Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Nutritional evaluation and human health-promoting potential of compounds biosynthesized by native microalgae from the Peruvian Amazon.

A plausible strategy to mitigate socioeconomic problems in the Peruvian Amazon is through the sustainable exploitation of biodiversity resources, such as native microalgae. Several studies worldwide affirm that these microorganisms are excellent sources of higher value products for human nutrition and possess health-promoting biochemicals, but these attributes are unknown for the native microalgae of Peru. Therefore, the aim of this investigation was to evaluate the nutritional and human health-promoting potential of compounds biosynthesized by native microalgae from the Peruvian Amazon. Ten native microalgae strains of the groups cyanobacteria and chlorophyta were cultured in BG-11 medium and their biomass harvested and dried. Standardized methods were then used to determine proximate composition, fatty acids and amino acids composition, antioxidant activity, and total phenolic content. All ten microalgae strains produce primary nutrients, the entire spectrum of essential amino acids, essential fatty acids, and 3 of the 10 microalgae strains produced eisosapentaenoic acid. Additionally, all microalgae strains exhibited antioxidant activities and contained phenolic compounds. In conclusion, native microalgae strains from the Peruvian Amazon analyzed in this study possess the ability to biosynthesize and accumulate several nutrients and compounds with human health-promoting potential.

De novo assembly and functional annotation of Myrciaria dubia fruit transcriptome reveals multiple metabolic pathways for L-ascorbic acid biosynthesis.

BACKGROUND: Myrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis. RESULTS: In total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs. CONCLUSIONS: This study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with the empirically observed capability of M. dubia to synthesize and accumulate AsA and other important molecules, and adds to our current knowledge of the molecular biology and biochemistry of their production in plants. By providing insights into the mechanisms underpinning these metabolic processes, these results can be used to direct efforts to genetically manipulate this organism in order to enhance the production of these bioactive phytochemicals. The accumulation of AsA precursor and discovery of genes associated with their biosynthesis and metabolism in M. dubia is intriguing and worthy of further investigation. The sequences and pathways produced here present the genetic framework required for further studies. Quantitative transcriptomics in concert with studies of the genome, proteome, and metabolome under conditions that stimulate production and accumulation of AsA and their precursors are needed to provide a more comprehensive view of how these pathways for AsA metabolism are regulated and linked in this species.

Genetic and biochemical strategies for regulation of L-ascorbic acid biosynthesis in plants through the L-galactose pathway.

Vitamin C (L-ascorbic acid, AsA) is an essential compound with pleiotropic functions in many organisms. Since its isolation in the last century, AsA has attracted the attention of the scientific community, allowing the discovery of the L-galactose pathway, which is the main pathway for AsA biosynthesis in plants. Thus, the aim of this review is to analyze the genetic and biochemical strategies employed by plant cells for regulating AsA biosynthesis through the L-galactose pathway. In this pathway, participates eight enzymes encoded by the genes PMI, PMM, GMP, GME, GGP, GPP, GDH, and GLDH. All these genes and their encoded enzymes have been well characterized, demonstrating their participation in AsA biosynthesis. Also, have described some genetic and biochemical strategies that allow its regulation. The genetic strategy includes regulation at transcriptional and post-transcriptional levels. In the first one, it was demonstrated that the expression levels of the genes correlate directly with AsA content in the tissues/organs of the plants. Also, it was proved that these genes are light-induced because they have light-responsive promoter motifs (e.g., ATC, I-box, GT1 motif, etc.). In addition, were identified some transcription factors that function as activators (e.g., SlICE1, AtERF98, SlHZ24, etc.) or inactivators (e.g., SlL1L4, ABI4, SlNYYA10) regulate the transcription of these genes. In the second one, it was proved that some genes have alternative splicing events and could be a mechanism to control AsA biosynthesis. Also, it was demonstrated that a conserved cis-acting upstream open reading frame (5'-uORF) located in the 5'-untranslated region of the GGP gene induces its post-transcriptional repression. Among the biochemical strategies discovered is the control of the enzyme levels (usually by decreasing their quantities), control of the enzyme catalytic activity (by increasing or decreasing its activity), feedback inhibition of some enzymes (GME and GGP), subcellular compartmentation of AsA, the metabolon assembly of the enzymes, and control of AsA biosynthesis by electron flow. Together, the construction of this basic knowledge has been establishing the foundations for generating genetically improved varieties of fruits and vegetables enriched with AsA, commonly used in animal and human feed.

Soil microbial diversity and functional profiling of a tropical rainforest of a highly dissected low hill from the upper Itaya river basin revealed by analysis of shotgun metagenomics sequencing data.

The tropical rainforest of a highly dissected low hill from the upper Itaya river basin belongs to the western Amazonia region. Some investigations on the biodiversity of these rainforests were more focused on animals and plants diversity. The soils of this region are composed of moderately fertile sediments deposited recently from the initiation of the Andean orogenesis in the Miocene until now. However, scientific information about the soil microbial and functional diversity is still missing. This report presents shotgun metagenomics sequencing data from soils of this rainforest type. A composite loamy soil sample was collected from a primary forest, and metagenomic DNA was purified with standardized methods. Furthermore, libraries were prepared and paired-end sequenced on the Illumina NextSeq 550 platform. Raw Illumina paired-end reads have been uploaded and analysed in the Metagenomics RAST server (MG-RAST). The raw sequence data in fastq format is available at NCBI's Sequence Read Archive (SRA) with accession number SRX12846710.

Genomic analysis and biochemical profiling of an unaxenic strain of Synechococcus sp. isolated from the Peruvian Amazon Basin region.

Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic resources. In this context, the main objective of this research was to determine the genome features and the biochemical profile of Synechococcus sp. UCP002. The cyanobacterium was isolated from the Peruvian Amazon Basin region and cultured in BG-11 medium. Growth parameters, genome features, and the biochemical profile of the cyanobacterium were determined using standardized methods. Synechococcus sp. UCP002 had a specific growth rate of 0.086 ± 0.008 µ and a doubling time of 8.08 ± 0.78 h. The complete genome of Synechococcus sp. UCP002 had a size of ∼3.53 Mb with a high coverage (∼200x), and its quality parameters were acceptable (completeness = 99.29%, complete and single-copy genes = 97.5%, and contamination = 0.35%). Additionally, the cyanobacterium had six plasmids ranging from 24 to 200 kbp. The annotated genome revealed ∼3,422 genes, ∼ 3,374 protein-coding genes (with ∼41.31% hypothetical protein-coding genes), two CRISPR Cas systems, and 61 non-coding RNAs. Both the genome and plasmids had the genes for prokaryotic defense systems. Additionally, the genome had genes coding the transcription factors of the metalloregulator ArsR/SmtB family, involved in sensing heavy metal pollution. The biochemical profile showed primary nutrients, essential amino acids, some essential fatty acids, pigments (e.g., all-trans-ß-carotene, chlorophyll a, and phycocyanin), and phenolic compounds. In conclusion, Synechococcus sp. UCP002 shows biotechnological potential to produce human and animal nutrients and raw materials for biofuels and could be a new source of genes for synthetic biological applications.

The complete mitochondrial genome of the oleaginous microalgae Ankistrodesmus falcatus strain UCP001 from the Peruvian Amazon.

Ankistrodesmus falcatus strain UCP001 is a native oleaginous microalgae isolated from the Peruvian Amazon basin. In this study we sequenced, de novo assembled, and functionally annotated the complete mitochondrial genome of the native oleaginous microalgae Ankistrodesmus falcatus strain UCP001 (Accesion number MT701044). This mitogenome is a typical circular double stranded DNA molecule of 41,048 bp in total length with G + C content of 37.4%. The mitogenome contains 49 genes, including 18 protein coding genes, 5 ribosomal (rRNA) genes and 26 transfer RNA (tRNA) genes. A phylogenetic analysis of 18 microalgae species indicated that Ankistrodesmus falcatus strain UCP001 was closely related to Ourococcus multisporus and Raphidocelis subcapitata. The complete mitochondrial genome sequence of Ankistrodesmus falcatus strain UCP001 enriches genomic resources of oleaginous native microalgae from the Peruvian Amazon for further basic and applied research.

Dataset of de novo assembly and functional annotation of the transcriptome during germination and initial growth of seedlings of Myrciaria Dubia "camu-camu".

Myrciaria dubia "camu-camu" is a native shrub of the Amazon that is commonly found in areas that are flooded for three to four months during the annual hydrological cycle. This plant species is exceptional for its capacity to biosynthesize and accumulate important quantities of a variety of health-promoting phytochemicals, especially vitamin C [1], yet few genomic resources are available [2]. Here we provide the dataset of a de novo assembly and functional annotation of the transcriptome from a pool of samples obtained from seeds during the germination process and seedlings during the initial growth (until one month after germination). Total RNA/mRNA was purified from different types of plant materials (i.e., imbibited seeds, germinated seeds, and seedlings of one, two, three, and four weeks old), pooled in equimolar ratio to generate the cDNA library and RNA paired-end sequencing was conducted on an Illumina HiSeq™2500 platform. The transcriptome was de novo assembled using Trinity v2.9.1 and SuperTranscripts v2.9.1. A total of 21,161 transcripts were assembled ranging in size from 500 to 10,001 bp with a N50 value of 1,485 bp. Completeness of the assembly dataset was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) software v2/v3. Finally, the assembled transcripts were functionally annotated using TransDecoder v3.0.1 and the web-based platforms Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS), and FunctionAnnotator. The raw reads were deposited into NCBI and are accessible via BioProject accession number PRJNA615000 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615000) and Sequence Read Archive (SRA) with accession number SRX7990430 (https://www.ncbi.nlm.nih.gov/sra/SRX7990430). Additionally, transcriptome shotgun assembly sequences and functional annotations are available via Discover Mendeley Data (https://data.mendeley.com/datasets/2csj3h29fr/1).

Dataset of de novo assembly and functional annotation of the transcriptomes of three native oleaginous microalgae from the Peruvian Amazon.

Microalgae are photosynthetic organisms with cosmopolitan distribution (i.e., marine, freshwater and terrestrial habitats) and possess a great diversity of species [1] and consequently an immense variation in biochemical compositions [2]. To date genomic information is available mainly from the model green microalga Chlamydomonas reinhardtii[3]. Here we provide the dataset of a de novo assembly and functional annotation of the transcriptomes of three native oleaginous microalgae from the Peruvian Amazon. Native oleaginous microalgae species Ankistrodesmus sp., Chlorella sp., and Scenedesmus sp. were cultured in triplicate using Chu-10 medium with or without a source of nitrate (NaNO3). Total RNA was purified, the cDNA libraries were constructed and sequenced as paired-end reads on an Illumina HiSeq™2500 platform. Transcriptomes were de novo assembled using Trinity v2.9.1. A total of 48,554 transcripts (range from 250 to 7966 bp; N50 = 1047) for Ankistrodesmus sp., 108,126 transcripts (range from 250 to 8160 bp; N50 = 1090) for Chlorella sp., and 77,689 transcripts (range from 250 to 8481 bp; N50 = 1281) for Scenedesmus sp. were de novo assembled. Completeness of the assembled transcriptomes were evaluated with the Benchmarking Universal Single-Copy Orthologs (BUSCO) software v2/v3. Functional annotation of the assembled transcriptomes was conducted with TransDecoder v3.0.1 and the web-based platforms Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS) and FunctionAnnotator. The raw reads were deposited into NCBI and are accessible via BioProject accession number PRJNA628966 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA628966) and Sequence Read Archive (SRA) with accession numbers: SRX8295665 (https://www.ncbi.nlm.nih.gov/sra/SRX8295665), SRX8295666 (https://www.ncbi.nlm.nih.gov/sra/SRX8295666), SRX8295667 (https://www.ncbi.nlm.nih.gov/sra/SRX8295667), SRX8295668 (https://www.ncbi.nlm.nih.gov/sra/SRX8295668), SRX8295669 (https://www.ncbi.nlm.nih.gov/sra/SRX8295669), and SRX8295670 (https://www.ncbi.nlm.nih.gov/sra/SRX8295670). Additionally, transcriptome shotgun assembly sequences and functional annotations are available via Discover Mendeley Data (https://data.mendeley.com/datasets/47wdjmw9xr/1).

Metagenomic 16S rDNA amplicon data on bacterial diversity profiling and its predicted metabolic functions of varillales in Allpahuayo-Mishana National Reserve.

The white-sands forests or varillales of the Peruvian Amazon are characterized by their distinct physical characteristics, patchy distribution, and endemism [1, 2]. Much research has been conducted on the specialized plant and animal communities that inhabit these ecosystems, yet their soil microbiomes have yet to be studied. Here we provide metagenomic 16S rDNA amplicon data of soil microbiomes from three types of varillales in Allpahuayo-Mishana National Reserve near Iquitos, Peru. Composite soil samples were collected from very low varillal, high-dry varillal, and high-wet varillal. Purified metagenomic DNA was used to prepare and sequence 16S rDNA metagenomic libraries on the Illumina MiqSeq platform. Raw paired-endsequences were analyzed using the Metagenomics RAST server (MG-RAST) and Parallel-Meta3 software and revealed the existence of a high percentage of undiscovered sequences, potentially indicating specialized bacterial communities in these forests. Also, were predicted several metabolic functions in this dataset. The raw sequence data in fastq format is available in the public repository Discover Mendeley Data (https://data.mendeley.com/datasets/syktzxcnp6/2). Also, is available at NCBI's Sequence Read Archive (SRA) with accession numbers SRX7891206 (very low varillal), SRX7891207 (high-dry varillal), and SRX7891208 (high-wet varillal).

Structural characterization of L-Galactose Dehydrogenase: an essential enzyme for vitamin C biosynthesis

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

In Silico Characterization and Expression Analysis Of The Alpha Subunit Of The Heteromeric Acetyl-Coenzyme A Carboxylase From Two Microalgae / Caracterización in silico y análisis de la expresión de la subunidad alfa de la acetil-coenzima a carboxilasa heteromérica de dos microalgas

RESUMEN Las microalgas son microorganismos fotosintéticos con gran potencial para abastecer las demandas energéticas mundiales. Sin embargo, los limitados conocimientos que se tienen de estos organismos, en particular a nivel molecular de los procesos metabólicos, han limitado su uso con estos propósitos. En esta investigación se ha realizado el análisis in silico de la subunidad alfa de la acetil-Coenzima A carboxilasa heteromérica (αACCasa), una enzima clave en la biosíntesis de lípidos de las microalgas Chlorella sp. y Scenedesmus sp. Asimismo, se ha medido la expresión de este gen en ambas especies cultivadas en medios deficientes de nitrógeno. Los resultados indican que la αACCasa muestra conservación estructural y funcional en ambas especies de microalgas y su mayor similitud genética con otras especies de microalgas. Asimismo, se ha mostrado que el nivel de expresión del gen se incrementa significativamente cuando las microalgas son cultivadas en ausencia de nitrógeno, lo cual se relaciona a su vez con una mayor acumulación de lípidos microalgales. En conclusión, el análisis in silico de la αACCasa de Chlorella sp. y Scenedesmus sp. presentan características estructurales, funcionales y evolutivas muy similares con otras especies de microalgas y plantas. Asimismo, el estudio revela que en ambas especies el gen se sobreexpresa cuando las microalgas son sometidas a estrés por deficiencia de nitrógeno, el cual se relaciona significativamente con la acumulación de lípidos totales en estas células.

ABSTRACT Microalgae are photosynthetic microorganisms with great potential to supply the world's energy demands. However, the limited knowledge of these organisms, particularly at the molecular level of metabolic processes, has limited their use to these purposes. In this investigation, the in silico analysis of the alpha subunit of the heteromeric acetyl-coenzyme A carboxylase (αACCase), a key enzyme in lipid biosynthesis of microalgae Chlorella sp. and Scenedesmus sp. was carried out. Also, the expression of this gene has been measured in both species cultivated in nitrogen-depleted media. Results indicate that αACCase shows structural and functional conservation in both species of microalgae and their greater genetic similarity with other species of microalgae. Also, it has been shown that the expression levels of this gene are significantly increased when the microalgae are cultured in the absence of nitrogen, which in turn is related to a greater accumulation of microalgal lipids. In conclusion, the in silico analysis of the Chlorella sp. and Scenedesmus sp. αACCase reveals structural, functional and evolutionary characteristics very similar to other microalgae and plant species. Also, the study reveals that in both species the gene is overexpressed when microalgae are subjected to nitrogen deficiency stress, which is significantly related to total lipids accumulation in these cells.

Plants used by native Amazonian groups from the Nanay River (Peru) for the treatment of malaria.

AIM OF THE STUDY: In order to evaluate the antimalarial potential of traditional remedies used in Peru, Indigenous and Mestizo populations from the river Nanay in Loreto were interviewed about traditional medication for the treatment of malaria. MATERIALS AND METHODS: The survey took place on six villages and led to the collection of 59 plants. 35 hydro-alcoholic extractions were performed on the 21 most cited plants. The extracts were then tested for antiplasmodial activity in vitro on Plasmodium falciparum chloroquine resistant strain (FCR-3), and ferriprotoporphyrin inhibition test was also performed in order to assume pharmacological properties. RESULTS: Extracts from 9 plants on twenty-one tested (Abuta rufescens, Ayapana lanceolata, Capsiandra angustifolia, Citrus limon, Citrus paradise, Minquartia guianensis, Potalia resinífera, Scoparia dulcis, and Physalis angulata) displayed an interesting antiplasmodial activity (IC(50)<10 µg/ml) and 16 remedies were active on the ferriprotoporphyrin inhibition test. CONCLUSIONS: The results give scientific validation to the traditional medical knowledge of the Amerindian and Mestizo populations from Loreto and exhibit a source of potentially active plants.