Browsing by Subject "biosynthesis"

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  • Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling (2017)
    The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3'-hydroxylase (NMCH), and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This transcriptomic database provides new directions for future studies on clarifying the aporphine alkaloid pathway.
  • Jokela, Jouni; Heinilä, Lassi M. P.; Shishido, Tania K.; Wahlsten, Matti; Fewer, David P.; Fiore, Marli F.; Wang, Hao; Haapaniemi, Esa; Permi, Perttu; Sivonen, Kaarina (2017)
    Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolandia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A-F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid, which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins, and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 mu M after 1 h) in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic nodularin has the potential to cause the death of eukaryotic organisms.
  • Saukkonen, K.; Hagström, J.; Mustonen, H.; Lehtinen, Laura; Carpen, O.; Andersson, L.C.; Seppänen, H.; Haglund, C. (2018)
    Expression of regenerating islet-derived protein 4 (REG4), a secretory protein involved in cell differentiation and proliferation, is upregulated in inflammatory bowel diseases and in many gastrointestinal malignancies. The prognostic significance of its expression in pancreatic ductal adenocarcinoma is unknown. Our aim was to investigate tumor tissue and serum REG4 expression in pancreatic ductal adenocarcinoma patients. We also evaluated as a control the diagnostic value of serum REG4 level in patients with chronic pancreatitis. Immunohistochemical expression of REG4 was evaluated in 154 surgical specimens and serum REG4 level in 130 samples from pancreatic ductal adenocarcinoma patients treated at Helsinki University Hospital, Finland, in 2000–2011. REG4 tissue and serum expression was assessed in relation to clinicopathological parameters and patient survival. A chronic pancreatitis control group comprised 34 patients who underwent pancreatic resection because of suspicion of malignancy. Significant survival differences were detectable in subgroups: in tumor stages IA–IIA, high serum REG4 level predicted worse survival (p=0.046). In patients with grade I tumor, positive tissue REG4 expression predicted better survival (p=0.006). In multivariate analysis, neither tissue nor serum REG4 expression was independent prognostic factors. Serum REG4 levels were higher in pancreatic ductal adenocarcinoma than in chronic pancreatitis (p=0.002), with diagnostic sensitivity of 45% and specificity of 91%. In logistic regression analysis, a multivariate model with REG4, CA19-9, and age provided sensitivity of 82% and specificity of 79%. REG4 tissue expression is a prognostic marker in subgroups of pancreatic ductal adenocarcinoma patients. Serum REG4 level might be useful in differential diagnosis between pancreatic ductal adenocarcinoma and chronic pancreatitis. © 2018, © The Author(s) 2018.
  • Heinilä, Lassi; Fewer, David; Jokela, Jouni; Wahlsten, Matti; Jortikka, Anna Elisabeth; Sivonen, Kaarina (2020)
    Cyanobacteria produce a wide range of lipopeptides that exhibit potent membrane-disrupting activities. Laxaphycins consist of two families of structurally distinct macrocyclic lipopeptides that act in a synergistic manner to produce antifungal and antiproliferative activities. Laxaphycins are produced by range of cyanobacteria but their biosynthetic origins remain unclear. Here, we identified the biosynthetic pathways responsible for the biosynthesis of the laxaphycins produced by Scytonema hofmannii PCC 7110. We show that these laxaphycins, called scytocyclamides, are produced by this cyanobacterium and are encoded in a single biosynthetic gene cluster with shared polyketide synthase enzymes initiating two distinct non-ribosomal peptide synthetase pathways. The unusual mechanism of shared enzymes synthesizing two distinct types of products may aid future research in identifying and expressing natural product biosynthetic pathways and in expanding the known biosynthetic logic of this important family of natural products.
  • Ahmed, Muhammad Nouman (Helsingfors universitet, 2016)
    Natural products have enormous structural and chemical diversity and are either the source or direct inspiration for many drugs in use today. Cyanobacteria are prolific producers of complex natural products with serine protease inhibiting activity. Many of these natural products are the product of non-ribosomal peptide synthetase (NRPS) modular enzyme complexes. Suomilide is a complex tetrapeptide produced by strains of the benthic cyanobacterium Nodularia sphaerocarpa. It has a highly complicated structure and contains an unusual azabicyclononane moiety, a methylglyceric acid, a xylose unit with hexanoic acids and a terminal 1-amidino-3-(2-aminoethyl)-3-pyrroline moiety. Suomilide inhibits thrombin, plasmin and trypsin in low micro-molar concentrations. The biosynthetic of this unusual glycoside remain unclear. However, suomilide is long predicted to be part to the aeruginosin family of protease inhibitors. A 5.4 Mb draft genome of Nodularia sphaerocarpa HKVV was obtained in order to identify the suomilide biosynthetic. The 43.7 kb suomilide gene cluster was identified on a single contig by performing tBLASTn searches on the draft genome of Nodularia HKVV using aerDEF genes from aeruginosins gene cluster as query. This gene cluster encodes 27 genes including two complex NRPS enzymes and a set of tailoring enzymes for the assembly of suomilide. The suomilide gene cluster shares extensive homology to known aeruginosin gene clusters including two aerB and aerG genes encoding NRPS enzymes, 12 genes (aerC, aerD, aerE, aerF, aerI, aerK, two copies of aerN and four copies of aerH) encoding for the enzymes responsible for synthesis of precursor non-proteinogenic amino acids and 13 other tailoring enzymes. The suomilide gene cluster was much larger and encoded a greater number of biosynthetic enzymes reflecting the structural complexity of suomilide. We identified 10 aeruginosin gene clusters and 2 suomilide gene clusters from 12 strains of cyanobacteria by genome mining. Bioinformatics analyses suggested these gene clusters encoded an unanticipated chemical diversity of aeruginosins and suomilides. LC-MS and Q-TOF analysis detected aeruginosins or suomilide variants from 12 of the 15 strains. Surprisingly, inhibition assays with the crude extracts using all three isoforms of human trypsin suggest that these compounds may have potent and selective inhibition of human trypsin isoforms. Further work is required to prove that suomilide alone can carry out selective inhibition of trypsin isoforms or is it a result of synergism between the compounds produce by cyanobacteria. Phylogenetic analysis demonstrated that the aeruginosin evolved through the acquisition of multiple loading mechanisms and tailoring enzymes through horizontal gene transfers. Our results support the hypothesis that suomilides are a part of aeruginosin family as they are made through the same genetic pathway, however have gained a greater degree of structural diversity due to the acquisition of tailoring enzymes. These results together suggest that cyanobacteria produce an unexpected wealth of complex natural products belonging to the aeruginosin family and that some of these may be potent and selective inhibitors of isoforms of human trypsin.
  • Hovilehto, Emmi (Helsingfors universitet, 2014)
    The literature review of this thesis concentrated on vitamin B12 (Clb), its forms, vitamin B12 anologues, and the biosynthetic route of Clb. In addition, the roles of ribolfavin (RF) and niacin (NAM) in this biosynthetic route were discussed. Cereals were also evaluated as a matrix for vitamin B12 synthesis based on their RF and NAM concentrations. The aims of the experimental part were to prepare a malt extract medium, to study the effects of RF and NAM, and to compare three Propionibacterium freudenreichii strains. The medium (pH 6.40) consisted of 10% of malt extract (ME) and 0.1 M potassium phos-phate buffer. Sterile filtered precursor solutions (e.g. CoCl2) were added to the autoclaved broth. The final composition of the broth was decided based on a preliminary test, where lactate (L; 8 g/L) and/or tryptone (T; 5 g/L) supplements were compared. Thus, the impact of RF and NAM to Clb yield was studied in ME+L+T medium. In these tests five conditions were used: ME+L+T, ME+L+T with DMBI (5,6-dimethylbenzimidazole), and either 1) RF levels of 1, 3, and 38 ?M (27 mM of NAM) or 2) NAM levels of 0.1, 0.6, and 27 mM (3 ?M of RF). The RF concentrations were also tested with strain 3 in ME+T broth. Strains were incubated anaerobically at 30 °C for 3 days and microaerobically for 4 days. Optical densities, cell masses, and pH values were measured. Intracellular Clb was extracted as cy-ano-Clb and quantified using an UHPLC/UV method. From medium RF and niacin were analyzed with an UHPLC/FL method, and sugars and acids with an HPLC/RI/UV method. In ME+L+T strain 1 produced 1.0 ± 0.2 ?g/mL of vitamin B12, strain 2 synthesized 1.2 ± 0.2 ?g/mL, and strain 3 yielded 0.82 ± 0.2 ?g/mL of Clb. DMBI increased Clb synthesis most in strains 1 and 3, while with strain 2 the 27 mM NAM level together with RF resulted in the highest yields. Alone low NAM concentrations did not affect Clb yields, but RF increased Clb production by strains 2 and 3 (p < 0.05). On the other hand, high RF concentration may have inhibited its intake. Thus, RF levels in cereals should be well suited for Clb synthesis. However, strains 1 and 2 had higher Clb yields and they salvaged RF more than strain 3. Furthermore, the results indicated that with these two strains NAM may stimulate Clb synthesis or growth. However, these results should be confirmed. Moreover, further studies are needed especially on the role of NAM and nicotinic acid, the salvage routes of niacin and RF, and other nutritional requirement of the strains in cereal matrices.