Overproduction Of Secondary Metabolites Research Articles

Upregulated synthesis and production of bioactive compounds in Lotus arabicus L. by in vitro feeding with dried powder of date palm seeds

BackgroundPlants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L.ResultsThe presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds.ConclusionThe marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

Co-Expression of Transcriptional Regulators and Housekeeping Genes in Streptomyces spp.: A Strategy to Optimize Metabolite Production.

The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

Ozone exposure response on physiological and biochemical parameters vis-a-vis secondary metabolites in a traditional medicinal plant Sida cordifolia L.

Tropospheric ozone is a phytotoxic air pollutant that alters the production of bioactive compounds and key metabolites of plants. A field study was established on a medicinal plant, Sida cordifolia L. (Bala) under ambient (AO; 43.3 ppb) and elevated level of ozone (ambient+20 ppb; EO) at different developmental stages. The objective of study was to analyze the changes in physiology and biochemical parameters with secondary metabolites of S. cordifolia under EO treatment using open-top chambers. Physiological parameters such as photosynthesis, chlorophyll fluorescence, water use efficiency, stomatal conductance, and intercellular CO2 concentration demonstrated a negative response at all growth stages in EO-treated plants. Trichome density increased by 83.56%, whereas photosynthetic pigments decreased under EO exposure. Total phenolics and thiols increased at 90 days after treatment (DAT) under EO, while non-enzymatic antioxidants such as flavonoids and proline induced at all growth stages. Reactive oxygen species showed a differential response at different growth stages under EO as compared with AO treatment. Enzymatic antioxidants such as ascorbate peroxidase, catalase, peroxidase, glutathione reductase, and phenylalanine ammonia-lyase increased at all growth stages under EO. However, superoxide dismutase was induced only at 30 DAT. A significant increase was observed in the contents of lignin, tannin, saponin, and alkaloids in leaves and roots of the test plant under EO exposure. Phytosterol increased under EO at all the growth stages in roots, and at 90 DAT in leaves. Despite diverse physiological, ROS, and antioxidant responses, the results demonstrated that elevated ozone boosted the production of secondary metabolites, medicinally important compounds in Sida cordifolia. The overproduction of secondary metabolites under futuristic levels of ozone could be a useful resource for the exploitation of bioactive, medicinally important compounds from S. cordifolia.

Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana

Nowadays, overproduction of secondary metabolites in remedial herbs through giving biotic/abiotic stresses is an interesting area of research. In the current study, the influences of various concentrations of silver nanoparticles (Ag NPs) were evaluated on several morphological and physio-biochemical traits, such as the steviol glycosides level in Stevia. The findings showed that the herbs incubated with 400-ppm Ag NPs own the highest dry and fresh weight of shoot, while those incubated with 80- up to 200- ppm Ag NPs own the highest steviol glycosides content. As a result, we successfully improve the content of stevioside glycoside up to 1.75-fold by applying the 80- up to 200-ppm Ag NPs in Stevia medicinal plant. Moreover, our findings revealed that low concentrations the Ag NPs lead to an increase of glutathione content and total antioxidant capacity, and a decrease of MDA, whereas treatments at higher concentrations induced adverse effects for the plant. As a result, the treatment with Ag NPs low concentrations had a favorable efficacy on physio-biochemical and morphological characteristics of Stevia. These achievements are very promising, because they revealed a considerable capability for the Ag NPs application in enhancing the secondary metabolites in Stevia remedial herb. The present study is the first case assessing the desirable influences of Ag NPs on the Stevia, in regard with shifting of biosynthetic pathway of steviol glycosides in a concentration-dependent manner.

Improvement of Rimocidin Biosynthesis by Increasing Supply of Precursor Malonyl-CoA via Over-expression of Acetyl-CoA Carboxylase in Streptomyces rimosus M527.

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Summary Verticillium dahliae is a broad host‐range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved.Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed.Population genomics previously revealed that G‐LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G‐LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N‐lauroylethanolamine (N‐acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase.The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

The Global Regulators Lrp, LeuO, and HexA Control Secondary Metabolism in Entomopathogenic Bacteria.

Photorhabdus luminescens TTO1 and Xenorhabdus nematophila HGB081 are insect pathogenic bacteria and producers of various structurally diverse bioactive natural products. In these entomopathogenic bacteria we investigated the role of the global regulators Lrp, LeuO, and HexA in the production of natural products. Lrp is a general activator of natural product biosynthesis in X. nematophila and for most compounds in TTO1. Microarray analysis confirmed these results in X. nematophila and enabled the identification of additional biosynthesis gene clusters (BGC) regulated by Lrp. Moreover, when promoters of two X. nematophila BGC were analyzed, transcriptional activation by Lrp was observed. In contrast, LeuO in X. nematophila and P. luminescens has both repressing and activating features, depending on the natural product examined. Furthermore, heterologous overexpression of leuO from X. nematophila in the closely related Xenorhabdus szentirmaii resulted in overproduction of several natural products including novel compounds. The presented findings could be of importance for establishing a tool for overproduction of secondary metabolites and subsequent identification of novel compounds.

Changing growth behavior of heavy‐metal tolerant bacteria: Media optimization using droplet‐based microfluidics

Overproduction of secondary metabolites requires know‐how for media optimization and design of effective screening procedures. Micro‐segmented flow was applied to test the growth characteristics of heavy‐metal tolerant soil bacteria (Bacillus sporothermodurans and Streptomyces tendae) toward changing media composition. The applied microfluidic technique was shown to be suitable for media optimization, with effects of media solutes like phosphorus and nitrogen, and a trace metal solution. For inorganic phosphate and trace metals, the synthesis of secondary metabolites had a much narrower tolerance for concentration ranges than vegetative cells. As we worked with metal‐tolerant strains, some tests were done in the presence of copper‐II ions in the bacteria‐specific critical concentration of 0.25 mM. In particular, for the Bacillus strain, the macronutrient phosphorus resulted in a threefold increase in growth with rising concentration. Copper led to a prolonged lag phase. For S. tendae, no similarly enhancing effect was observed. The trace metal solution promoted bacterial growth only in the case of S. tendae, for higher concentration ranges. For the nitrogen source, especially S. tendae responded with changes between the light scattering and the fluorescence signal. This could be a hint for the production of fluorescent secondary metabolites.

Enhanced intracellular Ca2+ concentrations in Escherichia coli and Bacillus subtilis after addition of oligosaccharide elicitors

Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca(2+) levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide elicitors on intracellular Ca(2+) levels. The apoaequorin gene was cloned into Escherichia coli DH5α and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase in cytosolic Ca(2+) in E. coli and tenfold increase in B. subtilis. These increases in Ca(2+) levels could therefore contribute to the enhancement of secondary metabolite levels.

Effects of oligosaccharides and polysaccharides on the generation of reactive oxygen species in different biological systems

Use of carbohydrates as elicitors is a novel technique for enhancement of the production of industrially important microbial products. The relation between the levels of ROS (reactive oxygen species) and overproduction of antibiotics in microbial cultures has already been established. In the present study, we aimed to exploit the ROS response to develop a fast technique to assess the potential of oligosaccharides [oligoguluronate, oligomannuronate and MO (mannan oligosaccharides)] and polysaccharides [alginate and LBG (locust-bean gum)] as elicitors for overproduction of secondary metabolites in Streptomyces rimosus and Penicillium chrysogenum. We have also investigated changes in the production of ROS in neutrophils as a result of the action of the same elicitors. LBG-derived oligosaccharides (MO) were most potent inhibitors of ROS in all systems investigated. This correlates with overproduction of secondary metabolites in microbes and enhancement of a number of mammalian systems. We believe that the effects of oligosaccharides and polysaccharides on ROS production by mammalian and microbial cells can be correlated predicatively with overproduction. The underlying methodology offers a fast screening of elicitors that can be applied across the different systems.

Strain improvement for fermentation and biocatalysis processes by genetic engineering technology.

Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?

Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans

A DNA fragment that caused pigment production in Streptomyces lividans was isolated from a gene library of PstI-digested chromosomal fragments of S. coelicolor A3(2). Subcloning and nucleotide sequencing proved the identity of the cloned gene to ptpA encoding a low-molecular-mass phosphotyrosine protein phosphatase. The S. lividans transformant containing ptpA on pIJ41 with a copy number of 3–4 per genome produced large amounts of undecylprodigiosin and A-factor, in addition to the pigmented antibiotic actinorhodin, whereas the transformant containing ptpA on an SCP2 ∗ derivative with a copy number of 1–2 did not. The PtpA protein produced as a fusion to the maltose binding protein in Escherichia coli showed phosphatase activity toward o-phosphotyrosine, but not toward o-phosphoserine or o-threonine. Introduction of a mutant ptpA gene encoding an inactive protein with serine instead of the 9th cysteine caused no pigmentation. Disruption of the chromosomal ptpA gene of S. coelicolor A3(2), however, appeared to cause no detectable effect on the production of the pigmented antibiotics or A-factor and the ptpA disruptants developed aerial mycelium and spores normally.

Some aspects of overproduction of secondary metabolites

Different approaches used to increase production of secondary metabolites and construct overproducing strains of microorganisms are reviewed. Overproduction of secondary metabolites incuudes the physiological control,e.g. feed-back inhibition, carbon and energy source regulation, nitrogen source regulation, phosphate regulation and the effect of autoregulatory compounds. The genetic control of overproduction of secondary metabolites includes mechanisms similar to those controlling the expression of primary metabolism coding genes, although the genes specifying biosynthesis of secondary metabolites and their expression have some particular features. Possible future trends in the study of overproduction of secondary metabolites are discussed.

5-Fluorouracil stimulation of naphthoquinone synthesis in Nectria haematococca (Berk. & Br.) Wr.

A large number of growth inhibitors were tested for their effects on the pigmentation of Nectria haematococca. Some of them (5-fluorouracil, 5-fluorouridine, 6-azauracil, cycloheximide, nystatin, benomyl, some amino acid analogs) stimulated the synthesis of naphthoquinones. The others (fluoroacetate, dl-ethionine, puromycin, methothrexate, N-phosphonacetyl- l-aspartate, 5-fluorocytosine, and other pyrimidine analogs) were ineffective. The highest amounts of pigments were detected in cultures containing 5-fluorouracil (5FU). Maximal synthesis was obtained only on adding 5FU during early vegetative growth. The action of 5FU was neither as a mutagen nor as a naphthoquinone precursor. Uracil and thymine decreased the pigmentation-enhancing effect of 5FU. Incorporation of [2- 14C]5FU was determined and part of the radioactivity was found in nucleic acids. The significance of these observations is discussed in the light of knowledge about (1) the mechanisms of action of 5FU and (2) the relationships between growth rate and overproduction of secondary metabolites in micro-organisms.