Decoding Lusichelins A-E: An In-Depth Look at the Metallophores of Lusitaniella coriacea LEGE 07167-Structure, Production, and Functionality

Abstract

Essential trace metals are vital for cellular processes, such as respiration, DNA replication, and photosynthesis. Cyanobacteria must tightly regulate metal homeostasis to prevent deficiency or toxicity, yet their metallophores remain overlooked. Here, we report lusichelins A-E (1-5), new metallophores isolated from the marine cyanobacterium Lusitaniella coriacea LEGE 07167. Their structures and configurational assignments were determined by using NMR, mass spectrometry, TD-DFT calculations, and retrobiosynthetic insights. Lusichelins feature a unique structural arrangement with thiazoline/thiazole rings connected via a vinyl group, an aliphatic carbon chain, or directly enabling the potential for metal coordination. Genomic analysis identified a hybrid PKS/NRPS biosynthetic gene cluster consistent with the lusichelin structure, bearing traits characteristic of metallophore biosynthesis. Functionally, lusichelins act as metallophores capable of chelating both iron and copper. Lusichelin C (3) consistently bound iron under both metal-rich and metal-limited culture conditions, while copper complexation was only observed under elevated copper levels. At physiologically relevant pH values, no significant metal-binding preference was detected. Moreover, compound production was maximized under metal-rich conditions and in response to copper limitation. Lusichelin B (2) exhibited cytotoxicity against colon carcinoma cells while reversing multidrug resistance via ABCB1 efflux pump modulation. These findings expand our understanding of cyanobacterial metallophores in microbial metal homeostasis and highlight their biological potential.

1

Structures of lusichelins A–E (1–5) isolated from Lusitaniella coriacea LEGE 0716.

2

Structure determination of lusichelins A and B (1 and 2, respectively). (A) Four independent ¹H-¹H spin systems (highlighted in bold blue) deduced from their ¹H-¹H COSY spectra were connected by key HMBC correlations (indicated by black arrows). (B) MS² fragmentation pattern with major ions annotated (m/z), deduced from (+)-HRESIMS/MS analysis.

3

Proposed biosynthetic pathway of lusichelins in Lusitaniella coriacea LEGE 07167. (A) Bioinformatics-based annotation of lus BGC. ACP (acyl-carrier protein); PKS (polyketide synthase); PP (phospho­pantetheinyl transferase); NRPS (non-ribosomal peptide synthetase). (B) Biosynthetic model for the assembly of lusichelins A–C (1–3) along with domain annotation. C/Cy (condensation/heterocyclization); A (adenylation); T (thiolation); KS (ketosynthase); AT (acyltransferase); KR (ketoreductase); DH (dehydratase); ER (enoylreductase); Ox (oxidase); cMT (carbon methyltransferase); TE (thioesterase).

4

(A) Configuration determination approach for lusichelin C (3). (B) Newman projections of conformers I and II of 3 around the C-18–C-19 and C-17–C-18 bonds, respectively, along with key experimental ³ J _CH and ³ J _HH coupling constants and NOESY correlations around these positions.

5

Comparison of the experimental ECD spectrum (dashed red line) of lusichelin C (3) to the calculated TD-DFT ECD spectra for 9R,17R,19R,22R (magenta line), 9R,17S,19S,22R (green line), 9S,17S,19S,22R (yellow line), and 9S,17R,19R,22R (cyan line).

6

Lusichelin production in response to: different salt sources (TM; NaCl); iron limitation (TM -Fe; NaCl -Fe); copper limitation (NaCl -Cu), dual metal restriction (NaCl -Fe -Cu); high copper concentration (NaCl [200 nM Cu]; NaCl [450 nM Cu]) and high copper concentration with iron restriction (NaCl -Fe [200 nM Cu]; NaCl -Fe [450 nM Cu]). Relative quantification in biomass and culture media was determined by the peak area of the extracted ion chromatogram for (A) lusichelins A–C (1–3) and the copper–lusichelin C (3-Cu) complex and (B) the iron–lusichelin C (3-Fe) complex. The statistical differences (total amount of compound) of the different experimental conditions versus NaCl were tested using an unpaired t test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; n = 3).

7

UV–vis absorbance spectra of compound 3, along with binding experiments with FeCl₃ (Fe³⁺) and CuSO₄ (Cu²⁺) in equimolar amounts at pH 6.0, 7.5, and 8.5: compound 3 (yellow line), 3:Fe ³⁺ (green line), 3:Cu ²⁺ (cyan line), 3:Fe ³⁺ → Cu ²⁺ (FeCl₃ added first, followed by CuSO₄; green dashed), 3:Cu ²⁺ → Fe ³⁺ (CuSO₄ added first, followed by FeCl₃; cyan dashed), 3:Fe ³⁺ :Cu ²⁺ (simultaneous addition of FeCl₃ and CuSO₄; purple line), 3-Fe (preisolated 3-Fe complex; orange line), and 3-Fe:Cu ²⁺ (preisolated 3-Fe complex with CuSO₄ added; orange dashed).