, the production of light, a stilbene antibiotic and an anthraquinone pigment. In this chapter, the role of secondary metabolism during the life cycle of Photorhabdus will be discussed. Recent work has shown that secondary metabolism in Photorhabdus is required for the mutualistic association between the bacteria and its nematode partner, in particular bacterial secondary metabolism is required to support normal nematode growth and development. An isogenic population of Photorhabdus is phenotypically heterogenous and this facilitates functional partitioning within the population. The relationship between secondary metabolism and the various phenotypic and phase variants that exist in populations of Photorhabdus will also be discussed. Finally, this chapter will also describe the various regulatory nodes that have been identified as being part of the complex regulatory network that is used to control the temporal expression of secondary metabolism in Photorhabdus.

Interspecies Complementation of the LuxR Family Pathway-Specific Regulator Involved in Macrolide Biosynthesis.

PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.

Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum.

BackgroundPenicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β–lactams. The high β–lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β–lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown.ResultsTo examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β–lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum.ConclusionsThe classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β–lactams.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2154-4) contains supplementary material, which is available to authorized users.

Cloning and expression analysis of 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Reciprocal Regulation of GlnR and PhoP in Response to Nitrogen and Phosphate Limitations in Saccharopolyspora erythraea.

Nitrogen and phosphate source sensing, uptake, and assimilation are essential for the growth and development of microorganisms. In this study, we demonstrated that SACE_6965 encodes the phosphate regulator PhoP, which controls the transcription of genes involved in phosphate metabolism in the erythromycin-producing Saccharopolyspora erythraea. We found that PhoP and the nitrogen regulator GlnR both regulate the transcription of glnR as well as other nitrogen metabolism-related genes. Interestingly, both GlnR- and PhoP-binding sites were identified in the phoP promoter region. Unlike the nonreciprocal regulation of GlnR and PhoP observed in Streptomyces coelicolor and Streptomyces lividans, GlnR negatively controls the transcription of the phoP gene in S. erythraea. This suggests that GlnR directly affects phosphate metabolism and demonstrates that the cross talk between GlnR and PhoP is reciprocal. Although GlnR and PhoP sites in the glnR and phoP promoter regions are located in close proximity to one another (separated by only 2 to 4 bp), the binding of both regulators to their respective region was independent and noninterfering. These results indicate that two regulators could separately bind to their respective binding sites and control nitrogen and phosphate metabolism in response to environmental changes. The reciprocal cross talk observed between GlnR and PhoP serves as a foundation for understanding the regulation of complex primary and secondary metabolism in antibiotic-producing actinomycetes.