Co-factor independent oxidases ncnN and actVA-3 are involved in the dimerization of benzoisochromanequinone antibiotics in naphthocyclinone and actinorhodin biosynthesis

Abstract Streptomyces produce complex bioactive secondary metabolites with remarkable chemical diversity. Benzoisochromanequinone polyketides actinorhodin and naphthocyclinone are formed through dimerization of half-molecules via single or double carbon-carbon bonds, respectively. Here we sequenced the genome of S. arenae DSM40737 to identify the naphthocyclinone gene cluster and established heterologous production in S. albus J1074 by utilizing direct cluster capture techniques. Comparative sequence analysis uncovered ncnN and ncnM gene products as putative enzymes responsible for dimerization. Inactivation of ncnN that is homologous to atypical co-factor independent oxidases resulted in the accumulation of fogacin, which is likely a reduced shunt product of the true substrate for naphthocyclinone dimerization. In agreement, inactivation of the homologous actVA-3 in S. coelicolor M145 also led to significantly reduced production of actinorhodin. Previous work has identified the NAD(P)H-dependent reductase ActVA-4 as the key enzyme in actinorhodin dimerization, but surprisingly inactivation of the homologous ncnM did not abolish naphthocyclinone formation and the mutation may have been complemented by an endogenous gene product. Our data suggests that dimerization of benzoisochromanequinone polyketides require two-component reductase-oxidase systems.


Introduction
Streptom yces ar e soil-dwelling pr okaryotes with a capability to gener ate numer ous bioactiv e secondary metabolites that ar e harnessed for medical usage as antibiotics, anticancer agents and imm unosuppr essants (Ne wman and Cr a gg 2020 ).A k e y feature of micr obial natur al pr oducts is their c hemical complexity and div ersity.Sur prisingl y, m uc h of this c hemodiv ersity is gener ated via a limited number of biosynthetic systems that classify secondary metabolites to the main classes of polyketides, non-ribosomal peptides, ribosomally synthesized, and post-translationally modified peptides, and terpenes (Fewer and Metsä-Ketelä 2020 ).
Ar omatic pol yketides ar e a lar ge subgr oup of secondary metabolites that harbour a wide range of pharmaceutical functions (Medema et al. 2015 ), which include the antibacterial tetracycline (Pickens and Tang 2009 ) and the anticancer agent doxorubicin (Hulst et al. 2021 ).One extensiv el y studied type-II pol yketide is the benzoisoc hr omanequinone (BIQ) antibiotic actinorhodin synthesized by the model organism Streptomyces coelicolor A3(2) (Okamoto et al. 2009 ;Ta guc hi et al. 2013 ;Hashimoto et al. 2023 ).Other notable examples of BIQ polyketide antibiotics in-clude naphthocyclinones, aln um ycin, granaticin and medermycin (Metsä-Ketelä et al. 2013 ) (Fig. 1 ).Naphthocyclinones pertinent to this study were originally discovered in 1974 from Streptomyces arenae DSM 40737 (Zeeck andMardin 1974 , Zeeck et al. 1974 ) and subsequently confirmed to be asymmetrical dimers (Krone et al. 1982, Ando et al. 2015 ).Three different conformations of naphthocyclinones, the α, β-, and γ -forms, have been identified, but only the βand γ -forms show bioactivities a gainst Gr am-positiv e bacteria (Brünker et al. 2001 ).One notable difference between actinorhodin and naphthocyclinone is the mode of dimerization, since the two monomers are joined together either by one or two C-C bonds, respectiv el y.
Earl y 13 C labelling experiments hav e indicated that both actinorhodin and naphthocyclinone are formed via dimerization of two 16-carbon pol yketides (Sc hröder andFloss 1978 , Gorst-Allman et al. 1981 ).The molecular genetics of actinorhodin biosynthesis have been studied since the 1970s (Rudd and Hopwood 1979 ).The carbon chain is synthesized from eight malonyl-CoA units via iter ativ e Claisen condensations by the ketosynthase α (KS α)/KS β heterodimer.The highly reactive polyketide In contrast, the biosynthesis of naphthocyclinone has received consider abl y less attention to date.Earlier work identified two DNA fr a gments fr om the biosynthetic gene cluster (BGC) (Sherman et al. 1989, Brünker et al. 1999, 2001 ) and v ery r ecentl y the genes were identified from the genome sequence of S. eurocidicus CGMCC 4.108 (Li et al. 2023 ).Here we have sequenced the genome of S. arenae DSM 40737 and ca ptur ed the BGC for heter ologous pr oduction of naphthocyclinone in S. albus J1074.We provide experimental evidence that ncnN and act VA-3 are involved in the dimerization of naphthocyclinone and actinorhodin, respectively.

Strains, oligonucleotides and chemicals
Streptomyces arenae DSM 40737 was used as the source of the ncn BGC, while Streptomyces albus J1074 was used as a host for heterologous expr ession studies.Streptom yces coelicolor M145 was used as a host to study actinorhodin biosynthesis.Cloning vectors and Esc heric hia coli cell lines (Supporting information text) were obtained kindl y fr om Pr of.A. Fr ancis Ste wart, Genomics, Biotec hnology Center , T ec hnisc he Univ ersität Dr esden, German y.Oligonucleotide primers used in our experiment were purchased from Eur ofins Genomics GmbH German y.All the c hemicals, endonucleases, and r ea gents used in our experiment w ere pur c hased fr om Merck, USA, unless otherwise stated.

Gener al DN A techniques and gene cloning
The strategy for cloning and recombineering of the ncn BGC was carried out as devised by Wang et al. ( 2016 ) with minor modifications as described in the Supporting information text.The inactivation of act VA-3 was carried out by homologous recombination using vector pWHM3 (Vara et al. 1989 ), including an additional oriT sequence to allow conjugation to Stre ptom yces, as described in the Supporting information text.

Production and purification of α-naphthocyclinone acid
The S. albus /SA-naphtho cells w ere gro wn in 4 L NoS-soyE1, which is modified E1 without starc h (Ylihonk o et al. 1994, Oja et al. 2008 ) F igure 2. Naphthoc yclinone biosynthetic gene cluster and heterologous expression trials.(A) Gene organization of the naphthocyclinone ncn biosynthetic cluster and comparison to actinorhodin act and aln um ycin aln BGCs .T he genes involved in naphthocyclinone dimerization are highlighted.Genes with significant sequence similarity (66% to 100%) in the different gene clusters are connected with grey arrows.(B) Heterologous expression of the ncn BGC in S. albus and inactivation of ncnN .HPLC chromatogram traces of (i) S. albus /pSA-naphtho harbouring the intact ncn BGC demonstr ates pr oduction of α-na phthocyclinone acid, while (ii) inactiv ation of ncnN leads to pr oduction of fogacin in S. albus /pSA-na phtho ncnN, (iii) authentic α-naphthocyclinone acid NMR standard and (iv) complementation of the ncnN mutation with an intact copy r estor es α-na phthocyclinone acid production in S. albus /pSA-naphtho ncnN/pEN-SV1-ncnN.Chemical structures of α-naphthocyclinone acid and fogacin.(C) Comparison of the ncn BGC to the fogacin fog BGC r e v eals conserv ed genes for pol yketide biosynthesis (br o wn), while genes inv olv ed in r egulation (y ello w) and transport (dark blue) differ.In addition, the fog BGC harbors genes for glycosylation (light blue) and beta-alkylation (purple).(D) Comparative metabolic profiling r e v eals that inactivation of ncnM does not alter α-naphthocyclinone acid production in (i) S. albus /pSA-naphtho and (ii) S. albus /pSA-naphtho ncnM.
For this, a seed culture was prepared a day before the main culture, and 10% of it was inoculated to the main production culture.To the knockout mutants, Apra (50 μg/mL) was added to the growth media.After 8 d, the gr owth cultur e was centrifuged to obtain the supernatant, and the cells were discarded.The supernatant was incubated with an adsorbent (LXA1180, Sunresin

Production of actinorhodin
Spores (1 × 10 7 ) of the S. coelicolor M145 wild type and knock-out mutant S. coelicolor M145 act VA-3 were used to inoculate 8 cm plate containing 25 mL of R5 solid medium (consisting of 10 g/L glucose, 5 g/L yeast extract (Difco TM ), 0.1 g/L casamino acids, 0.25 g/L K 2 SO 4 , 10.12 g/L MgCl 2 .6H 2 O , 5.73 g/L TES buffer , 2 ml/L trace element solution, 20 g/L agar (pH 7.2)) at 30 • C for 3 da ys .T he culture was homogenized and metabolites were extracted with ethyl acetate.Actinorhodin production was calculated based on ar ea percenta ge at 520 nm.

Analysis of metabolites
Shimadzu's SCL-10Avp HPLC with an SPD-M10Avp diode array detector was used to perform analytical HPLC analyses .T he analyses to detect naphthocyclinones were performed with a reversedphase column (Phenomenex, Kinetex, 2.6 μm, 4.6 × 100 mm) us-ing gr adients fr om 15% acetonitrile containing 0.1% formic acid to 100% acetonitrile.Production of actinorhodin was analysed using a r e v ersed-phase column (Phenomenex Luna Phenyl-Hexyl 100, 10μm, 250-by 10-mm column) using a gradient from 0.1% Trifluoroacetic acid in water to 100% acetonitrile.MS analyses were carried out with either a low-resolution MS with an HPLC system (Agilent 1260 Infinity 6120 Quadropole LC-MS) with similar conditions and column as the analytical HPLC, or with a MicrO T OF-Q high-resolution MS with direct injection (Bruker Daltonics).NMR samples wer e pr epar ed fr om ov ernight desiccated compounds with deuterated methanol ( α-naphthocyclinone acid) and deuterated acetone, methanol, and DMSO for fogacin.NMR analysis was performed with a 600 MHz Bruker A V ANCE-III NMR-system equipped with liquid nitrogen cooled Prodigy TCI (inverted Cry-oProbe) at 298-300 K.The experiments included 1D ( 1 H, 13 C) and 2D measurements (COSY, HMBC, HSQC and additionally NOESY for fogacin).Topspin (Bruker Biospin) was used for spectral analysis.

Genome sequencing of S. arenae DSM 40737
We initiated the study by extracting high quality genomic DNA from S. arenae DSM 40737 and sequencing the genome using MiSeq resulting in 9560148 reads, which were normalized, error corrected, and trimmed down to 7644475 reads .T he final de novo draft genome assembly consisted of 111 contigs covering 10.5 Mbp with an N50 of 205 194.Annotation of the genome and analysis by an-tiSMASH (Blin et al. 2021 ) r e v ealed 42 BGCs involved in secondary metabolism ( Table S1 ), 27 of which are similar to known clusters.BiG-FAM analysis (Kautsar et al. 2021 ) identified 36 gene cluster families r epr esenting the wide c hemical div ersity of the str ain.
Mor eov er, this str ain contains 10 complete BGCs having a distance greater than 900 in BiG-FAM analysis indicating that they may encode novel molecules ( Table S1 ).Finally, we identified three type II polyketide BGCs encoding genes for biosynthesis of naphthocyclinone (Fig. 2 A), a putative fluostatin-type compound and spore pigment from the sequencing data.

Bioinforma tic anal ysis of earl y steps in naphthocyclinone biosynthesis
Sequence anal ysis r e v ealed a total of 19 genes that could be responsible for the biosynthesis of naphthocyclinones with eight genes for r egulation, self-r esistance and tr ansport (Table 1 ).The biosynthetic logic could be inferred by comparison to related BIQ pathways (Metsä-Ketelä et al. 2013 ).We propose that naphthocyclinone biosynthesis initiates with the assembly of an octak etide polyk etide on the NcnC ACP through condensation of eight malonyl-CoA units by the NcnA/NcnB KS α/KS β heterodimer (Scheme 1 ).The ACP-bound polyketide is subsequently modified by the NcnQ 9-KR, the NcnD ARO, and the NcnE CYC to generate the common enzyme-free bicyclic intermediate on BIQ pathwa ys .Pyran ring formation is preceded by 3-ketoreduction, which determines the ster eoc hemical outcome of cyclization.The naphthoc yclinone pathw ay is likely to follo w the paradigm established for granaticin biosynthesis, since the pathways harbours the SDR enzyme NcnP that is homologous to the 3-KR Gr a-6 (Ic hinose et al. 1998 ).The equivalent step in actinorhodin biosynthesis is catalysed by a protein of the 3-hydroxy ac yl-CoA dehydrogenase famil y (Metsä-K etelä et al. 2013 ) for whic h no homologous enzymes are encoded by the naphthocyclinone BGC.The NcnG CYC is the likely candidate for pyran ring cyclization and formation of ( R )-DNPA (4-dih ydro-9-h ydroxy-1-meth yl-10-oxo-3-H -na phtho[2,3-c]pyr an-3-acetic acid) (Scheme 1 ).An interesting de viation fr om canonical BIQ pathways is that the naphthocyclinone BGC does not contain any gene products of the NAD(P)H dependent medium-chain alcohol dehydrogenases that are typically associated with enoyl reduction and therefore the gene for this step is unknown (Metsä-Ketelä et al. 2013 ).Howe v er, quinone formation is likely to be catalysed by the two-component monooxygenases NcnH/NcnT that are homologous to experimentally c har acterized pr oteins fr om the actinorhodin (Valton et al. 2006 ;Hashimoto et al. 2020 ) and aln um ycin (Gr oc holski et al. 2012 ) systems.

Insight into naphthocyclinone dimerization
In order to obtain experimental evidence for genes involved in the dimerization of naphthoc yclinone, w e cloned the BGC using Rec ET direct cloning (Wang et al. 2016 ) to facilitate recombineering efforts ( Fig. S1 ).The main metabolite (Fig. 2 B) produced by the gener ated str ain S. albus /pSA-na phtho was found to be α-naphthocyclinone acid (Zeeck and Mardin 1974 ).The compound was isolated from the culture broth with acidic ethyl acetate and purified further by silica column and pr epar ativ e HPLC using a r e v ersed phase column.The structure was verified by NMR ( Figs S2 -S7 , Table S2 ) and MS analysis (ESI m/z [M-H] − obs.651.1353 calc.651.1355), which correlated to previously published data (Zeeck and Mardin 1974 ).
Sequence anal ysis r e v ealed two candidate gene pr oducts for dimerization.NcnM (Table 1 , Fig. S8 ) is a member of the NmrA family of NAD(P)H dependent reductases and homologous to ActVA-4, whic h is r esponsible for dimerization in actinorhodin biosynthesis (Ta guc hi et al. 2012 ).In addition, we considered that NcnN (Table 1 , Fig. S9 ) could be involved in the reaction, since it shares sequence identity to the co-factor independent oxidase Aln6 from the aln um ycin pathway that catalyses C-C bond cleavage (Oja et al. 2008(Oja et al. , 2012 ) ).
We proceeded to disrupt ncnN by recombineering in E. coli and conjugated the resulting plasmid pSA-naphtho ncnN to S. albus .Anal ysis of cultur e extr acts (Fig. 2 B) r e v ealed loss of αnaphthocyclinone acid production and accumulation of another metabolite, which was identified as the octaketide fogacin based on NMR ( Figs S10 -S16 , Table S3 ) and MS (ESI m/z [M-H] − obs.305.1019, calc.305.1031) analyses.Fogacin has pr e viousl y been isolated from S. lividans (Santamaría et al. 2018 ), Streptomyces sp.Tü 6319 (Radzom et al. 2006 ) and S. violaceoruber (Lu et al. 2014 ).The r elativ e ster eoc hemistry was v erified to be trans by NOESY ( Figs S15 and S16 ), where H-3 was coupled with the methyl group H-13, but not with H-1.Complementation of ncnN with an intact copy in strain S. albus /SA-naphtho ncnN/pEN-SV1-ncnN restored pr oduction of α-na phthocyclinone acid (Fig. 2 B).A fogacin fog BGC has r ecentl y been identified fr om Actinoplanes missouriensis (Sato et al. 2019 ) and comparison to the ncn BGC r e v ealed a homologous gene set for polyketide biosynthesis (Fig. 2 C).Ho w ever, significant differ ences wer e also a ppar ent, since the ncn BGC contains additional tailoring genes for dimerization and further modification (see below), while the fog BGC harbors genes for production of the glycosylated fogacin B and the β-alkylated fogacin C (Sato et al. 2019 ).
Next, we used recombineering to inactivate ncnM resulting in str ain S. albus /pSA-na phtho ncnM.Sur prisingl y, the gene deletion did not have an effect in the production profile of the strain (Fig. 2 D) and α-naphthocyclinone acid (Fig. 2 D) was detected as the main secondary metabolite .T hese r esults ar e in contr ast to experiments on the actinorhodin pathway, where the homologous act VA-4 has been shown to be essential for dimerization (Ta guc hi et al. 2012 ).We surmise that the result may be due to endogenous complementation of the ncnM deletion by the S. albus J1074 host str ain, whic h contains an unc har acterized SDR gene (accession number WP_033240217.1)with 40.0%sequence identity to ncnM .

Bioinforma tic anal ysis of la te-stage tailoring steps in naphthocyclinone biosynthesis
After dimerization, we propose that the flavoenzyme NcnO would catalyse asymmetrical quinone formation (Scheme 1 ).Late steps in naphthocyclinone biosynthesis have very recently been investigated in the nap BGC from strain S. eurocidicus CGMCC 4.108, where experimental data indicates that acylation by NcnL may be the next biosynthetic step (Li et al. 2023 ).The key difference in the two naphthocyclinone BGCs is that the ncn pathway encodes a F 420de pendent o xidor eductase NcnJ, for whic h ther e is no equiv alent gene on the nap pathway.We suggest that NcnJ is involved in formation of α-na phthocyclinone, whic h has not been detected from S. eurocidicus CGMCC 4.108, and is responsible for late-stage pyran ring opening and oxidation.The final biosynthetic step is likely to be conversion of α-naphthocyclinone acid to α-naphthocyclinone by the methyltr ansfer ase NcnI.

Mechanism of dimerization in the biosynthesis of benzoisochromanequinone antibiotics
Next, we turned our attention to act VA-3 from the actinorhodin pathway, which is homologous to ncnN (Fig. 2 A), in order to determine if the gene product is required for dimerization along with act VA-4 product.We generated a knock-out mutant in S. coelicolor M145 based on classical homologous recombination.The target gene act VA-3 was replaced by an a pr amycin-r esistance (a pr r ) cassette resulting in strain S. coelicolor M145 act VA-3 ( Fig. S18 ).
Compar ativ e metabolic pr ofiling r e v ealed se v er el y impair ed actinorhodin production, since the mutant strain accumulated only a ppr oximatel y 8% of actinorhodin in comparison to the wild type (Fig. 3 A and B).This result is in agreement with very recent data from the Ichinose laboratory, where the authors demonstrated similar reduction in actinorhodin biosynthesis in the act VA-3 mutant and in vitro dimerization activity with ActVA-3 and ActVA-4 (Hashimoto et al. 2023 ).LC-MS analysis was carried out to analyse the mass spectrum of the metabolite .T he metabolite displa yed the m/z value at 629.0 in a negative mode, agreeing to data for authentic γ actinorhodin (Fig. 3 C and D; Fig. S18C ).
Our data is in a gr eement with the r ecentl y pr oposed mec hanism for dimerization on the actinorhodin pathway (Hashimoto et al. 2023 ) (Scheme 2 A), where the tetrahydroxynaphthalene product (T4HN) formed by ActVA-5/ActVB, is converted into hydroxytetr ahydr okalafungin (THK-OH) thr ough keto-enol tautomerization.ActVA-3 would then use molecular oxygen to oxidize THK-OH into 8-h ydroxy-dih ydrokalafungin (DHK-OH) by abstracting two adjacent hydrogen atoms and forming hydrogen peroxide in the process .T hen ActVA-4 catalyses the dimerization of DHK-OH through a hydride transfer from the NADPH cofactor to the r eactiv e double bond at position 9 of DHK-OH, which together with protonation of the carbonyl at position 11 leads to the formation of an 10/11 enolic intermediate .T he π -electrons of the enol then function as a Michael donor attacking the 9'/10' double bond (Michael acceptor) of another molecule of DHK-OH, forming the THK-OH dimer, which is then oxidized into actinorhodin by ActVA-3 by again abstracting two hydrogens and reducing molecular oxygen into hydrogen peroxide.
We propose that naphthocyclinone dimerization proceeds through a similar mechanism (Scheme 2 B).In an orthologous manner to ActVA-3, keto/enol tautomerization of pr ena phthocyclinone would allow its oxidation into the corresponding quinone (dehydr opr ena phthoc yclinone, DHPN) b y NcnN using molecular oxygen.The differences in the two pathways would occur in the dimerization reaction, although the initial first step might proceed through similar chemistry.A C-C bond would be formed between the carbon atoms at positions 8 and 8' of two copies of DHPN thr ough Mic hael addition.The Mic hael donor is activ ated by a hydride tr ansfer fr om NAD(P)H to the carbon at position 7 of DHPN, which would facilitate adjacent π -electrons to perform a nucleophilic attack on another DHPN molecule (DHPN'), which functions as the Michael acceptor (Scheme 2 B).A ketone at position 9 could be involved in resonance stabilization of the formed transient carbanion intermediate, as described for actinorhodin (Scheme 2 A).The electrophilicity of the Michael acceptor is increased by a simultaneous protonation of the carbonyl oxygen at position 6'.The unique second step in the naphthoc yclinone dimerization w ould be an aldol reaction betw een the formed Michael acceptor and the carbonyl carbon at position 6 (Scheme 2 B).This reaction would be catalysed by deprotonation of the 6'-enol and protonation of the 6-ketone functional groups .T he resulting bicyclic dimer would tautomerize into the corresponding hydroquinone and no additional oxidations by NcnN, in contrast to what has been demonstrated for ActVA-3 in actinorhodin biosynthesis, would be r equir ed.
The S. albus /pSA-na phtho ncnN str ain pr oduced fogacin, which is likely a shunt product.The diketone tautomer of prena phthocyclinone, whic h is normally oxidised by NcnN into DHPN, is instead reduced into fogacin in the absence of NcnN in the heterologous host.Possibly the same enzyme that would normally use NAD(P)H to catalyse the dimerization of DHPN, would catal yse this ketor eduction.Fogacin lac ks the necessary 7/8 double bond, and is ther efor e an unsuitable substrate for dimerization.

Conclusions
In this study, we have confirmed that ncnN and act VA-3 are involved in the dimerization of naphthocyclinone and actinorhodin, r espectiv el y.Our heter ologous expr ession studies of the na phthocyclinone ncn cluster in S. albus led to production of αnaphthocyclinone acid.Bioinformatic analysis allo w ed us to propose a tentative scheme for the generation of α-naphthocyclinone acid.We provide the first insight into the unique asymmetrical naphthocyclinone dimerization via gene inactivation experiments and propose that NcnN is an oxidase that primes naphthocyclinone monomer units for dimerization.Furthermore, our r esults corr obor ate r ecent findings (Hashimoto et al. 2023 ) that ActVA-3 is dir ectl y r ele v ant to actinorhodin biosynthesis in S. coelicolor .Deletion of a ct VA-3 led to a significant decrease (about 92%) in actinorhodin biosynthesis.NcnN and ActVA-3 belong to a poorly characterized enzyme family that has not been structur all y c har acterized, but members of the famil y a ppear to be cofactor inde pendent o xidases that utilize molecular o xygen without metal ions or organic co-factors (Oja et al. 2012 ).Collectively, our r esults pav e the way for mor e detailed mec hanistic studies in order to understand how NcnN and ActVA-3 are able to break the spin barrier between organic molecules and molecular oxygen possibly in a co-factor independent manner.

Figure 3 .
Figure 3. Functional analysis of act VA-3.(A) Phenotypic effect of the act VA3 deletion on ACT biosynthesis demonstrates reduced production of blue pigmented actinorhodin in the mutant strain in comparison to the wild type.Strains were inoculated on R5 solid medium and grown at 30 • C for 3 da ys .T he r e v erse side of plates is shown for (i) S. coelicolor M145 actVA-3 m utant and (ii) S. coelicolor M145 wild type.(B) HPLC c hr omatogr am tr aces recorded at 520 nm of culture extracts from S. coelicolor M145 wild type (top) and S. coelicolor M145 actVA-3 mutant (bottom) demonstrates reduced production of actinorhodin in the mutant.(C) Chemical structure of γ actinorhodin and (D) UV/Vis spectrum of γ actinorhodin.

Table 1 .
Sequence analysis of genes residing in the naphthocyclinone ncn gene cluster of S. arenae DSM 40737.
vested and discarded.The supernatant was subjected to ethyl acetate extraction with 1% (v/v) of acetic acid.The extraction was repeated 2-4 times .T he ethyl acetate fractions were combined and dried.Fractions were stored at −20 • C. Part of the sample was solubilized in c hlor oform and loaded to the column and the part that did not solubilize was dissolved in acetone and dried with silica and dry-loaded to the column.The silica columns were equilibrated with 10:90 acetone:chloroform.A gradient from 10 to 100% of acetone was run, but the main compound did not elute from the column.Finally, 100% methanol was used to elute the main compound, which was collected and dried.The dried sample was solubilized with methanol and subjected to pr epar ativ e HPLC, LC-20 AP with a diode array detector SPD-M20A (Shimadzu) with a C-18 column (Kinetex Prep C18, 5 μm, 250 × 21.2 mm; Phenomenex).The fractions containing the main compound were combined and extracted with c hlor oform, dried, and stored at −20 • C. The yield of α-naphthoc yclinone acid w as 2 mg per liter culture.Scheme 1. Pr oposed sc heme for the biosynthesis of α-na phthocyclinone acid in S. arenae.