A Promiscuous Halogenase for the Derivatization of Flavonoids

Kolling, Dominik; Stierhof, Marc; Lasch, Constanze; Myronovskyi, Maksym; Luzhetskyy, Andriy

doi:10.3390/molecules26206220

Cited by 6 publications

(3 citation statements)

References 60 publications

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“…[5] If other halogenated salts are used instead of sea salt in the production medium, isocoumarin containing the added halogen might be obtained under the action of halogenase. [6] Since some compounds containing bromine exhibited good biological activity, [7] it is worth trying to add bromine salt. In this work, based on the one strain many compounds strategy, we found that this strain could produce more abundant and more active secondary metabolites when adding bromine salt.…”

Section: Introductionmentioning

confidence: 99%

“…And several compounds including a new chlorinated isocoumarin named 5‐chloro‐6,7‐dihydroxy‐3,8‐dimethylisocoumarin produced by this strain have been reported by us [5] . If other halogenated salts are used instead of sea salt in the production medium, isocoumarin containing the added halogen might be obtained under the action of halogenase [6] . Since some compounds containing bromine exhibited good biological activity, [7] it is worth trying to add bromine salt.…”

Section: Introductionmentioning

confidence: 99%

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A New Brominated Isocoumarin from the Marine Starfish‐Associated Fungus Aspergillus sp. WXF1904**

Li,

Lin,

Wang

et al. 2023

Chemistry & Biodiversity

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Based on the one strain many compounds strategy, a new brominated isocoumarin, 5‐bromo‐6,8‐dihydroxy‐3,7‐dimethylisocoumarin (1), along with four new natural products, methyl 3‐bromo‐2,4‐dihydroxy‐6‐methylbenzoate (2), methyl 2‐bromo‐4,6‐dihydroxybenzoate (3), (E)‐3‐(3‐bromo‐4‐hydroxyphenyl) acrylic acid (4) and 4‐hydroxy‐3‐methyl‐6‐phenyl‐2H‐pyran‐2‐one (5), and four known compounds, methyl orsellinate (6), 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐1‐propenyl)‐2H‐pyran‐2‐one (7), pilobolusate (8) and cis‐ferulic acid (9), were isolated from the ethyl acetate extract of the fungus Aspergillus sp. WXF1904 under the condition of adding bromine salt to the production medium. The structures of the new compounds were established by analysis of NMR and MS data. Compounds (1–9) were evaluated for inhibitory activity of acetylcholinesterase and pancreatic lipase, the new compound 1, known compounds 6 and 7 displayed weak inhibitory activity against acetylcholinesterase, compounds 2, 5, 7 and 8 showed weak inhibitory activity against pancreatic lipase.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A New Brominated Isocoumarin from the Marine Starfish‐Associated Fungus Aspergillus sp. WXF1904**

Li,

Lin,

Wang

et al. 2023

Chemistry & Biodiversity

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“…The optimization of the IPS production in TT01 leads to the opportunity to also produce high amounts of stilbene derivatives. However, the production of halogenated CA or IPS without supplementation of the cultures with CA-derivatives was not successful although genes encoding for enzymes incorporating phenol modifying tailoring enzymes like the halogenases radH from the fungus Chaetomium chiversii and dklH from the Grampositive bacterium Frankia alni were expressed (data not shown) 166,167 . One crucial part of the optimization of IPS titers relied on the overproduction of phenylalanine and CA which would not be a sufficient approach, if the production of derivatives like the halogenated IPS are desired since so far no suitable halogenase is known.…”

Section: Outlook On Further Optimizationmentioning

confidence: 99%

Investigation and optimization of biosynthetic pathways from entomopathogenic bacteria

Kavakli¹

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This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii. The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug. The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered. The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains. The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches. The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.

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