ECHINULIN

Two new indole alkaloids, identified as (E)-1-(5,6-dihydroxy-1H-indol-1-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one, named oleraindole A (1), and (E)-1-(5,6-dihydroxy-1H-indol-1-yl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, named oleraindole B (2), together with five known indole alkaloids, (-)-neoechinulin A (3), neoechinulin D (4), isoechinulin A (5), MT-6 (6) and echinulin (7) were isolated from the aqueous extract of Portulaca oleracea L. for the first time, using various chromatographic techniques. The structures of seven alkaloids were elucidated by the 1D and 2D NMR and HR-ESI-TOF-MS spectroscopic methods. Oleraindole A (1) and oleraindole B (2) exhibited a relatively high anticholinesterase activity with IC50 values of 55.12 ± 0.20 μΜ and 46.76 ± 0.08 μM, and antioxidant activities with the IC50 values of 16.20 ± 0.11 μM and 13.88 ± 0.06 μM among seven alkaloids.

The echinulin family alkaloids can be grouped into three series depending on the number of the exo double bonds adjacent to the diketopiperazine core structure. Heterologous expression of the putative echinulin biosynthetic gene cluster from Aspergillus ruber in Aspergillus nidulans led to accumulation of echinulin without a double bond and neoechinulin A with one double bond (Δ10) as major products. Their analogues with a different number of prenyl moieties were detected as minor products. Neoechinulin B and analogues with two double bonds (Δ10,14) were not observed. Feeding experiments confirmed that the cytochrome P450 enzyme EchP450 only catalyzes the formation of the double bond between C10 and C11. Coincubation and substrate concentration dependent assays with the prenyltransferase EchPT2 revealed that the reversely C2-prenylated preechinulin without a double bond is a much better substrate than neoechinulin A. These results prove that preechinulin serves as a common substrate for the formation of echinulin by two regiospecific prenylation steps with EchPT2 or for EchP450 to introduce one double bond and subsequent prenylations with low regioselectivity.

Background: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. Results: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. Conclusions: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.