Microbial Production of Melanin Pigments from Caffeic Acid and L-tyrosine Using Streptomyces glaucescens and FCS-ECH-Expressing Escherichia coli.

In this study, synthetic allomelanin was prepared from wild-type Streptomyces glaucescens and recombinant Escherichia coli BL21(DE3) strains. S. glaucescens could produce 125.25 ± 6.01 mg/L of melanin with a supply of 5 mM caffeic acid within 144 h. The ABTS radical scavenging capacity of S. glaucescens melanin was determined to be approximately 7.89 mg/mL of IC50 value, which was comparable to L-tyrosine-based eumelanin. The isolated melanin was used in cotton fabric dyeing, and the effect of copper ions, laccase enzyme treatment, and the dyeing cycle on dyeing performance was investigated. Interestingly, dyeing fastness was greatly improved upon treatment with the laccase enzyme during the cotton dyeing process. Besides, the supply of C5-diamine, which was reported to lead to more complex crosslinking between melanin units, to caffeic acid-based melanin synthesis was also investigated for higher production and novel functionalities. To facilitate the supply of caffeic acid and C5-diamine, E. coli strains expressing each or combinations of tyrosine ammonia lyase/p-coumarate 3-hydroxylase, feruloyl-CoA synthetase/enoyl-CoA hydratase/aldolase, and tyrosinase/lysine decarboxylase enzymes were prepared and investigated for their eumelanin, C5-diamine, and allomelanin production from L-tyrosine and L-lysine, respectively. Finally, H-NMR, FT-IR, and MALDI-TOF analysis of the synthetic melanin pigments were attempted to obtain the chemical information.

The Endophytic Microbiome as a Hotspot of Synergistic Interactions, with Prospects of Plant Growth Promotion.

The plant root is the primary site of interaction between plants and associated microorganisms and constitutes the main components of plant microbiomes that impact crop production. The endophytic bacteria in the root zone have an important role in plant growth promotion. Diverse microbial communities inhabit plant root tissues, and they directly or indirectly promote plant growth by inhibiting the growth of plant pathogens, producing various secondary metabolites. Mechanisms of plant growth promotion and response of root endophytic microorganisms for their survival and colonization in the host plants are the result of complex plant-microbe interactions. Endophytic microorganisms also assist the host to sustain different biotic and abiotic stresses. Better insights are emerging for the endophyte, such as host plant interactions due to advancements in 'omic' technologies, which facilitate the exploration of genes that are responsible for plant tissue colonization. Consequently, this is informative to envisage putative functions and metabolic processes crucial for endophytic adaptations. Detection of cell signaling molecules between host plants and identification of compounds synthesized by root endophytes are effective means for their utilization in the agriculture sector as biofertilizers. In addition, it is interesting that the endophytic microorganism colonization impacts the relative abundance of indigenous microbial communities and suppresses the deleterious microorganisms in plant tissues. Natural products released by endophytes act as biocontrol agents and inhibit pathogen growth. The symbiosis of endophytic bacteria and arbuscular mycorrhizal fungi (AMF) affects plant symbiotic signaling pathways and root colonization patterns and phytohormone synthesis. In this review, the potential of the root endophytic community, colonization, and role in the improvement of plant growth has been explained in the light of intricate plant-microbe interactions.

Bacillus subtilis as a robust host for biochemical production utilizing biomass.

Bacillus subtilis is regarded as a suitable host for biochemical production owing to its excellent growth and bioresource utilization characteristics. In addition, the distinct endogenous metabolic pathways and the suitability of the heterologous pathways have made B. subtilis a robust and promising host for producing biochemicals, such as: bioalcohols; bioorganic acids (lactic acids, α-ketoglutaric acid, and γ-aminobutyric acid); biopolymers (poly(γ-glutamic acid, polyhydroxyalkanoates (PHA), and polysaccharides and monosaccharides (N-acetylglucosamine, xylooligosaccharides, and hyaluronic acid)); and bioflocculants. Also for producing oligopeptides and functional peptides, owing to its efficient protein secretion system. Several metabolic and genetic engineering techniques, such as target gene overexpression and inactivation of bypass pathways, have led to the improvement in production titers and product selectivity. In this review article, recent progress in the utilization of robust B. subtilis-based host systems for biomass conversion and biochemical production has been highlighted, and the prospects of such host systems are suggested.

Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from ricinoleic acid by utilizing crude glycerol as sole carbon source in engineered Escherichia coli expressing BVMO-ADH-FadL.

Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from recinoleic acid was achieved by whole-cell biotransformation by Escherichia coli, utilizing crude glycerol as the sole carbon source. Whole-cell biotransformation resulted in ∼93% conversion of the substrate ricinoleic acid to (Z)-11-(heptanoyloxy)undec-9-enoic acid. We replaced the inducer-dependent promoter system (T7 and Rhm promotors) with a constitutive promoter system. This resulted in successful expression of ADH, FadL, and E6-BVMO, without costly inducer addition. Efficacy evaluation of the whole-cell biotransformation by inducer-free system by five different E. coli strains revealed that the highest product titer was accumulated in E. coli BW25113 strain. The engineered inducer-free system using crude glycerol as the sole carbon source showed competitive performance with induction systems. Optimized conditions resulted in the accumulation of 7.38 ± 0.42 mM (Z)-11-(heptanoyloxy)undec-9-enoic acid, and when 10 mM substrate was used as feed concentration, the product titer reached 2.35 g/L. The inducer-free construct with constitutive promoter system that this study established, which utilizes the waste by-product crude glycerol, will pave the way for the economic synthesis of many industrially important chemicals, like (Z)-11-(heptanoyloxy)undec-9-enoic acid.

Y-doped zinc oxide (YZO) nanoflowers, microstructural analysis and test their antibacterial activity.

Self-assembled 3D flower-like yttrium-doped zinc oxide (YZO) microstructures composed of nanorods were prepared by hydrothermal-precipitation, and tested their antibacterial activity. The morphological, structural, and compositional properties of YZO nanoflowers were characterized by various techniques, which confirmed a well-crystallized wurtzite hexagonal phase. X-ray photoelectron spectroscopy (XPS) of YZO nanopowder showed the 3d core level spectra of yttrium (Y), which formed by two components at about 158.2 eV (3d5/2) and 160.4 eV (3d3/2). The antibacterial activity of YZO nanoflowers were investigated using both gram-positive and gram-negative microorganisms. Enhancement in antibacterial activity was observed by the incorporation of yttrium (Y: 2 at.%) of nanorod-based-flowers because of increased surface area. The prepared YZO nanocomposite showed potential as an antibacterial agent with applications in controlling the spread of infections and also the ability of fast antibacterial activity which can hinder the re-emergence of infection.

Phylogeography and molecular diversity analysis of Jatropha curcas L. and the dispersal route revealed by RAPD, AFLP and nrDNA-ITS analysis.

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity and its distribution. This study was aimed to study the diversity and deduce the phylogeography of Jatropha curcas L. which is said to be the most primitive species of the genus Jatropha. Here we studied the intraspecific genetic diversity of the species distributed in different parts of the globe. The study also focused to understand the molecular diversity at reported probable center of origin (Mexico), and to reveal the dispersal route to other regions based on random amplified polymorphic DNA, amplified fragment length polymorphism and nrDNA-ITS sequences data. The overall genetic diversity of J. curcas found in the present study was narrow. The highest genetic diversity was observed in the germplasm collected from Mexico and supports the earlier hypothesis based on morphological data and natural distribution, it is the center for origin of the species. Least genetic diversity found in the Indian germplasm and clustering results revealed that the species was introduced simultaneously by two distinct germplasm and subsequently distributed in different parts of India. The present molecular data further revealed that J. curcas might have spread from the center of the origin to Cape Verde, than to Spain, Portuguese to other neighboring countries and simultaneously to Africa. The molecular evidence supports the Burkill et al. (A dictionary of the economic products of the Malay Peninsula, Governments of Malaysia and Singapore by the Ministry of Agriculture and Co-operatives. Kuala Lumpur, Malaysia, 1966) view of Portuguese might have introduced the species to India. The clustering pattern suggests that the distribution was interfered by human activity.

Cyclization tag for the detection and facile purification of backbone-cyclized proteins.

Backbone-cyclized proteins, with their characteristic stability toward denaturants such as heat and chemicals, are becoming increasingly significant in many applications. Intein-mediated protein cyclization is the most efficient and frequently used method of choice and has been successfully applied to various targets, achieving stable proteins. However, the detection and isolation of the cyclic protein from the linear one after cyclization is very difficult because the backbone-cyclized protein and the linear one (a by-product formed during the cyclization reaction), which originated from the same molecule, are almost identical in terms of their size. Thus, we first developed a split c-myc tag system; the active c-myc tag was formed only in the backbone-cyclized protein and not in the linear by-product from the inactive precursor, and this helps both the detection and purification of the backbone-cyclized proteins. This tag system, which we called a cyclization tag, was further engineered in its sequence to develop an engineered c-myc (e-myc) tag with enhanced efficiency in the backbone cyclization reaction while keeping its specificity toward the commercial antibody intact. Using two different proteins as models, we show that the cyclization tag developed here can be used as a specific tag for the backbone-cyclized protein, thereby facilitating detection and purification.

Development of SCAR marker specific to non-toxic Jatropha curcas L. and designing a novel multiplexing PCR along with nrDNA ITS primers to circumvent the false negative detection.

Jatropha curcas L., a multipurpose shrub, has acquired significant economic importance for its seed oil which can be converted to biodiesel an emerging alternative to petro-diesel. In addition to the commercial value, it is also having medicinal and even high nutritional value to use as animal fodder which is limited due to the toxicity. Development of molecular marker will enable to differentiate non-toxic from toxic variety of J. curcas in a mixed population and also for quality control since the toxic components of J. curcas has deleterious effect on animals. In the present study, the efforts were made to generate the specific SCAR marker for toxic and/or non-toxic J. curcas from RAPD markers. Among the markers specific for toxic and non-toxic varieties, four were selected, purified, cloned, sequenced, and designed primers out of which one set of primers NT-JC/SCAR I/OPQ15-F and R could able to discriminate the non-toxic with toxic Jatropha by giving expected 430 bp size amplification in non-toxic variety. Furthermore, novel multiplex PCR was designed using the nrDNA ITS primers to overcome the false negatives. Present work also demonstrates utility of the conserved regions of nrDNA coding genes in ruling out the artifacts in PCR-like false negatives frequently occur in SCAR due to various reasons. The specific SCAR markers generated in the present investigation will help to distinguish non-toxic from toxic varieties of J. curcas or vice versa, and isolated marker along with designed multiplex protocol has applications in quality control for selective cultivation of non-toxic variety and will also assist in breeding and molecular mapping studies.

Molecular characterization of intra-population variability of Jatropha curcas L. using DNA based molecular markers.

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity of J. curcas germplasm. In the present study, efforts were made to analyze the genetic diversity among the elite germplasms of J. curcas, selected on the basis of their performance in field using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The plants were selected on the basis of height, canopy circumference, number of seeds per fruit, weight of 100 seeds, seed yield in grams per plant and oil content. Out of 250 RAPD (with 26 primers), 822 AFLP (with 17 primers) and 19 SSR band classes, 141, 346 and 7 were found to be polymorphic, respectively. The percentage polymorphism among the selected germplasms using RAPD, AFLP and SSR was found to be 56.43, 57.9, and 36.84, respectively. The Jaccard's similarity coefficient was found 0.91, 0.90 and 0.91 through RAPD, AFLP and SSR marker systems, respectively. Principle component analysis (PCA) and dendrogarm analysis of genetic relationship among the germplasm using RAPD, AFLP and SSR data showed a good correlation for individual markers. The germplasm JCC-11, 12, 13, 14 and 15 whose yield found to be high were clustered together in dendrogram and PCA analysis though JCC11 is geographically distinct from others. In overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and JCC 6 and JCC10 (in SSR) were found genetically diverse. Characterization of geographically distinct and genetically diverse germplasms with varied yield characters is an important step in marker assisted selection (MAS) and it can be useful for breeding programs and QTL mapping.

Cross species amplification ability of novel microsatellites isolated from Jatropha curcas and genetic relationship with sister taxa: cross species amplification and genetic relationship of Jatropha using novel microsatellites.

The present investigation was undertaken with an aim to check the ability of cross species amplification of microsatellite markers isolated from Jatropha curcas--a renewable source of biodiesel to deduce the generic relationship with its six sister taxa (J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis). Out of the 49 markers checked 31 markers showed cross species amplification in all the species studied. JCDS-30, JCDS-69, JCDS-26, JCMS-13 and JCMS-21 amplified in J. curcas. However, these markers did not show any cross species amplification. Overall percentage of polymorphism (PP) among the species studied was 38% and the mean genetic similarity (GS) was found to be 0.86. The highest PP (24) and least GS (0.76) was found between J. curcas/J. podagrica and J. curcas/J. multifida and least PP (4.44) and highest GS (0.96) was found between J. integerrima/J. tanjorensis. Dendrogram analysis showed good congruence to RAPD and AFLP than nrDNA ITS data reported earlier. The characterized microsatellites will pave way for intraspecies molecular characterization which can be further utilized in species differentiation, molecular identification, characterization of interspecific hybrids, exploitation of genetic resource management and genetic improvement of the species through marker assisted breeding for economically important traits.

Isolation of novel microsatellites using FIASCO by dual probe enrichment from Jatropha curcas L. and study on genetic equilibrium and diversity of Indian population revealed by isolated microsatellites.

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petrodiesel. Very few attempts were made to isolate novel microsatellite markers and assessment of the extent of genetic equilibrium and diversity that exists in J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India using isolated microsatellites. The overall efficiency of the enrichment of microsatellite by dual probe in the present study found to be 54% and among the sequences obtained the percentage of sequences having suitable flanking regions for the primer designing was found to be 89.58%. The mean co-efficient of genetic similarity (CGS) was found to be 0.97. The overall diversity obtained by microsatellites was found to be low in comparison with the diversity reported by multilocus markers systems observed in earlier studies; however, the good allele polymorphism was observed. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in accordance to geographical area of collection. The present study, diversity analysis using microsatellite markers concludes the low genetic diversity and genetic disequlibrium of J. curcas in India and will provide pavement for further intra-population studies on narrow geographical areas to understand the population genetic structure, phylogeography and molecular ecological studies. The germplasm characterized, and the microsatellite markers isolated and characterized in the present study can be employed efficiently in breeding programs for genetic improvement of the species through marker assisted selection and QTL analysis, for further genetic resource management and help in making the J. curcas as potential crop with superior agronomical traits.

Molecular characterization and identification of markers for toxic and non-toxic varieties of Jatropha curcas L. using RAPD, AFLP and SSR markers.

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).

Genetic divergence and phylogenetic analysis of genus Jatropha based on nuclear ribosomal DNA ITS sequence.

The genus Jatropha belongs to the family Euphorbiaceae having significant economic importance. The present investigation was undertaken with an aim to understand phylogenetic relationships among seven species (J. curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis.) which are widely distributed in India, using nuclear ribosomal DNA ITS sequence (nrDNA ITS) and to compare the results with multilocus marker analysis systems reported earlier for the same genus. The size variation obtained among sequenced nrDNA ITS regions was narrow and ranged from 647 to 654 bp. The overall mean genetic distance (GD) of genus Jatropha was found to be 0.385. Highest interspecific GD (0.419) was found between J. glandulifera and J. multifida. The least interspecific GD (0.085) was found between J. gossypifolia and J. tanjorensis. The highest intraspecific GD was observed in J. podagrica (0.011) and least in J. gossypifolia (0.002). The phylogram obtained using nrDNA ITS sequence showed congruence with the phylograms obtained using multilocus markers system reported earlier with minor variations. The present study also strongly supports high phylogenetic closeness of J. curcas and J. integerrima. The only exception found was J. podagrica which clustered with J. multifida in earlier based on multilocus marker analysis, was clustered with J. curcas in the present analysis. The sequence data generated in the present investigation will help for further studies in intraspecies population, and their phylogenetic analysis, biogeographical, molecular evolution studies and also pave way for future phylogenetic and/or evolution studies among the other groups belongs to the family Euphorbiaceae.