Mining raw plant transcriptomic data for new cyclopeptide alkaloids.

In recent years, genome and transcriptome mining have dramatically expanded the rate of discovering diverse natural products from bacteria and fungi. In plants, this approach is often more limited due to the lack of available annotated genomes and transcriptomes combined with a less consistent clustering of biosynthetic genes. The recently identified burpitide class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products offer a valuable opportunity for bioinformatics-guided discovery in plants due to their short biosynthetic pathways and gene encoded substrates. Using a high-throughput approach to assemble and analyze 700 publicly available raw transcriptomic data sets, we uncover the potential distribution of split burpitide precursor peptides in Streptophyta. Metabolomic analysis of target plants confirms our bioinformatic predictions of new cyclopeptide alkaloids from both known and new sources.

Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants.

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta. To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta. We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana, we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

Transcriptome Sequencing of the Diatom Asterionellopsis thurstonii and In Silico Identification of Enzymes Potentially Involved in the Synthesis of Bioactive Molecules.

Microalgae produce a plethora of primary and secondary metabolites with possible applications in several market sectors, including cosmetics, human nutrition, aquaculture, biodiesel production and treatment/prevention of human diseases. Diatoms, in particular, are the most diversified microalgal group, many species of which are known to have anti-cancer, anti-oxidant, anti-diabetes, anti-inflammatory and immunomodulatory properties. Compounds responsible for these activities are often still unknown. The aim of this study was to de novo sequence the full transcriptome of two strains of the diatom Asterionellopsis thurstonii, sampled from two different locations and cultured in both control and phosphate starvation conditions. We used an RNA-sequencing approach to in silico identify transcripts potentially involved in the synthesis/degradation of compounds with anti-cancer and immunomodulatory properties. We identified transcript coding for L-asparaginase I, polyketide cyclase/dehydrase, bifunctional polyketide phosphatase/kinase, 1-deoxy-D-xylulose-5-phosphate synthase (fragment), inositol polyphosphate 5-phosphatase INPP5B/F, catechol O-Methyltransferase, digalactosyldiacylglycerol synthase (DGD1), 1,2-diacylglycerol-3-beta-galactosyltransferase and glycerolphosphodiester phosphodiesterase. Differential expression analysis also allowed to identify in which culturing condition these enzymes are more expressed. Overall, these data give new insights on the annotation of diatom genes, enzymatic pathways involved in the generation of bioactive molecules and possible exploitation of Asterionellopsis thurstonii.

Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a Painless homolog.

The transient receptor potential (TRP) family of cation channels are evolutionarily conserved proteins with critical roles in sensory physiology. Despite extensive studies in model species, knowledge of TRP channel functional diversity and physiological impact remains limited in many non-model insect species. To assess the TRP channel repertoire in a non-model agriculture pest species (Lygus hesperus), publicly available transcriptomic datasets were mined for potential homologs. Among the transcripts identified, 30 are predicted to encompass complete open reading frames that encode proteins representing each of the seven TRP channel subfamilies. Although no homologs were identified for the Pyrexia and Brivido channels, the TRP complement in L. hesperus exceeded the 13-16 channels reported in most insects. This diversity appears to be driven by a combination of alternative splicing, which impacted members of six subfamilies, and gene expansion of the TRPP subfamily. To validate the in silico data and provide more detailed analyses of L. hesperus TRP functionality, the putative Painless homolog was selected for more in depth analysis and its functional role in thermosensation examined in vitro. RT-PCR expression profiling revealed near ubiquitous expression of the Painless transcript throughout nymphal and adult development. Electrophysiological data generated using a Xenopus oocyte recombinant expression system indicated activation parameters for L. hesperus Painless homolog that are consistent with a role in noxious heat (40°-45 °C) thermosensation.

Whole-body transcriptome mining for candidate effectors from Diuraphis noxia.

BACKGROUND: Proteins within aphid saliva play a crucial role as the molecular interface between aphids and their host plants. These salivary effectors modulate plant responses to favour aphid feeding and facilitate infestation. The identification of effectors from economically important pest species is central in understanding the molecular events during the aphid-plant interaction. The Russian wheat aphid (Diuraphis noxia, Kurdjumov) is one such pest that causes devastating losses to wheat and barley yields worldwide. Despite the severe threat to food security posed by D. noxia, the non-model nature of this pest and its host has hindered progress towards understanding this interaction. In this study, in the absence of a salivary gland transcriptome, whole-body transcriptomics data was mined to generate a candidate effector catalogue for D. noxia. RESULTS: Mining the transcriptome identified 725 transcripts encoding putatively secreted proteins amongst which were transcripts specific to D. noxia. Six of the seven examined D. noxia putative effectors, termed DnE's (Diuraphis noxia effectors) exhibited salivary gland-specific expression. A comparative analysis between whole-body D. noxia transcriptome data versus the head and body transcriptomes from three other aphid species allowed us to define a catalogue of transcripts putatively upregulated in D. noxia head tissue. Five of these were selected for RT-qPCR confirmation, and were found to corroborate the differential expression predictions, with a further three confirmed to be highly expressed in D. noxia salivary gland tissue. CONCLUSIONS: Determining a putative effector catalogue for D. noxia from whole-transcriptome data, particularly the identification of salivary-specific sequences potentially unique to D. noxia, provide the basis for future functional characterisation studies to gain further insight into this aphid-plant interaction. Furthermore, due to a lack of publicly available aphid salivary gland transcriptome data, the capacity to use comparative transcriptomics to compile a list of putative effector candidates from whole-body transcriptomics data will further the study of effectors in various aphid species.

Transcriptome Mining to Identify Genes of Interest: From Local Databases to Phylogenetic Inference.

The advancement in next-generation sequencing technologies and the dropping of sequencing costs have seen an increase in the amount of transcriptome data generated each year. These data are of big potential for identifying genes and molecular pathways of interest across a plethora of organisms. However, navigating these resources requires some bioinformatics and evolutionary skills. Here, we describe a protocol of transcriptome data mining for genes of interest, from the creation of a protein database to the inference of phylogenetic trees, which was used for marine protists, but can be used as general pipeline across different taxa.

Discovery of a Novel Species of Trichomonasvirus in the Human Parasite Trichomonas vaginalis Using Transcriptome Mining.

Trichomonas vaginalis is the most common non-viral cause of sexually transmitted infections globally. Infection by this protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory disease with acute and chronic consequences. Half or more isolates of this parasite are themselves infected with one or more dsRNA viruses that can exacerbate the inflammatory syndrome. At least four distinct viruses have been identified in T. vaginalis to date, constituting species Trichomonas vaginalis virus 1 through Trichomonas vaginalis virus 4 in genus Trichomonasvirus. Despite the global prevalence of these viruses, few complete coding sequences have been reported. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-Seq accessions representing at least 13 distinct T. vaginalis isolates. The results led to sequence assemblies for 27 novel trichomonasvirus strains across all four recognized species. Using a strategy of de novo sequence assembly followed by taxonomic classification, we additionally discovered six strains of a newly identified fifth species, for which we propose the name Trichomonas vaginalis virus 5, also in genus Trichomonasvirus. These additional strains exhibit high sequence identity to each other, but low sequence identity to strains of the other four species. Phylogenetic analyses corroborate the species-level designations. These results substantially increase the number of trichomonasvirus genome sequences and demonstrate the utility of mining publicly available transcriptomes for virus discovery in a critical human pathogen.

First Report of OvoA Gene in Marine Arthropods: A New Candidate Stress Biomarker in Copepods.

Ovothiol is one of the most powerful antioxidants acting in marine organisms as a defense against oxidative stress during development and in response to environmental cues. The gene involved in the ovothiol biosynthesis, OvoA, is found in almost all metazoans, but open questions existed on its presence among arthropods. Here, using an in silico workflow, we report a single OvoA gene in marine arthropods including copepods, decapods, and amphipods. Phylogenetic analyses indicated that OvoA from marine arthropods separated from the other marine phyla (e.g., Porifera, Mollusca) and divided into two separate branches, suggesting a possible divergence through evolution. In the copepod Calanus finmarchicus, we suggest that OvoA has a defense role in oxidative stress as shown by its high expression in response to a toxic diet and during the copepodite stage, a developmental stage that includes significant morphological changes. Overall, the results of our study open possibilities for the use of OvoA as a biomarker of stress in copepods and possibly also for other marine holozooplankters. The finding of OvoA in copepods is also promising for the drug discovery field, suggesting the possibility of using copepods as a new source of bioactive compounds to be tested in the marine biotechnological sector.

Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus.

Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5' terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.

A novel positive single-stranded RNA virus from the crustacean parasite, Probopyrinella latreuticola (Peracarida: Isopoda: Bopyridae).

A positive, single-stranded RNA virus is identified from the transcriptome of Probopyrinella latreuticola Gissler, 1882; a bopyrid isopod parasite of the Sargassum shrimp, Latreutes fucorum Fabricius, 1789. The viral sequence is 13,098 bp in length (including polyA), encoding four open reading frames (ORF). ORF-1 encodes a polyprotein, with three computationally discernible functional domains: viral methyltransferase; viral helicase; and RNA-directed RNA polymerase. The remaining ORFs encode a transmembrane protein, a capsid protein and a protein of undetermined function. The raw transcriptomic data reveal a low level of background single nucleotide mutations within the data. Comparison of the protein sequence data and synteny with other viral isolates reveals that the greatest protein similarity (<39%) is shared with the Negevirus group, a group that exclusively infects insects. Phylogenetic assessment of the individual polyprotein domains revealed a mixed prediction of phylogenetic origins, suggesting with low confidence that the novel +ssRNA virus could be present in multiple places throughout the individual gene trees. A concatenated approach strongly suggested that this new virus is an early diverging isolate, branching before the Negevirus and Cilevirus groups. Alongside the new isolate are other marine viruses, also present toward the base of the tree. The isopod virosphere, with the addition of this novel virus, is discussed relative to viral genomics/systematics. A great diversity of nege-like viruses appears to be present in marine invertebrate hosts, which require greater efforts for discovery and identification.

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans.

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.

Assessment and comparison of putative amine receptor complement/diversity in the brain and eyestalk ganglia of the lobster, Homarus americanus.

In decapods, dopamine, octopamine, serotonin, and histamine function as locally released/hormonally delivered modulators of physiology/behavior. Although the functional roles played by amines in decapods have been examined extensively, little is known about the identity/diversity of their amine receptors. Recently, a Homarus americanus mixed nervous system transcriptome was used to identify putative neuronal amine receptors in this species. While many receptors were identified, some were fragmentary, and no evidence of splice/other variants was found. Here, the previously predicted proteins were used to search brain- and eyestalk ganglia-specific transcriptomes to assess/compare amine receptor complements in these portions of the lobster nervous system. All previously identified receptors were reidentified from the brain and/or eyestalk ganglia transcriptomes, i.e., dopamine alpha-1, beta-1, and alpha-2 (Homam-DAα2R) receptors, octopamine alpha (Homam-OctαR), beta-1, beta-2, beta-3, beta-4, and octopamine-tyramine (Homam-OTR-I) receptors, serotonin type-1A, type-1B (Homam-5HTR1B), type-2B, and type-7 receptors; and histamine type-1 (Homam-HA1R), type-2, type-3, and type-4 receptors. For many previously partial proteins, full-length receptors were deduced from brain and/or eyestalk ganglia transcripts, i.e., Homam-DAα2R, Homam-OctαR, Homam-OTR-I, and Homam-5HTR1B. In addition, novel dopamine/ecdysteroid, octopamine alpha-2, and OTR receptors were discovered, the latter, Homam-OTR-II, being a putative paralog of Homam-OTR-I. Finally, evidence for splice/other variants was found for many receptors, including evidence for some being assembly-specific, e.g., a brain-specific Homam-OTR-I variant and an eyestalk ganglia-specific Homam-HA1R variant. To increase confidence in the transcriptome-derived sequences, a subset of receptors was cloned using RT-PCR. These data complement/augment those reported previously, providing a more complete picture of amine receptor complement/diversity in the lobster nervous system.

Identification of putative neuropeptidergic signaling systems in the spiny lobster, Panulirus argus.

Members of the decapod infraorder Achelata, specifically species from the genus Panulirus, have storied histories as models for investigating the basic principles governing the generation, maintenance, and modulation of rhythmic motor behavior, including modulation by locally released and circulating peptides. Despite their contributions to our understanding of peptidergic neuromodulation, little is known about the identity of the native neuropeptides and neuronal peptide receptors present in these crustaceans. Here, a Panulirus argus nervous system-specific transcriptome was used to help fill this void, providing insight into the neuropeptidome and neuronal peptide receptome of this species. A neuropeptidome consisting of 266 distinct peptides was predicted using the P. argus assembly, 128 having structures placing them into a generally recognized arthropod peptide family: agatoxin-like peptide, allatostatin A (AST-A), allatostatin B, allatostatin C, bursicon, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31 (DH31), ecdysis-triggering hormone (ETH), FMRFamide-like peptide (FLP), glycoprotein hormone (GPH), GSEFLamide, inotocin, leucokinin, myosuppressin, natalisin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, periviscerokinin, pigment-dispersing hormone, pyrokinin, red pigment-concentrating hormone, RYamide, short neuropeptide F (sNPF), SIFamide, sulfakinin, tachykinin-related peptide (TRP), and trissin. Twenty-five putative neuronal receptors, encompassing 15 peptide groups, were also identified from the P. argus transcriptome: AST-A, bursicon, CCHamide, DH31, diuretic hormone 44, ETH, FLP, GPH, inotocin, insulin-like peptide, myosuppressin, natalisin, periviscerokinin, sNPF, and TRP. Collectively, the reported data provide a powerful resource for expanding studies of neuropeptidergic control of physiology and behavior in members of the genus Panulirus specifically, and decapods generally.

Identification of putative amine receptor complement in the eyestalk of the crayfish, Procambarus clarkii.

In decapod crustaceans, the amines dopamine, octopamine, serotonin, and histamine are known to serve as locally released and/or circulating neuromodulators. While many studies have focused on determining the modulatory actions of amines on decapod nervous systems, comparatively little is known about the identity of the receptors through which they exert their actions. Here, a crayfish, Procambarus clarkii, tissue-specific transcriptome was used to identify putative amine receptors in the eyestalk, a structure composed largely of the eyestalk ganglia, including the neuroendocrine X-organ-sinus gland system, and retina. Transcripts encoding 17 distinct putative amine receptors, three dopamine (one dopamine 1-like, one dopamine 2-like, and one dopamine/ecdysteroid-like), five octopamine (one alpha-like, three beta-like, and one octopamine/tyramine-like), three serotonin (two type-1-like and one type-7-like), and six histamine (five histamine-gated chloride channel A-like and one histamine-gated chloride channel B-like) were identified in the assembly. Comparison of the nucleotide sequence of the transcript encoding one predicted type-1-like serotonin receptor with that cloned previously from the P. clarkii nervous system shows the two sequences to be essentially identical, providing increased support for the validity of the transcripts used to deduce the proteins reported here. Reciprocal BLAST and structural/functional domain analyses support the protein family annotations ascribed to the putative P. clarkii receptors. These data represent the first large-scale description of amine receptors from P. clarkii, and as such provide a new resource for initiating gene-based studies of aminergic control of physiology/behavior at the level of receptors in this species.

What can transcriptomics reveal about the phylogenetic/structural conservation, tissue localization, and possible functions of CNMamide peptides in decapod crustaceans?

Over the past several years, in silico analyses of arthropod genomes/transcriptomes have led to the identification of several previously unknown peptide families. The CNMamides are one such peptide group, having been discovered via computational analyses of the fruit fly, Drosophila melanogaster, genome; both a CNMamide precursor and receptor were identified. Recently, a CNMamide family member, VMCHFKICNLamide (disulfide bridging between the cysteine residues), was predicted via in silico mining of a crayfish, Procambarus clarkii, transcriptome, suggesting the presence of this peptide group in members of the Decapoda. Here, using publically accessible transcriptomic data, the phylogenetic/structural conservation, tissue localization, and possible functions of the CNMamide family in decapods were explored. Evidence for CNMamide precursors was found for members of each decapod infraorder for which significant sequence data are available, suggesting a ubiquitous conservation of the CNMamide family in the Decapoda. For the Penaeoidea, Caridea, Astacidea and Achelata, the isoform of CNMamide originally identified from P. clarkii appears to be ubiquitously conserved; in members of the Brachyura, VMCHFKICNMamide (disulfide bridging between the cysteine residues) is the native isoform. Interestingly, the decapod CNMamide gene appears to also have a splice variant in which the carboxy-terminal portion of the preprohormone containing the CNMamide peptide is replaced by one containing a different disulfide bridged peptide that is structurally unrelated to it; this second peptide shows considerable conservation within, but variation among, decapod infraorders. A highly conserved putative CNMamide receptor was identified from members of the Penaeoidea, Astacidea and Brachyura. Phylogenetic analyses support the annotation of the decapod receptor as a true member of the CNMamide receptor family. The presence of precursor and receptor transcripts in both nervous system- and reproductive tissue-specific transcriptomes suggests CNMamides serve as modulators of decapod neural and reproductive control systems.

Identification of putative amine biosynthetic enzymes in the nervous system of the crab, Cancer borealis.

Amines function as neuromodulators throughout the animal kingdom. In decapod crustaceans, the amines serving neuromodulatory roles include dopamine, octopamine, serotonin and histamine. While much work has focused on examining the physiological effects of amines on decapod nervous systems, the identity of the native enzymes involved in their biosynthesis remains largely unknown. In an attempt to help fill this void, a transcriptome generated from multiple portions of the crab, Cancer borealis, nervous system, a species that has long served as a model species for investigating the neuromodulatory control of rhythmically active neural networks, was used to identify putative amine biosynthetic enzyme-encoding transcripts, and by proxy, proteins. Transcripts encoding full complements of the enzymes involved in the production of dopamine, octopamine, serotonin, and histamine were deduced from the C. borealis assembly, i.e., tryptophan-phenylalanine hydroxylase, tyrosine hydroxylase, DOPA decarboxylase, tyrosine decarboxylase, tyramine ß-hydroxylase, tryptophan hydroxylase, and histidine decarboxylase. All proteins deduced from the C. borealis transcripts appear to be full-length sequences, with reciprocal BLAST and structural domain analyses supporting the protein family annotations ascribed to them. These data provide the first descriptions of the native amine biosynthetic enzymes of C. borealis, and as such, serve as a resource for initiating gene-based studies of aminergic control of physiology and behavior at the level of biosynthesis in this important biomedical model.

Molecular characterization of putative neuropeptide, amine, diffusible gas and small molecule transmitter biosynthetic enzymes in the eyestalk ganglia of the American lobster, Homarus americanus.

The American lobster, Homarus americanus, is a model for investigating the neuromodulatory control of physiology and behavior. Prior studies have shown that multiple classes of chemicals serve as locally released/circulating neuromodulators/neurotransmitters in this species. Interestingly, while many neuroactive compounds are known from Homarus, little work has focused on identifying/characterizing the enzymes responsible for their biosynthesis, despite the fact that these enzymes are key components for regulating neuromodulation/neurotransmission. Here, an eyestalk ganglia-specific transcriptome was mined for transcripts encoding enzymes involved in neuropeptide, amine, diffusible gas and small molecule transmitter biosynthesis. Using known Drosophila melanogaster proteins as templates, transcripts encoding putative Homarus homologs of peptide precursor processing (signal peptide peptidase, prohormone processing protease and carboxypeptidase) and immature peptide modifying (glutaminyl cyclase, tyrosylprotein sulfotransferase, protein disulfide isomerase, peptidylglycine-α-hydroxylating monooxygenase and peptidyl-α-hydroxyglycine-α-amidating lyase) enzymes were identified in the eyestalk assembly. Similarly, transcripts encoding full complements of the enzymes responsible for dopamine [tryptophan-phenylalanine hydroxylase (TPH), tyrosine hydroxylase and DOPA decarboxylase (DDC)], octopamine (TPH, tyrosine decarboxylase and tyramine ß-hydroxylase), serotonin (TPH or tryptophan hydroxylase and DDC) and histamine (histidine decarboxylase) biosynthesis were identified from the eyestalk ganglia, as were those responsible for the generation of the gases nitric oxide (nitric oxide synthase) and carbon monoxide (heme oxygenase), and the small molecule transmitters acetylcholine (choline acetyltransferase), glutamate (glutaminase) and GABA (glutamic acid decarboxylase). The presence and identity of the transcriptome-derived transcripts were confirmed using RT-PCR. The data presented here provide a foundation for future gene-based studies of neuromodulatory control at the level of neurotransmitter/modulator biosynthesis in Homarus.

Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.