Transcriptomic Analysis of the Response of the Toxic Dinoflagellate Prorocentrum lima to Phosphorous Limitation.

Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Phosphorus (P) is a limiting macronutrient for dinoflagellate growth in the ocean. Previous studies have been focused on the physiological response of dinoflagellates to ambient P changes. However, the whole-genome's molecular mechanisms are poorly understood. In this study, RNA-Seq was utilized to compare the global gene expression patterns of a marine diarrheic shellfish poisoning (DSP) toxin-producing dinoflagellate, Prorocentrum lima, grown in inorganic P-replete and P-deficient conditions. A total of 148 unigenes were significantly up-regulated, and 30 unigenes were down-regulated under 1/4 P-limited conditions, while 2708 unigenes were significantly up-regulated, and 284 unigenes were down-regulated under 1/16 P-limited conditions. KEGG enrichment analysis of the differentially expressed genes shows that genes related to ribosomal proteins, glycolysis, fatty acid biosynthesis, phagosome formation, and ubiquitin-mediated proteolysis are found to be up-regulated, while most of the genes related to photosynthesis are down-regulated. Further analysis shows that genes encoding P transporters, organic P utilization, and endocytosis are significantly up-regulated in the P-limited cells, indicating a strong ability of P. lima to utilize dissolved inorganic P as well as intracellular organic P. These transcriptomic data are further corroborated by biochemical and physiological analyses, which reveals that under P deficiency, cellular contents of starch, lipid, and toxin increase, while photosynthetic efficiency declines. Our results indicate that has P. lima evolved diverse strategies to acclimatize to low P environments. The accumulation of carbon sources and DSP toxins could provide protection for P. lima to cope with adverse environmental conditions.

Identification of a (+)-cubenene synthase from filamentous fungi Acremonium chrysogenum.

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.

Systematic Mining and Evaluation of the Sesquiterpene Skeletons as High Energy Aviation Fuel Molecules.

Sesquiterpenes have been identified as promising ingredients for aviation fuels due to their high energy density and combustion heat properties. Despite the characterization of numerous sesquiterpene structures, studies testing their performance properties and feasibility as fuels are scarce. In this study, 122 sesquiterpenoid skeleton compounds, obtained from existing literature reports, are tested using group contribution and gaussian quantum chemistry methods to assess their potential as high-energy aviation fuels. Seventeen sesquiterpene compounds exhibit good predictive performance and nine compounds are further selected for overproduction in yeast. Through fed-batch fermentation, all compounds achieve the highest reported titers to date. Subsequently, three representative products, pentalenene, presilphiperfol-1-ene, and α-farnesene, are selected, produced, purified in large quantities, and tested for use as potential fuels. The performance of pentalenene, presilphiperfol-1-ene, and their derivatives reveals favorable prospects as high-energy aviation fuels.

A novel, genetically encoded whole-cell biosensor for directed evolution of myrcene synthase in Escherichia coli.

ß-myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite.

Transcriptomic analysis of polyketide synthesis in dinoflagellate, Prorocentrum lima.

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

Design and synthesis of a clickable, photoreactive amino acid p-(4-(but-3-yn-1-yl)benzoyl)-l-phenylalanine for peptide photoaffinity labeling.

Photoaffinity labeling is a powerful technique to investigate the interactions between bioactive peptides and their targets. To construct a peptide-derived photoaffinity probe, at least two amino acids need to be modified or replaced, increasing experimental difficulties and negatively affecting activity. Herein, we report the synthesis of a clickable, photoreactive amino acid p-(4-(but-3-yn-1-yl)benzoyl)-l-phenylalanine (Abpa) and its Fmoc-protected version from 3-(4-bromophenyl)-1-propanol in 11 steps with an overall 12.5% yield. The amino acid contains both a photoreactive benzophenone and a clickable terminal alkyne which acts like a reporter tag by fast attachment to other functional groups via 'click' reaction, and a photoaffinity probe could be created by one single amino acid substitution during peptide synthesis. And its small size helps to retain bioactivity. The efficiency of Abpa was demonstrated by photoaffinity labeling experiments using photoactivatable probes of α-conotoxin MI.

Identification of the sesquiterpene synthase AcTPS1 and high production of (-)-germacrene D in metabolically engineered Saccharomyces cerevisiae.

BACKGROUND: The sesquiterpene germacrene D is a highly promising product due to its wide variety of insecticidal activities and ability to serve as a precursor for many other sesquiterpenes. Biosynthesis of high value compounds through genome mining for synthases and metabolic engineering of microbial factories, especially Saccharomyces cerevisiae, has been proven to be an effective strategy. However, there have been no studies on the de novo synthesis of germacrene D from carbon sources in microbes. Hence, the construction of the S. cerevisiae cell factory to achieve high production of germacrene D is highly desirable. RESULTS: We identified five putative sesquiterpene synthases (AcTPS1 to AcTPS5) from Acremonium chrysogenum and the major product of AcTPS1 characterized by in vivo, in vitro reaction and NMR detection was revealed to be (-)-germacrene D. After systematically comparing twenty-one germacrene D synthases, AcTPS1 was found to generate the highest amount of (-)-germacrene D and was integrated into the terpene precursor-enhancing yeast strain, achieving 376.2 mg/L of (-)-germacrene D. Iterative engineering was performed to improve the production of (-)-germacrene D, including increasing the copy numbers of AcTPS1, tHMG1 and ERG20, and downregulating or knocking out other inhibitory factors (such as erg9, rox1, dpp1). Finally, the optimal strain LSc81 achieved 1.94 g/L (-)-germacrene D in shake-flask fermentation and 7.9 g/L (-)-germacrene D in a 5-L bioreactor, which is the highest reported (-)-germacrene D titer achieved to date. CONCLUSION: We successfully achieved high production of (-)-germacrene D in S. cerevisiae through terpene synthase mining and metabolic engineering, providing an impressive example of microbial overproduction of high-value compounds.

A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Structural and Functional Characterization of Conotoxins from Conus achatinus Targeting NMDAR.

Conotoxin-Ac1 and its variant conotoxin-Ac1-O6P, were isolated from the venom duct of Conus achatinus, a fish-hunting cone snail species collected in the Sea of Hainan, China. Conotoxin-Ac1 is linear peptide that contain 15 amino acids. In the present study, we synthesized and structurally and functionally characterized conotoxin-Ac1 as well as 19 variants. Electrophysiological results showed that conotoxin-Ac1 inhibited N-methyl-D-aspartate receptor subunit 2B (NR2B) with an IC50 of 8.22 ± 0.022 µM. Further structure-activity studies of conotoxin-Ac demonstrated that polar amino acid residues were important for modulating its active, and the replacement of N1, O9, E10, and S12 by Ala resulted in a significant decrease in potency to NR2B. °Furthermore, conotoxin-Ac1 and conotoxin-Ac1-O6P were tested in hot-plate and tail-flick assays to measure the potential analgesic activity to an acute thermal stimulus in a dose-dependent manner. Subsequently, the analgesic activity of conotoxin-Ac1 mutants was analyzed by the hot-plate method. The results show that N1, Y2, Y3, E10, N11, S12, and T15 play an important role in the analgesic activity of conotoxin-Ac1. N1 and S12 have significant effects on conotoxin-Ac1 in inhibiting NR2B and analgesic activity. In conclusion, we have discovered that conotoxin-Ac1 is an inhibitor of NMDAR and displays antinociceptive activity.

Research Progress in the Biosynthetic Mechanisms of Marine Polyether Toxins.

Marine polyether toxins, mainly produced by marine dinoflagellates, are novel, complex, and diverse natural products with extensive toxicological and pharmacological effects. Owing to their harmful effects during outbreaks of marine red tides, as well as their potential value for the development of new drugs, marine polyether toxins have been extensively studied, in terms of toxicology, pharmacology, detection, and analysis, structural identification, as well as their biosynthetic mechanisms. Although the biosynthetic mechanisms of marine polyether toxins are still unclear, certain progress has been made. In this review, research progress and current knowledge on the biosynthetic mechanisms of polyether toxins are summarized, including the mechanisms of carbon skeleton deletion, pendant alkylation, and polyether ring formation, along with providing a summary of mined biosynthesis-related genes. Finally, future research directions and applications of marine polyether toxins are discussed.

The Upregulation of TRAF1 Induced by Helicobacter pylori Plays an Antiapoptotic Effect on the Infected Cells.

BACKGROUND: Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a member of the TRAF family and is dysregulated in diseases, such as atheroma, lymphoma, and solid tumors, but the role of TRAF1 in gastric cancer remains unknown. This study was aimed to investigate the role of TRAF1 in Helicobacter pylori (H. pylori)-related cell apoptosis and gastric carcinogenesis. MATERIALS AND METHODS: The mRNA and protein expression levels of TRAF1 were measured in a panel of gastric cancer cell lines and in H. pylori -infected mice by quantitative real-time PCR (qPCR) and Western blotting. The transcription factor that mainly affects transcription of TRAF1 during H. pylori infection was identified. The roles of H. pylori virulence factors that regulate TRAF1 expression were analyzed using isogenic cagA-, vacA-, and cagE-null mutants. The effects of TRAF1 on gastric cell viability and apoptosis during H. pylori infection were detected using the standard MTS (cell viability) assay and flow cytometry, respectively. RESULTS: H. pylori infection induced TRAF1 overexpression in both gastric epithelial cells and mice. The expression of TRAF1 in response to H. pylori infection was majorly regulated by the activation of NF-κB and was strongly related to H. pylori virulence factor CagA. The upregulation of TRAF1 inhibited cell apoptosis and increased the viability of infected cells. CONCLUSIONS: H. pylori infection induces the overexpression of TRAF1 in gastric epithelial cells. The upregulation of TRAF1 plays an antiapoptotic role in H. pylori -infected gastric cells and may contribute to the gastric carcinogenesis.

Helicobacter pylori inhibits the cleavage of TRAF1 via a CagA-dependent mechanism.

AIM: To study the impact on cleavage of tumor necrosis factor receptor-associated factor 1 (TRAF1) regulated by Helicobacter pylori (H. pylori). METHODS: Cleavage of TRAF1 was detected by western blotting in the human gastric cancer cell line AGS following treatment with an apoptosis inducer. Cleavage of TRAF1 mediated by caspase was examined in vitro using specific caspase inhibitors. The effect of the COOH-terminal TRAF1 fragment on gastric cell apoptosis during H. pylori infection was measured using flow cytometry. The impact of H. pylori infection on TRAF1 cleavage was detected in the presence of apoptosis inducer. The roles of H. pylori virulence factors that may regulate TRAF1 cleavage were analyzed using isogenic cagA-, vacA- and cagE-null mutants. RESULTS: TRAF1 was found to be cleaved in AGS cells treated with the apoptosis inducer, and caspase-8 was the major caspase involved in the cleavage of TRAF1. The COOH-terminal TRAF1 fragment significantly induced cell apoptosis (P < 0.05) as well as promoted H. pylori-induced cell apoptosis (P < 0.05). H. pylori infection was found to significantly inhibit the cleavage of TRAF1 and to inhibit the activation of caspase-8 in the presence of the apoptosis inducer at specific infection times and different cell/bacteria ratios. We also found that the effects of cagE- and cagA-null mutants on the inhibition of TRAF1 cleavage and activation of caspase-8 were significantly attenuated, compared with wild-type H. pylori, in the presence of the apoptosis inducer, showing that the virulence factor CagA was mainly involved in the inhibition of TRAF1 cleavage. CONCLUSION: H. pylori infection significantly inhibits the cleavage of TRAF1 via a CagA-dependent mechanism, which would increase the relative amounts of full-length TRAF1 and exert an antiapoptotic effect on H. pylori-infected cells.

[Advances in CagA protein and CagAmediated pathogenesis of Helicobacter pylori-A review].

Helicobacter pylori (H. pylori) is a strong risk factor for gastric disease ranging from chronic gastritis to gastric cancer. But the mechanisms underlying the pathogenesis of H. pylori are still not completely understood.The cytotoxin-associated gene A (CagA) of H. pylori, an important virulence factor and the only bacterial oncoprotein, is extensively studied. CagA is delivered into gastric epithelial cells via type IV secretion of H. pylori. Upon delivery, CagA perturbs multiple host signaling pathways by interacting with the host signaling molecules, resulting in cytopathic effects and subsequent cell transformation. Some animal experiments also provide in vivo evidence for the oncogenic capacity of CagA. In this review, recent advances in the structural property, delivery manner and pathogenesis of CagA are summarized, which we hope could better explain the CagA-mediated pathogenesis of Helicobacter pylori and provide directions for the future approach.