Genomics-Driven Discovery of Benzoxazole Alkaloids from the Marine-Derived Micromonospora sp. SCSIO 07395

Benzoxazole alkaloids exhibit a diverse array of structures and interesting biological activities. Herein we report the identification of a benzoxazole alkaloid-encoding biosynthetic gene cluster (mich BGC) in the marine-derived actinomycete Micromonospora sp. SCSIO 07395 and the heterologous expression of this BGC in Streptomyces albus. This approach led to the discovery of five new benzoxazole alkaloids microechmycin A–E (1–5), and a previously synthesized compound 6. Their structures were elucidated by HRESIMS and 1D and 2D NMR data. Microechmycin A (1) showed moderate antibacterial activity against Micrococcus luteus SCSIO ML01 with the minimal inhibitory concentration (MIC) value of 8 μg mL−1.

Heterologous expression of natural products biosynthetic gene clusters (BGCs) is of increasing interest in the discovery of novel bioactive compounds, which facilitates genome mining in the post-genomic era [19][20][21]. Our group previous research on genome mining by heterologous expression led to the isolation of twelve new polycyclic tetramate macrolactams from marine-derived Streptomyces pactum SCSIO 02999 and Streptomyces sp. SCSIO 40010 [22,23]. A rare actinomycete Micromonospora sp. SCSIO 07395, collected from the South China Sea at a depth of 96 m, was proven to be capable of production of a series of everninomicin derivatives with antimicrobial activity [24]. The genome sequence of this strain revealed the presence of more than twenty BGCs, suggesting a far greater

Results and Discussion
In the course of genome mining to explore natural products from marine-derived actinomycetes, a BGC (mich, GenBank Accession NO. OP432093) encoded in Micromonospora sp. SCSIO 07395 predicated by antiSMASH [25] attracted our attention (Table S1 and Figure 1A). It contains five conserved genes (michABCDE) that are proposed to encode biosynthetic machinery to synthesize benzoxazole-containing compounds. The three enzymes MichBCD are homologous to BomOPQ that are involved in the biosynthesis of 3-hydroxyanthranilic acid (3-HAA) [12]; MichA and MichE (homologs of BomN and BomJ) are likely responsible for catalyzing dimerization of 3-HAA by ester formation and subsequent heterocyclization [17].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 11 of everninomicin derivatives with antimicrobial activity [24]. The genome sequence of this strain revealed the presence of more than twenty BGCs, suggesting a far greater potential to produce specialized metabolites than we have isolated. In this work, a dormant benzoxazole alkaloids encoding BGC from Micromonospora sp. SCSIO 07395 was activated through heterologous expression in Streptomyces albus, leading to five new compounds (1-5) and one previously synthesized compound (6).

Results and Discussion
In the course of genome mining to explore natural products from marine-derived actinomycetes, a BGC (mich, GenBank Accession NO. OP432093) encoded in Micromonospora sp. SCSIO 07395 predicated by antiSMASH [25] attracted our attention (Table S1 and Figure 1A). It contains five conserved genes (michABCDE) that are proposed to encode biosynthetic machinery to synthesize benzoxazole-containing compounds. The three enzymes MichBCD are homologous to BomOPQ that are involved in the biosynthesis of 3hydroxyanthranilic acid (3-HAA) [12]; MichA and MichE (homologs of BomN and BomJ) are likely responsible for catalyzing dimerization of 3-HAA by ester formation and subsequent heterocyclization [17]. We attempted to investigate whether benzoxazole-containing compounds could be produced by the mich cluster by heterologous expression studies, since no such metabolites were detected in the fermentation of Micromonospora sp. SCSIO 07395. A pMSBBAC2based [26] bacterial artificial chromosomal (BAC) genomic library of Micromonospora sp. SCSIO 07395 was constructed to screen positive clones (Table S2) covering the entire mich BGC by PCR with two primers pairs 8-F1/R1 and 8-F2/R2 (Table S3)  We attempted to investigate whether benzoxazole-containing compounds could be produced by the mich cluster by heterologous expression studies, since no such metabolites were detected in the fermentation of Micromonospora sp. SCSIO 07395. A pMSBBAC2based [26] bacterial artificial chromosomal (BAC) genomic library of Micromonospora sp. SCSIO 07395 was constructed to screen positive clones (Table S2) covering the entire mich BGC by PCR with two primers pairs 8-F1/R1 and 8-F2/R2 (Table S3). Subsequently, the positive plasmid pCSG8103 was introduced into Streptomyces albus J1074 for heterologous expression. A new compound 1 was detected when compared to the control strain transformed with the empty BAC vector pMSBBAC2 ( Figure 1C, trace i and ii), but the Molecules 2023, 28, 821 3 of 10 production of 1 was extremely low. S. albus Del14, the derivative of S. albus J1074, with deletion of 15 endogenous gene clusters has shown to be superior to the parental strain for heterologous production of natural products [27,28]. Then, the plasmid pCSG8103 was introduced into S. albus Del14. To our delight, in addition to the increased production of 1, five additional compounds 2-6 were detected in S. albus Del14 harboring pCSG8103 ( Figure 1C, trace iv). Therefore, a 15 L fermentation of S. albus Del14/pCSG8103 were performed for the isolation of compounds 1-6 ( Figure 1B). As expected, compounds 1-6 were structurally established to be benzoxazole-containing compounds by extensive analysis of HRESIMS, 1D and 2D NMR data, named microechmycin A-F, respectively.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 11 expression. A new compound 1 was detected when compared to the control strain transformed with the empty BAC vector pMSBBAC2 ( Figure 1C, trace i and ii), but the production of 1 was extremely low. S. albus Del14, the derivative of S. albus J1074, with deletion of 15 endogenous gene clusters has shown to be superior to the parental strain for heterologous production of natural products [27,28]. Then, the plasmid pCSG8103 was introduced into S. albus Del14. To our delight, in addition to the increased production of 1, five additional compounds 2-6 were detected in S. albus Del14 harboring pCSG8103 ( Figure  1C, trace iv). Therefore, a 15 L fermentation of S. albus Del14/pCSG8103 were performed for the isolation of compounds 1-6 ( Figure 1B). As expected, compounds 1-6 were structurally established to be benzoxazole-containing compounds by extensive analysis of HRESIMS, 1D and 2D NMR data, named microechmycin A-F, respectively. Microechmycin A (1) was isolated as yellow oil. The molecular formula of 1 was determined by HRESIMS m/z: [ Figure 2). Subsequently, two 1,2,3-substituted benzene moieties were deduced from HMBC correlations from H-3 to C-5/C-7, H-4 to C-2/C-6, H-5 to C-3/C-7, H-12 to C-10/C-14, H-13 to C-9/C-11, and H-14 to C-10/C-12 ( Figure 2). Comparing the NMR data of 1 with the intermediate 2-(2-amino-3-hydroxyphenyl)benzoxazole-4-carboxylic acid in the biosynthetic pathway of A33853 [12,17] showed their high structural similarity. The only difference was the presence of signals for an additional methoxy group in 1. The methoxy group was located at C-11 by the HMBC correlation from H3-15 to C-11. Finally, the structure of 1 was determined ( Figure 2).    Figure S3). Compound 2 displayed 1 H and 13 C NMR data that were similar to those of 1 (Table 1 and Figure S3). The major difference was the presence of NMR signals for an additional glycerol moiety in 2. The presence of the glycerol moiety was supported by COSY correlations between H 2 -16 (δ H 4.39, 4.27)/H-17 (δ H 3.88) and H-17/H2-18 (δ H 3.53). The glycerol moiety was linked to the carboxy group at C-1 in 1 to form an ester bond by HMBC correlations from H 2 -16 to C-1 ( Figure 2). Finally, 2 was established as glycerolester derivative of 1. The C-17 configuration of compound 2 failed to be established. Given that 2 is intermolecular esterification product between 1 and glycerol, it might be a racemic mixture, which was proved by chiral phase HPLC analysis with a mixture of two enantiomers with a ratio of 1:1 (2a:2b) ( Figure S3A).
Microechmycin C (3) was isolated as yellow powder. The molecular formula of 3 was assigned to be C 23 Figure S4). Analysis of NMR data of 3 revealed that 3 was highly similar to 1. The difference was that an additional phenylethyl group was present in 3 (Table 1 and Figure S4 Figure S5). Comprehensive analysis of the 1 H, 13 C and HSQC NMR data of 4 (Table 2 and Figure S5) revealed a pair of signals for eight sp 2 nonprotonated carbons, six sp 2 methines, one oxidized sp 3 methylene group, one methoxy group, and the signal for an oxidized sp 3 methine. Further analysis of 2D NMR data indicated that 4 was a dimeric compound connecting two monomers of

-diester bonds. This assignment was confirmed by the COSY correlations between H 2 -16(H-18)/H-17 and HMBC correlations from H 2 -16(H-18)
to C-1(C-1') ( Figure 2). Therefore, the structure of compound 4 was established ( Figure 2).  Figure S6), which is similar to that of 4. A detailed comparison of the NMR data of 5 and 4 (Table 2 and Figure S6) indicated that they were structurally highly similar and only differed from each other at the position forming the diester bonds. In 5, the two monomers of 1 were connected by glyceryl bridge through 1,2-diester bonds, instead of the 1,3-diester bonds in 4. The assignment was suggested by the COSY correlations between H 2 -16/H-17 and H-17/H 2 -18, and the HMBC correlations from H 2 -16 to C-1 and H-17 to C-1' (Figure 2). Thus, the structure of compound 5 was determined. Although bearing a stereogenic center at C-17, 5 was shown to have an almost straight line in the electronic circular dichroism (ECD) spectrum ( Figure S6A), thus indicating the presence of a racemate of 5. A chiralphase HPLC analysis of 5 further indicated that it was a mixture of two enantiomers with a ratio of 1:1 (5a:5b) (Figure S6A).
Microechmycin G (6) Figure S7). Careful comparison of the NMR data demonstrated that the structures of 6 and 1 were highly similar (Table 1 and Figure S7). The only difference was the presence of an additional methoxy group (δ C /δ H 52.4/4.03) in 6, which was linked to the carboxy group at C-1 to form an ester bond. This assignment was supported by the HMBC correlations from H 3 -16 to C-1 (Figure 2). The NMR data of 6 was almost identical to those of a chemically synthesized compound [29], designated herein as Microechmycin G.
Based on the conserved biosynthetic mechanism for the formation of initial building block 3-HAA and heterocycle benzoxazole ( Figure S1) [12,13,15,17], a putative pathway for microechmycins is outlined in Figure 1B. The 3-deoxy-D-arabino-heptulosonate 7phosphate (DAHP) synthase MichG condenses phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to yield DAHP, the first intermediate in the shikimate pathway leading to chorismate. 3-HAA is proposed to be derived from chorismate catalyzed by the anthranilate synthase MichD, isochorismatase MichC and the 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase MichB. Then, 3-HAA is condensed with a second 3-HAA by the ATPdependent coenzyme A ligase MichE via an unstable ester intermediate followed by MichA-mediated benzoxazole formation. The methyltransferase MichF is responsible for the C-11 O-methylation to from compound 1. The tailoring steps of microechmycins biosynthesis involve the attachment of glycerol and phenethyl alcohol moiety to the benzoxazole core structure of 1 via esterification at the C-1 carboxyl leading to compounds 2, 3 or O-methylation to form 6. The second esterification between 3 and 1 to produce 4 and 5 through 1,3 diester or 1,2 diester. However, no genes encoding enzymes for esterification were identified in the mich BGC, suggested the esterification could be catalyzed by endogenous enzymes from the heterologous host S. albus Del14.

Bacterial Strains and Reagents
Bacterial strains and plasmids used and constructed in this study are listed in Table S2. The strain Streptomyces albus Del14 [27] was obtained from Prof. Andriy Luzhetskyy in University of Saarland, Germany and Prof. Hailong Wang in Shandong University, China. The plasmid pMSBBAC2 [26] was obtained from Prof. Jing He in Huazhong Agricultural University, China. Chemicals and molecular biological reagents were purchased from standard commercial sources and used according to the manufacturer's recommendations.

General DNA Manipulation Techniques
All DNA manipulations in this study were performed according to manufacturers' protocols. PCR was performed according to recommended protocol using EasyTaq (Trans-Gen Biotech, Beijing, China). DNA sequencing was performed at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Primers used in this study (Table S3) were synthesized at IGE biotech (Guangzhou, China).

Heterologous Expression of Mich BGC
The BAC library of Micromonospora sp. SCSIO 07395 was constructed using the BAC vector pMSBBAC2 [26] by Eight Star Bio-tech company (Eight Star Bio-tech Co., Ltd. Wuhan China). The positive clone pCSG8103 containing the mich BGC was screened out by PCR with primers 8-F1/R1 and 8-F2/R2 (Table S3). S. albus Del14 [27] and S. albus J1074 [31] ( Table S2) were used as the hosts for heterologous expression. E. coil ET12567/pUB307 [32] was used as the helper strains for the triparental mating. The heterologous hosts were grown at 28 • C in MS medium (soybean powder 20 g·L −1 , mannitol 20 g·L −1 , agar powder 20 g·L −1 , pH 7.2) for growth and sporulation. The E. coli strains were grown in Luria-Bertani medium at 37 • C. The plasmid pCSG8103 was introduced into the host strain S. albus J1074 and S. albus Del14 by conjugation with the help of E. coli ET12567/pUB307. Three positive clones were randomly selected for small scale fermentation and subjected to metabolite analyses by HPLC.

Large-Scale Fermentation and Isolation of Compounds from Heterologous Expression Strain
The 15 L of culture broth of S. albus Del14/pCSG8103 were pooled and centrifuged at 3900 rpm for 15 min at 25 • C. The mycelia were extracted three times, each with 2 L acetone. The acetone extracts were concentrated under reduced pressure to afford an aqueous residue, which was extracted three times with equal volume butanone. The supernatant was extracted three times with equal volume butanone. The butanone extracts were combined and concentrated under reduced pressure to afford crude extract (9.86 g). Subsequently, the crude extract was subjected to a (100-200 mesh) silica gel column eluting with a gradient of petroleum ether/acetone mixture from 50:1, 20:1, 10:1, 5:1 and 1:1 yielded 27 fractions (Fr.1-Fr.27). The fractions Fr.18 and Fr.19 were combined further separated by silica gel column (100-200 mesh) and eluted with CHCl 3 /CH 3 OH (50:1, v/v) to obtain 7 subfractions (Fr.A1 to Fr.A7). The fractions Fr.A2 and Fr.A3 were combined (0.83 g) and further separated by Sephadex LH-20 and eluted with petroleum ether/CHCl 3 /CH 3 OH (5:5:1, be synthesized through the actions of esterification between glycerol and two molecules of 1. This glycerol esterification has been observed in acautalides C discovery from Acaulium sp H-JQSF [33]. The discovery of microechmycins adds to the inventory of secondary metabolites that can be biosynthesized by Micromonospora sp. SCSIO 07395, and highlights the potential of searching for novel natural products from marine rare actinomycetes by heterologous expression.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28020821/s1, Table S1. Deduced functions of individual orf s in the mich gene cluster. Table S2. Strains and plasmids were used and generated in this study. Table S3. Primers were used in this study. Table S4. The antibacterial activities of compound 1-6. Figure S1. The representative biosynthetic pathway of benzoxazoles. Figure S2. The spectroscopic data of 1. Figure S3. The spectroscopic data of 2. Figure S4. The spectroscopic data of 3. Figure S5. The spectroscopic data of 4. Figure S6. The spectroscopic data of 5. Figure S7. The spectroscopic data of 6. Refs [26,27,31,32]