Assessment of fungal diversity in soil rhizosphere associated with Rhazya stricta and some desert plants using metagenomics.

This study aimed to compare the fungal rhizosphere communities of Rhazya stricta, Enneapogon desvauxii, Citrullus colocynthis, Senna italica, and Zygophyllum simplex, and the gut mycobiota of Poekilocerus bufonius (Orthoptera, Pyrgomorphidae, "Usherhopper"). A total of 164,485 fungal reads were observed from the five plant rhizospheres and Usherhopper gut. The highest reads were in S. italica rhizosphere (29,883 reads). Species richness in the P. bufonius gut was the highest among the six samples. Ascomycota was dominant in all samples, with the highest reads in E. desvauxii (26,734 reads) rhizosphere. Sordariomycetes and Dothideomycetes were the dominant classes detected with the highest abundance in C. colocynthis and E. desvauxii rhizospheres. Aspergillus and Ceratobasidium were the most abundant genera in the R. stricta rhizosphere, Fusarium and Penicillium in the E. desvauxii rhizosphere and P. bufonius gut, Ceratobasidium and Myrothecium in the C. colocynthis rhizosphere, Aspergillus and Fusarium in the S. italica rhizosphere, and Cochliobolus in the Z. simplex rhizosphere. Aspergillus terreus was the most abundant species in the R. stricta and S. italica rhizospheres, Fusarium sp. in E. desvauxii rhizosphere, Ceratobasidium sp. in C. colocynthis rhizosphere, Cochliobolus sp. in Z. simplex rhizosphere, and Penicillium sp. in P. bufonius gut. The phylogenetic results revealed the unclassified species were related closely to Ascomycota and the species in E. desvauxii, S. italica and Z. simplex rhizospheres were closely related, where the species in the P. bufonius gut, were closely related to the species in the R. stricta, and C. colocynthis rhizospheres.

Soil compartment is a major determinant of the impact of simulated rainfall on desert microbiota.

Although desert soils support functionally important microbial communities that affect plant growth and influence many biogeochemical processes, the impact of future changes in precipitation patterns on the microbiota and their activities is largely unknown. We performed in-situ experiments to investigate the effect of simulated rainfall on bacterial communities associated with the widespread perennial shrub, Rhazya stricta in Arabian desert soils. The bacterial community composition was distinct between three different soil compartments: surface biological crust, root-attached, and the broader rhizosphere. Simulated rainfall had no significant effect on the overall bacterial community composition, but some population-level responses were observed, especially in soil crusts where Betaproteobacteria, Sphingobacteria, and Bacilli became more abundant. Bacterial biomass in the nutrient-rich crust increased three-fold one week after watering, whereas it did not change in the rhizosphere, despite its much higher water retention. These findings indicate that between rainfall events, desert-soil microbial communities enter into stasis, with limited species turnover, and reactivate rapidly and relatively uniformly when water becomes available. However, microbiota in the crust, which was relatively enriched in nutrients and organic matter, were primarily water-limited, compared with the rhizosphere microbiota that were co-limited by nutrients and water.

Molecular and cytogenetic assessment of Dipterygium glaucum genotoxicity.

The aim of the present study is to assess the genotoxicity of Dipterygium glaucum grows widely in Saudi Arabia desert to produce safety herbal products. This work is considered the first and pioneer report so far due to the lack and poor evaluated reports of the plant species for their mutagensity, genotoxicity and cytogenetics effects. Cytogenetic effects of D. glaucum on mitotic in roots of Vicia faba showed reduction in mitotic activity using three extracts; water, ethanol and ethyl acetate. Chromosomal abnormalities were recorded that included stickiness of chromosomes, chromatin bridge, fragments, lagging chromosome and micronuclei. Protein bands and RAPD analyses of V. faba treated with three D. glaucum extracts revealed some newly induced proteins and DNA fragments and other disappeared. Chemical constitution of the plant species should be identified with their biological activities against human and animal cells like HeLa cancer cell line. We are recommending using additional genotoxicity tests and other toxicity tests on animal culture with different concentrations and also utilizing several drought and heat tolerant genes of the plant species in gene cloning to develop and improve other economical crop plants instead of using the species as oral herbal remedy.

Complete sequences of organelle genomes from the medicinal plant Rhazya stricta (Apocynaceae) and contrasting patterns of mitochondrial genome evolution across asterids.

BACKGROUND: Rhazya stricta is native to arid regions in South Asia and the Middle East and is used extensively in folk medicine to treat a wide range of diseases. In addition to generating genomic resources for this medicinally important plant, analyses of the complete plastid and mitochondrial genomes and a nuclear transcriptome from Rhazya provide insights into inter-compartmental transfers between genomes and the patterns of evolution among eight asterid mitochondrial genomes. RESULTS: The 154,841 bp plastid genome is highly conserved with gene content and order identical to the ancestral organization of angiosperms. The 548,608 bp mitochondrial genome exhibits a number of phenomena including the presence of recombinogenic repeats that generate a multipartite organization, transferred DNA from the plastid and nuclear genomes, and bidirectional DNA transfers between the mitochondrion and the nucleus. The mitochondrial genes sdh3 and rps14 have been transferred to the nucleus and have acquired targeting presequences. In the case of rps14, two copies are present in the nucleus; only one has a mitochondrial targeting presequence and may be functional. Phylogenetic analyses of both nuclear and mitochondrial copies of rps14 across angiosperms suggests Rhazya has experienced a single transfer of this gene to the nucleus, followed by a duplication event. Furthermore, the phylogenetic distribution of gene losses and the high level of sequence divergence in targeting presequences suggest multiple, independent transfers of both sdh3 and rps14 across asterids. Comparative analyses of mitochondrial genomes of eight sequenced asterids indicates a complicated evolutionary history in this large angiosperm clade with considerable diversity in genome organization and size, repeat, gene and intron content, and amount of foreign DNA from the plastid and nuclear genomes. CONCLUSIONS: Organelle genomes of Rhazya stricta provide valuable information for improving the understanding of mitochondrial genome evolution among angiosperms. The genomic data have enabled a rigorous examination of the gene transfer events. Rhazya is unique among the eight sequenced asterids in the types of events that have shaped the evolution of its mitochondrial genome. Furthermore, the organelle genomes of R. stricta provide valuable genomic resources for utilizing this important medicinal plant in biotechnology applications.

Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes.

Land plant plastid genomes (plastomes) provide a tractable model for evolutionary study in that they are relatively compact and gene dense. Among the groups that display an appropriate level of variation for structural features, the inverted-repeat-lacking clade (IRLC) of papilionoid legumes presents the potential to advance general understanding of the mechanisms of genomic evolution. Here, are presented six complete plastome sequences from economically important species of the IRLC, a lineage previously represented by only five completed plastomes. A number of characters are compared across the IRLC including gene retention and divergence, synteny, repeat structure and functional gene transfer to the nucleus. The loss of clpP intron 2 was identified in one newly sequenced member of IRLC, Glycyrrhiza glabra. Using deeply sequenced nuclear transcriptomes from two species helped clarify the nature of the functional transfer of accD to the nucleus in Trifolium, which likely occurred in the lineage leading to subgenus Trifolium. Legumes are second only to cereal crops in agricultural importance based on area harvested and total production. Genetic improvement via plastid transformation of IRLC crop species is an appealing proposition. Comparative analyses of intergenic spacer regions emphasize the need for complete genome sequences for developing transformation vectors for plastid genetic engineering of legume crops.

Cell factories for insulin production.

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

GC-MS Analysis of Bioactive Compounds Extracted from Plant Rhazya stricta Using Various Solvents.

Worldwide, human beings have traditionally employed many folkloric herbal resources as complementary and alternative remedies, and these remedies have played a pivotal role in modern medicines for many decades, as scientists have used them to develop drugs. We studied the effects of employing solvents with varying polarity on the yields of phytochemical components extracted from the plant Rhazya stricta. We used chloroform-methanol (1:1), methanol, ethanol, diethyl ether, and ethyl acetate as extraction solvents. The results showed that the efficiencies of the solvents at extracting phytochemical compounds were in this order: chloroform-methanol < ethanol < methanol < diethyl ether < ethyl acetate extract. The chloroform-methanol extract produced the highest concentration of phenolic and flavonoid contents among the five solvents tested (13.3 mg GAE/g DM and 5.43 CE/g DM). The yields of the extracted phytochemical compounds ranged from 47.55 to 6.05%. The results revealed that the properties of the extraction solvents considerably impacted the extraction yield and the phytochemical components of the R. stricta extract. Furthermore, compared with the other solvents, the chloroform-methanol extraction led to the highest yield (47.55%) and to more phytochemical substances being extracted. The aim of this study is to investigate the phytochemical compounds extracted from R. stricta with different solvents that have different polarities.

Composition, Abundance, and Diversity of the Soil Microbiome Associated with the Halophytic Plants Tamarix aphylla and Halopeplis perfoliata on Jeddah Seacoast, Saudi Arabia.

The coast of the Red Sea in Jeddah City is home to a unique microbial community that has adapted to extreme environmental conditions. Therefore, it is essential to characterize the microbial community in this unique microbiome to predict how environmental changes will affect it. The aim of this study was to conduct metagenomic sequencing of 16S rRNA and ITS rRNA genes for the taxonomic classification of the microbial community in soil samples associated with the halophytic plants Tamarix aphylla and Halopeplis perfoliata. Fifteen soil samples were collected in triplicate to enhance robustness and minimize sampling bias. Firstly, to identify novel microbial candidates, the gDNAs were isolated from the saline soil samples surrounding each plant, and then bacterial 16S (V3-V4) and fungal ITS1 regions were sequenced utilizing a high-throughput approach (next-generation sequencing; NGS) on an Illumina MiSeq platform. Quality assessment of the constructed amplicon libraries was conducted using Agilent Bioanalyzer and fluorometric quantification methods. The raw data were processed and analyzed using the Pipeline (Nova Lifetech, Singapore) for bioinformatics analysis. Based on the total number of readings, it was determined that the phylum Actinobacteriota was the most prevalent in the soil samples examined, followed by the phylum Proteobacteria. Based on ITS rRNA gene analysis, the alpha and beta fungal diversity in the studied soil samples revealed that the fungal population is structured into various groups according to the crust (c) and/or rhizosphere (r) plant parts. Fungal communities in the soil samples indicated that Ascomycota and Basidiomycota were the two most abundant phyla based on the total amount of sequence reads. Secondly, heat-map analysis of the diversity indices showed that the bacterial alpha diversity, as measured by Shannon, Simpson, and InvSimpson, was associated with soil crust (Hc and Tc enclosing H. perfoliata and T. aphylla, respectively) and that the soil rhizosphere (Hr and Tr) was strongly correlated with bacterial beta diversity. Finally, fungal-associated Tc and Hc samples clustered together, according to observations made using the Fisher and Chao1 methods, and Hr and Tr samples clustered together according to Shannon, Simpson, and InvSimpson analyses. As a result of the soil investigation, potential agents that have been identified could lead to innovative agricultural, medical, and industrial applications.

Differential control of growth, apoptotic activity, and gene expression in human breast cancer cells by extracts derived from medicinal herbs Zingiber officinale.

The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale) on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A). Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose) polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4). On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT). These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas.

Evaluation of cytotoxicity and antiviral activity of Rhazya stricta Decne leaves extract against influenza A/PR/8/34 (H1N1).

Influenza viruses have developed resistance to the current classes of drugs, which means they could eventually become more virulent and cause more mortality and hospitalization. Our study aims to investigate the antiviral activity of Rhazya stricta Decne leaves extract in vitro and search for new promising drugs from R. stricta identified compounds in silico. The study was performed in vitro by utilizing Madin-Darby Canine Kidney cell line (MDCK) as a substrate for the influenza virus and estimating the inhibition performance of the plant leaves extract. Additionally, in silico screening was conducted to explore the antiviral activity of R. stricta phytochemicals. We investigated the cytotoxicity of R. stricta leaves extract and its antiviral activity against influenza virus (A/Puerto Rico/8/34 (H1N1)) using the MTT assay. The mode of action of the plant leaves extract during the viral life cycle was tested using time-of-addition assay. In silico analyses were performed, including molecular docking, drug-likeness analysis, and toxicity risk assessment, to state the leading compounds to be developed into an anti-influenza virus drug. The 50% cytotoxicity concentration of the leaves extract was CC50: 184.6 µg/mL, and the 50% inhibition concentration was CI50: 19.71 µg\mL. The time of addition assay revealed that R. stricta leaves extract exerted its activity in the late step of the influenza virus replication cycle. In comparison to Oseltamivir, the leading compounds showed better binding affinity and can be developed into oral drugs with low toxicity risk. Isolation and purification of the leading compounds and testing their antiviral activity in vitro and in vivo are required.

In silico screening of some compounds derived from the desert medicinal plant Rhazya stricta for the potential treatment of COVID-19.

The latest coronavirus pandemic (SARS-CoV-2) poses an exceptional threat to human health and society worldwide. The coronavirus (SARS-CoV-2) spike (S) protein, which is required for viral-host cell penetration, might be considered a promising and suitable target for treatment. In this study, we utilized the nonalkaloid fraction of the medicinal plant Rhazya stricta to computationally investigate its antiviral activity against SARS-CoV-2. Molecular docking and molecular dynamics simulations were the main tools used to examine the binding interactions of the compounds isolated by HPLC analysis. Ceftazidime was utilized as a reference control, which showed high potency against the SARS-CoV-2 receptor binding domain (RBD) in an in vitro study. The five compounds (CID:1, CID:2, CID:3, CID:4, and CID:5) exhibited remarkable binding affinities (CID:1, - 8.9; CID:2, - 8.7; and CID:3, 4, and 5, - 8.5 kcal/mol) compared to the control compound (- 6.2 kcal/mol). MD simulations over a period of 200 ns further corroborated that certain interactions occurred with the five compounds and the nonalkaloidal compounds retained their positions within the RBD active site. CID:2, CID:4, and CID:5 demonstrated high stability and less variance, while CID:1 and CID:3 were less stable than ceftazidime. The average number of hydrogen bonds formed per timeframe by CID:1, CID:2, CID:3, and CID:5 (0.914, 0.451, 1.566, and 1.755, respectively) were greater than that formed by ceftazidime (0.317). The total binding free energy calculations revealed that the five compounds interacted more strongly within RBD residues (CID:1 = - 68.8, CID:2 = - 71.6, CID:3 = - 74.9, CID:4 = - 75.4, CID:5 = - 60.9 kJ/mol) than ceftazidime (- 34.5 kJ/mol). The drug-like properties of the selected compounds were relatively similar to those of ceftazidime, and the toxicity predictions categorized these compounds into less toxic classes. Structural similarity and functional group analyses suggested that the presence of more H-acceptor atoms, electronegative atoms, acidic oxygen groups, and nitrogen atoms in amide or aromatic groups were common among the compounds with the lowest binding affinities. In conclusion, this in silico work predicts for the first time the potential of using five R. stricta nonalkaloid compounds as a treatment strategy to control SARS-CoV-2 viral entry.

A Review of Rhazya stricta Decne Phytochemistry, Bioactivities, Pharmacological Activities, Toxicity, and Folkloric Medicinal Uses.

The local medicinal plant Rhazya stricta Decne is reviewed for its folkloric medicinal, phytochemical, pharmacological, biological, and toxicological features. R. stricta has been used widely in different cultures for various medical disorders. The phytochemical studies performed on the R. stricta extract revealed many alkaloidal and fatty acid compounds. Moreover, several flavonoid and terpenoid compounds were also detected. Pharmacological activates of R. stricta extracts are approved to possess antimicrobial, antioxidant, anticancer, antidiabetic, and antihypertensive activities. Additionally, R. stricta extract was found to hold biological activates such as larvicidal and phytoremediation activates R. stricta extract was found to be toxic, genotoxic, and mutagenic. R. stricta contains novel phytochemical compounds that have not been investigated pharmacologically. Further research is needed through in vitro and in vivo experiments to pave the road for these compounds for medical, veterinary, and ecological uses.

ITS2: An Ideal DNA Barcode for the Arid Medicinal Plant Rhazya Stricta.

INTRODUCTION: In Saudi Arabia, Rhazya stricta is a widely used folkloric plant because of its various therapeutic properties. It is sold in herbal markets as a dried powder; however, the absence of visible phenotypic traits in the powder can mask its authenticity. Potential misidentification of this substance threatens consumer health. DNA barcoding could accurately identify this plant regardless of its physical state, however barcoding presents the challenge of variations in marker loci. OBJECTIVES: The objective of this work was to assess barcode markers from the chloroplast and nuclear regions to determine their taxonomic accuracy in R. stricta barcoding, and select the best marker for this species that could fulfill the authentication test for its fresh and dried samples. METHOD: In this study, we assessed seven barcode markers from the chloroplast (psbA-trnH, matK, rbcL, rpoB, and rpoC1) and nuclear regions (ITS1and ITS2). We compared DNA sequences of R. stricta from 50 fresh locally collected samples and 10 dried ground samples from the herbal market with the database sequences of R. stricta, R. orientalis, and eight other related species as controls. We utilized three methods (BLAST, nearest distance, and neighbor-joining tree) in this analysis. RESULT: With the exception of psbA-trnH, all the chloroplast markers determined high similarity with other taxa. However, nuclear ITS2 best distinguished between R. stricta, R. orientalis, and other related species because of its secondary structures, which allowed for more accurate distinctions. A two-locus marker of ITS1 + ITS2 sequences also showed promising results. A two-dimensional image of our proposed marker was generated to more easily handle DNA barcoding applications. CONCLUSION: Our study indicates that ITS2 is a cost-effective barcoding marker capable of verifying the authenticity of R. stricta and other medicinal plants in order to protect consumer health.

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr.

Bradyrhizobium elkanii USDA 76T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76T are described, together with its genome sequence information and annotation. The 9,484,767 bp high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76T to introduce various effector molecules into this host to enable nodulation.

Molecular and Morphological Investigations of the Stauros-bearing, Raphid Pennate Diatoms (Bacillariophyceae): Craspedostauros E.J. Cox, and Staurotropis T.B.B. Paddock, and their Relationship to the Rest of the Mastogloiales.

Several lineages of raphe-bearing diatoms possess a "stauros," which is a transverse, usually thickened area free of pores across the center of the valve. It has been suggested that this structure has evolved several times across the raphid diatoms, but we have noticed similarities beyond the stauros between two marine genera-Craspedostauros and Staurotropis-in the structure of their pore occlusions. We have isolated, cultured and extracted DNA from several strains of both genera to infer the phylogenetic relationship between these taxa, as well as test the suggested relationship of Craspedostauros to Achnanthes and Mastogloia based on plastid morphology. DNA sequence data (nuclear-encoded rRNA SSU, plastid-encoded rbcL and psbC) suggest that, except for Mastogloia, these genera are closely-related, though not sister taxa. The DNA phylogeny also suggests that the Mastogloiales are not monophyletic, with clades containing Achnanthes and Craspedostauros sister to clades containing taxa in the Bacillariales. Using evidence from molecular and morphological data, we describe the following new taxa: Craspedostauros alyoubii and C. paradoxa from the Red Sea and Guam, respectively; Staurotropis khiyamii and S. americana from the Red Sea and the Gulf of Mexico, respectively; and Dreuhlago cuneata n. gen., n. sp. from Guam.

High-quality draft genome sequence of Rhizobium mesoamericanum strain STM6155, a Mimosa pudica microsymbiont from New Caledonia.

Rhizobium mesoamericanum STM6155 (INSCD = ATYY01000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as an effective nitrogen fixing microsymbiont of the legume Mimosa pudica L.. STM6155 was isolated in 2009 from a nodule of the trap host M. pudica grown in nickel-rich soil collected near Mont Dore, New Caledonia. R. mesoamericanum STM6155 was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) genome sequencing project. Here we describe the symbiotic properties of R. mesoamericanum STM6155, together with its genome sequence information and annotation. The 6,927,906 bp high-quality draft genome is arranged into 147 scaffolds of 152 contigs containing 6855 protein-coding genes and 71 RNA-only encoding genes. Strain STM6155 forms an ANI clique (ID 2435) with the sequenced R. mesoamericanum strain STM3625, and the nodulation genes are highly conserved in these strains and the type strain of Rhizobium grahamii CCGE501T. Within the STM6155 genome, we have identified a chr chromate efflux gene cluster of six genes arranged into two putative operons and we postulate that this cluster is important for the survival of STM6155 in ultramafic soils containing high concentrations of chromate.

The nuclear genome of Rhazya stricta and the evolution of alkaloid diversity in a medically relevant clade of Apocynaceae.

Alkaloid accumulation in plants is activated in response to stress, is limited in distribution and specific alkaloid repertoires are variable across taxa. Rauvolfioideae (Apocynaceae, Gentianales) represents a major center of structural expansion in the monoterpenoid indole alkaloids (MIAs) yielding thousands of unique molecules including highly valuable chemotherapeutics. The paucity of genome-level data for Apocynaceae precludes a deeper understanding of MIA pathway evolution hindering the elucidation of remaining pathway enzymes and the improvement of MIA availability in planta or in vitro. We sequenced the nuclear genome of Rhazya stricta (Apocynaceae, Rauvolfioideae) and present this high quality assembly in comparison with that of coffee (Rubiaceae, Coffea canephora, Gentianales) and others to investigate the evolution of genome-scale features. The annotated Rhazya genome was used to develop the community resource, RhaCyc, a metabolic pathway database. Gene family trees were constructed to identify homologs of MIA pathway genes and to examine their evolutionary history. We found that, unlike Coffea, the Rhazya lineage has experienced many structural rearrangements. Gene tree analyses suggest recent, lineage-specific expansion and diversification among homologs encoding MIA pathway genes in Gentianales and provide candidate sequences with the potential to close gaps in characterized pathways and support prospecting for new MIA production avenues.

High-quality draft genome sequence of Flavobacterium suncheonense GH29-5(T) (DSM 17707(T)) isolated from greenhouse soil in South Korea, and emended description of Flavobacterium suncheonense GH29-5(T).

Flavobacterium suncheonense is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. Strain GH29-5(T) (DSM 17707(T)) was isolated from greenhouse soil in Suncheon, South Korea. F. suncheonense GH29-5(T) is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The 2,880,663 bp long draft genome consists of 54 scaffolds with 2739 protein-coding genes and 82 RNA genes. The genome of strain GH29-5(T) has 117 genes encoding peptidases but a small number of genes encoding carbohydrate active enzymes (51 CAZymes). Metallo and serine peptidases were found most frequently. Among CAZymes, eight glycoside hydrolase families, nine glycosyl transferase families, two carbohydrate binding module families and four carbohydrate esterase families were identified. Suprisingly, polysaccharides utilization loci (PULs) were not found in strain GH29-5(T). Based on the coherent physiological and genomic characteristics we suggest that F. suncheonense GH29-5(T) feeds rather on proteins than saccharides and lipids.

Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

Enzymatic Synthesis of Refined Olive Oil-Based Structured Lipid Containing Omega -3 and -6 Fatty Acids for Potential Application in Infant Formula.

Structured lipids (SLs) containing palmitic, docosahexaenoic (DHA), and gamma-linolenic (GLA) acids were produced using refined olive oil, tripalmitin, and ethyl esters of DHA single cell oil and GLA ethyl esters. Immobilized Lipozyme TL IM lipase was used as the biocatalyst. The SLs were characterized for fatty acid profile, triacylglycerol (TAG) molecular species, solid fat content, oxidative stability index, and melting and crystallization profiles and compared to physical blend of substrates, extracted fat from commercial infant formula (IFF), and milk fat. 49.28 mol% of palmitic acid was found at the sn-2 position of SL TAG and total DHA and GLA composition were 0.73 and 5.00 mol%, respectively. The total oleic acid content was 36.13 mol% which was very close to the 30.49% present in commercial IFF. Comparable solid fat content profiles were also found between SLs and IFF. The SLs produced have potential for use in infant formulas.