Genome Mining, Heterologous Expression, Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS+ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.


Introduction
Bacillus subtilis is nonpathogenic, and displays considerable genetic diversity, even among closely related strains. Its secondary metabolites have been studied for more than 50 years, and numerous studies have revealed that the secondary metabolites are characterized by antimicrobial and other biological activities. [1] B. subtilis has been widely used in the food industry and agriculture for inhibiting and eliminating foodborne and plant pathogens [2,3]. Genomic sequencing revealed that an average of 4-5% of the genome in each strain is devoted to the synthesis of bioactive compounds, giving the organism the potential to produce more than two dozen antibiotics with a great variety of structures [4].
Improvements in sequencing technology have made genome mining an important tool for the discovery of novel natural products [5,6]. During the last decade, the rapid development of bioinformatics tools, as well as improved sequencing and annotation of microbial genomes, has led to the discovery of novel bioactive compounds synthesized Thus, the hybrid NRPS/PKS cluster from B. subtilis fmb60 was predicted to synthesize amicoumacins.

Isolation and Characterization of Lipoamides
In searching for possible lipoamides, it was noticed that apart from fengycin, surfactin, and bacillibactin, another fraction of the crude extracts was eluted from the semipreparation HPLC. Three novel compounds were isolated from this fraction.
The structure of compound 1 was determined using 1 H and 13 CNMR spectroscopy and heteronuclear single quantum coherence spectroscopy (HSQC) data. The 13 C NMR and DEPT spectra displayed 35 carbon signals, classified as three carbonyls, two methyl, 10 methylene, and two methylidynes ( Table 1). The planar structure of compound 1 was established using two-dimensional (2D) NMR data ( Figure 1A). In the HSQC, 1 H− 1 H COSY, and heteronuclear multiple bond correlation (HMBC) spectra of compound 1, C-2' (δ C 50.5, δ H 4.70, t) was easily recognized and set as a starting point. The key HMBC correlations of H-2 (δ H 4.70, t) to C-1 (δ C 175.1), C-3 (δ C 37.9), C-4 (δ C 174.1), and C-1 (δ C 176.2), as well as H-3 (δ H 2.75, 1.31, m) to C-1 (δ C 175.1) and C-4 (δ C 174.1) indicated the existence of an Asn unit. Two methyl proton signals at δ H 0.85 and a broad peak at δ H 1.31 indicated the presence of an iso fatty acid side chain, a finding which was supported by the correlation spectroscopy (COSY, HSQC, and DEPT) data. Two 1 H− 1 H COSY spin subunits were recognized. These two subunits are linked through an amide bond, as evidenced by the HMBC correlation from H-2 (δ H 4.70, t) to C-1 (δ C 176.2). Further comprehensive analysis of 2D NMR data showed that the branched-chain methyl fatty acid of compound 1 is different from that of lipoamide A and other similar structures [20,21]. The position of the methyl group at C-13, which was manifested by the HMBC correlations of C-13 (19.7) to H-11 (δ H 1.31, 1.09), and C-12(δ C 11.8) was correlated with H-11 (δ H 1.31, 1.09). Thus, the structure of compound 2 was proposed, as shown in Figure 1B, and named lipoamide D. The structure of compound 2 was investigated using NMR spectroscopy. These data revealed that compound 2 possessed structural similarities to compound 1, but differed from the molecular formula of compound 1 by the addition of -CH 2 (δ C 30.4, δ H 1.31) ( Table 1). By analyzing the HSQC and HMBC, the position of the methyl group was confirmed at C-12. Thus, compound 2 s structure was proposed, as shown in Figure 1B, and it is first named lipoamide E. The structure of compound 2 was investigated using NMR spectroscopy. These data revealed that compound 2 possessed structural similarities to compound 1, but differed from the molecular formula of compound 1 by the addition of -CH2 (δC 30.4, δH 1.31) ( Table  1). By analyzing the HSQC and HMBC, the position of the methyl group was confirmed at C-12. Thus, compound 2′s structure was proposed, as shown in Figure 1B, and it is first named lipoamide E.
The structure of compound 3 was investigated using NMR spectroscopy. The 1D NMR spectra of compound 3 exhibited high similarities to those of compound 1, indicating that its structure was closely related to that of compound 1 ( Table 1). The only difference was that the molecular weight of compound 3 was higher than 14 amu of compound 1, while an additional carbon resonance signal (δC 30.3) could be observed. Meanwhile, the position of the methyl group was confirmed at C-14 according to HSQC and HMBC analysis. Thus, compound 3′s structure was proposed, as shown in Figure 1B, and it is named lipoamide F for the first time.

Isolation and Characterization of New Amicoumacins
The fermentation broth of strain B. subtilis fmb60 was extracted with ethyl acetate. Thirteen compounds were isolated using HPLC, all of which exhibited similar UV spectra, indicating that they were probably structurally related. Analysis of the 1 H, 13 C NMR, and ESI-HRMS/MS spectra of this extract revealed that the structure of the bioactive fractions was almost identical to that of isocoumarin-type compounds [22].  (Table 2) as well as 1 H− 1 H COSY and HSQC, suggested the presence of 21 carbons, including three carbonyl carbons, six aromatic carbons, four methylenes, a methine, two methyls, and five carbons bonded to nitrogen or oxygen. These data revealed that compound 4 possessed structural similarities to amicoumacin C but differed from the molecular formula of amicoumacin C by the addition of -CH2 [23]. This methylene group was identified by two proton signals observed The structure of compound 3 was investigated using NMR spectroscopy. The 1D NMR spectra of compound 3 exhibited high similarities to those of compound 1, indicating that its structure was closely related to that of compound 1 ( Table 1). The only difference was that the molecular weight of compound 3 was higher than 14 amu of compound 1, while an additional carbon resonance signal (δ C 30.3) could be observed. Meanwhile, the position of the methyl group was confirmed at C-14 according to HSQC and HMBC analysis. Thus, compound 3 s structure was proposed, as shown in Figure 1B, and it is named lipoamide F for the first time.

Isolation and Characterization of New Amicoumacins
The fermentation broth of strain B. subtilis fmb60 was extracted with ethyl acetate. Thirteen compounds were isolated using HPLC, all of which exhibited similar UV spectra, indicating that they were probably structurally related. Analysis of the 1 H, 13 C NMR, and ESI-HRMS/MS spectra of this extract revealed that the structure of the bioactive fractions was almost identical to that of isocoumarin-type compounds [22].  (Table 2) as well as 1 H− 1 H COSY and HSQC, suggested the presence of 21 carbons, including three carbonyl carbons, six aromatic carbons, four methylenes, a methine, two methyls, and five carbons bonded to nitrogen or oxygen. These data revealed that compound 4 possessed structural similarities to amicoumacin C but differed from the molecular formula of amicoumacin C by the addition of -CH 2 [23]. This methylene group was identified by two proton signals observed at δ H 2.36, 2.23 in the 1 H NMR spectrum, an observation which was supported by the associated carbon resonating at δ C  was connected with C-10 (δ C 57.8) and C-12 (δ C 31.1). The assignment of the methylene group at C-12 was confirmed by the HMBC correlation from H-11 (δ H 2.22, m; 2.16, m) and H-12 (δ H 2.36, 2.23) to carbonyl carbon C-13 (δ C 181.5). The HMBC correlation of H-10 (δ H 3.88, m) and C-13 (δ C 181.5) suggested the presence of an amide bond between C-10 and C-12 ( Figure 2C). The relative configuration of compound 4 was determined by NOESY correlations of H-3/H-4 , H-5 /H-8 , and H-8 /H-10 , and shares stereochemical configurations similar to those of amicoumacin C. Thus, compound 4 was assigned as a new amicoumacin derivative from the spectroscopic data analysis, and was named amicoumacin D.
gested that C-11´ (δC 22.7) was connected with C-10´ (δC 57.8) and C-12´ (δC 31.1). The assignment of the methylene group at C-12´ was confirmed by the HMBC correlation from H-11´ (δH 2.22, m; 2.16, m) and H-12´ (δH 2.36, 2.23) to carbonyl carbon C-13´ (δC 181.5). The HMBC correlation of H-10´ (δH 3.88, m) and C-13´ (δC 181.5) suggested the presence of an amide bond between C-10´ and C-12´ ( Figure 2C). The relative configuration of compound 4 was determined by NOESY correlations of H-3/H-4´, H-5´/H-8´, and H-8´/H-10´, and shares stereochemical configurations similar to those of amicoumacin C. Thus, compound 4 was assigned as a new amicoumacin derivative from the spectroscopic data analysis, and was named amicoumacin D.   Figure 2B). The 1D NMR data ( 1 H and 13 C NMR, Table 2) and 2D NMR ( 1 H-1 H COSY and HSQC data) suggested the presence of 26 carbons, including four carbonyl carbons, six aromatic carbons, two methylenes, two methines, five methyls, and seven carbons bonded to nitrogen or oxygen. These data revealed that compound 5 possessed structural similarities to the 14 , 15 -methyl-15 -hydroxypyrrolidine amicoumacin C, but differed from the molecular formula of 14 , 15 -methyl-15 -hydroxypyrrolidine amicoumacin C by the addition of -COCH 3 [21]. This acetyl group was identified by a distinct singlet peak observed at δ H 2.05 in the 1 H NMR spectrum and a carbonyl carbon (δ C 172.1) in the 13 C NMR spectrum, which was supported by the HMBC correlation between H-17 (δ H 2.05, s) and carbonyl carbon C-16 (δ C 172.1). The 1  15 -methyl-15 -hydroxypyrrolidine amicoumacin C. Thus, compound 5 was also assigned as a new amicoumacin derivative, and was named amicoumacin E for the first time.

Direct Cloning and Heterologous Expression of NRPS/PKS Gene Cluster
To connect the NRPS/PKS gene cluster with amicoumacin biosynthesis, we direct cloned these DNA regions into E. coli plasmids using ExoCET and LLHR. Using the cloning vector p15A-cm-tet R -ccdB-hyg as the template, we designed a pair of primers carrying the terminal 62-bp homology arms of the NRPS/PKS gene cluster. The 5 and 3 homology arms were selected at the locations of the restriction enzymes site SpeI and PacI flanking the gene cluster. Using ExoCET, this biosynthetic gene cluster was successfully cloned into the cloning vector, and the efficiency 2/13 verified by restriction enzyme analysis ( Figure  S19A). The resulting recombinant plasmids containing the NRPS/PKS gene cluster were designated as p15A-cm-ami. Consistent with the close genetic relationship between the native host and the heterologous host, the NRPS/PKS gene cluster was under the control of its original promoters.
The recombinant plasmid p15A-cm-ami was transferred into E. coli GB05-MtaA by electroporation, and verified by restriction enzyme analysis, to obtain E. coli GB05-MtaAami. The correct transformants were subsequently fermented, and crude extracts were analyzed using HPLC-MS to detect the products. The resulting transformants successfully produced two amicoumacins-amicoumacin A (m/z 424.2006 [M + H] + ) and amicoumacin C (m/z 407.17 [M + H] + -at levels 100-fold less than that in the native B. subtilis fmb60 strain ( Figure 3A). This result indicated that the NRPS/PKS locus encodes amicoumacin biosynthesis. However, only two amicoumacins were obtained, and the biosynthetic pathways of all other amicoumacins require further investigation.

Direct Cloning and Heterologous Expression of NRPS/PKS Gene Cluster
To connect the NRPS/PKS gene cluster with amicoumacin biosynthesis, we direct cloned these DNA regions into E. coli plasmids using ExoCET and LLHR. Using the cloning vector p15A-cm-tet R -ccdB-hyg as the template, we designed a pair of primers carrying the terminal 62-bp homology arms of the NRPS/PKS gene cluster. The 5´ and 3´ homology arms were selected at the locations of the restriction enzymes site SpeI and PacI flanking the gene cluster. Using ExoCET, this biosynthetic gene cluster was successfully cloned into the cloning vector, and the efficiency 2/13 verified by restriction enzyme analysis (Figure S19A). The resulting recombinant plasmids containing the NRPS/PKS gene cluster were designated as p15A-cm-ami. Consistent with the close genetic relationship between the native host and the heterologous host, the NRPS/PKS gene cluster was under the control of its original promoters.
The recombinant plasmid p15A-cm-ami was transferred into E. coli GB05-MtaA by electroporation, and verified by restriction enzyme analysis, to obtain E. coli GB05-MtaAami. The correct transformants were subsequently fermented, and crude extracts were analyzed using HPLC-MS to detect the products. The resulting transformants successfully

Direct Cloning and Heterologous Expression of NRPS/PKS Gene Cluster and ace Gene Cluster
Using the cloning vector pBR322-apra-OriT and genomic DNA as the template, we designed two pairs of primers to amplify the pBR322-apra cloning vector and the ace gene cluster. The resulting recombinant plasmids containing the ace gene cluster were designated as pBR322-apra-ace ( Figure S19B). The recombinant plasmid pBR322-apra-ace was transferred into E. coli GB05-MtaA-ami by electroporation and verified by restriction en-

Direct Cloning and Heterologous Expression of NRPS/PKS Gene Cluster and ace Gene Cluster
Using the cloning vector pBR322-apra-OriT and genomic DNA as the template, we designed two pairs of primers to amplify the pBR322-apra cloning vector and the ace gene cluster. The resulting recombinant plasmids containing the ace gene cluster were designated as pBR322-apra-ace ( Figure S19B). The recombinant plasmid pBR322-apra-ace was transferred into E. coli GB05-MtaA-ami by electroporation and verified by restriction enzyme analysis, to obtain E. coli GB05-MtaA-ami-ace [26]. It was found that after adding the ace gene cluster, compared with the NRPS/PKS gene cluster alone, bacilosarcin A (m/z 492.20 [M + H] + ) and N-butanonyl-amicoumacin C (m/z 477.20 [M + H] + ) were identified ( Figure 3B). Bacilosarcin B and bacilosarcin A, N-butanonyl-amicoumacin C can be synthesized using the same substrate, indicating that the ace gene cluster plays a key role in the biosynthesis of amicoumacins.

Antimicrobial Bioassay of Lipoamides and Amicoumacins
The antimicrobial activities of lipoamides, amicoumacins, and erythromycin gluceptate were evaluated in vitro using a broth microdilution method. As shown in Table 3, lipoamides D-F, amicoumacin A, and bacilosarcin B produced significant inhibition against S. aureus ATCC25923, M. luteus CMCC 28001, B. pumilus CMCC 63202, B. cereus ATCC 14579, L. monocytogenes CICC 21662, and S. aureus MRSA, with MIC values ranging from 1.56 to 25 µg/mL. In particular, the MIC values against S. aureus MRSA were 6.25 µg/mL for amicoumacin A and 3.13 µg/mL for bacilosarcin B. However, amicoumacins D, E and other isolated compounds were found to be weakly active or inactive at 100 µg/mL.

Discussion
B. subtilis has significant metabolic capabilities and versatile biochemical mechanisms, because of its production of structurally diverse bioactive chemical structures [27,28]. Recent microarray-based comparative genomic analyses have revealed that members of this species also have diverse genomic properties [29]. There are about 700 Bacillus sp. genomes available on the NCBI website. Thus, the genomes presented here can guide the in-depth investigation of other B. subtilis metabolites. The genome sequence and analysis of B. subtilis fmb60 showed that a variety of clusters were involved in the production of bioactive compounds.
In the present work, five novel lipoamides and amicoumacins were identified from B. subtilis fmb60 by genome-directed isolation. Amicoumacins belong to a family of 3, 4-

Discussion
B. subtilis has significant metabolic capabilities and versatile biochemical mechanisms, because of its production of structurally diverse bioactive chemical structures [27,28]. Recent microarray-based comparative genomic analyses have revealed that members of this species also have diverse genomic properties [29]. There are about 700 Bacillus sp. genomes available on the NCBI website. Thus, the genomes presented here can guide the in-depth investigation of other B. subtilis metabolites. The genome sequence and analysis of B. subtilis fmb60 showed that a variety of clusters were involved in the production of bioactive compounds.
In the present work, five novel lipoamides and amicoumacins were identified from B. subtilis fmb60 by genome-directed isolation. Amicoumacins belong to a family of 3, 4-dihydroisocoumarin derivatives produced by the genus Bacillus, which have shown antibacterial, anti-inflammatory, and antiulcer activities and potent gastroprotective and antiulcerogenic activities [30,31]. To identify the genetic determinants of lipoamide and amicoumacins biosynthesis in B. subtilis fmb60, the NRPS/PKS gene cluster of B. subtilis fmb60 genome was submitted to BLAST. Our analysis indicated that the hybrid NRPS/PKS gene cluster was similar to that of the ami gene cluster synthase. ami was identified in earlier studies as the amicoumacin A biosynthetic gene cluster from B. subtilis 1779 [32]. Thus, the hybrid NRPS/PKS gene cluster could be plausibly assigned to the biosynthetic gene cluster for amicoumacins and lipoamides, which were isolated from B. subtilis fmb60.
Heterologous expression is a strategy for natural product discovery, and Red/ET recombineering has been widely used for direct cloning of biosynthetic pathways. The Ex-oCET method was used to direct clone the NRPS/PKS gene cluster of B. subtilis fmb60 and subsequently expressed it in the heterologous host E. coli GB05-MtaA. Two amicoumacins were obtained, which further verified that NRPS/PKS gene cluster is crucial for the biosynthesis of the amicoumacins.
However, the synthesis mechanism of other amicoumacins has not been reported yet. Previous reports indicated that this may in part be due to the possibility that many of the amicoumacin analogues discussed here may be isolated artifacts [4]. The formation mechanism of the 2-hydroxymorpholine moiety, an unusual cyclic structure in bacilosarcin, cannot be elucidated only by bioinformatics analysis [33]. Previous studies reported that chemical synthesis of bacilosarcins has been accomplished from amicoumacin C, using diastereoselective reductive amination of amicoumacin C with acetoin to form the corresponding N-butanonyl-amicoumacin C [21,34]. Therefore, the diastereomeric mixture of N-butanonyl-amicoumacin C is the core intermediate in the synthesis of bacilosarcin. In this study, N-butanonyl-amicoumacin C was identified in B. subtilis fmb60 fermentation broth for the first time. Acetoin is also an important physiological metabolic product excreted from B. subtilis [35]. Thus, it is presumed that bacilosarcins from B. subtilis are also synthesized using the aforementioned pathway under the catalysis of different enzymes. We used exonuclease combined with ExoCET technology to clone the NRPS/PKS and ace gene clusters into E. coli. We found that heterologous co-expression strains can synthesize bacilosarcin A and N-butanonyl-amicoumacin C, bacilosarcin B, bacilosarcin A, and Nbutanonyl-amicoumacin C from the same substrate, indicating that the ace gene cluster is related to the biosynthesis of bacilosarcins.
For amicoumacin D, Gln was recognized by the amiI domain of the NRPS/PKS gene cluster instead of Asn, resulting in the synthesis of bacillmacin A. The synthesis of Nacetylamicoumacin A-C from X. boviensis was mainly related to N-acetyltransferase, and B. subtilis also synthesized metabolites with a structure similar to that of N-acetylamicoumacin A-C. This study also assumed that N-acetyltransferase in B. subtilis may take part in the synthesis process [36]. However, focusing on the hybrid NRPS/PKS gene cluster of B. subtilis fmb60, no domain related to N-acetyltransferase synthesis was found near the genome. Therefore, it is presumed that the synthesis of N-acetylamicoumacin B and C in B. subtilis may be mediated by the isomerase or through post-modification. Furthermore, the biosynthetic pathways of amicoumacin E still need further investigation.

Bacterial Strains and Culture Conditions
B. subtilis fmb60 was isolated from the straw compost of Nanjing. B. subtilis fmb60 colonies and transferred to solid medium nutrient agar. B. subtilis fmb60 was cultured in beef peptone yeast medium at 37 • C for 16 h as seeds. One milliliter of seed culture was inoculated into 300 mL of Landy medium in a 1 L shake-flask and cultured in a shaking incubator (speed: 180 rpm) at 33 • C for 36 h.

Genome Sequencing, Annotation, and Bioinformatics Analysiszz
The genome of B. subtilis fmb60 was sequenced using the third-generation singlemolecule sequencing machine PacBio RS II (PacBio, Menlo Park, CA, USA) by Shanghai Hanyu Bio-Tech (Shanghai, China). Total genomic DNA was extracted using bacterial DNA kits (Omega Bio-Tek, USA). The extracted DNA was fragmented to 10 kb using Covaris ® g-TUBE ® (Covaris, Woburn, MA, USA). Subsequently, the DNA fragment library used as the template for sequencing was constructed using PacBio ® SMRTbell™ Template Prep Kits (PacBio, Menlo Park, CA, USA). After sequencing, the fragments were assembled to reconstruct the complete genome of B. subtilis fmb60. The bioinformatics program antiSMASH 5.2.0 (Kai Blin, Kgs. Lyngby, Denmark) was initially used to analyze the whole draft genome sequence [37].

Isolation and Purification of Lipoamides and Amicoumacins
To isolate the lipoamides from B. subtilis fmb60, the fermentation broth was centrifuged for 15 min at 10,000× g. The supernatant was collected and adjusted to pH 2.0 with HCl. After acid precipitation at 4 • C for 12 h, the supernatant was collected by centrifuging at 10,000× g for 15 min. Then, the supernatant was extracted twice with the same volume of ethyl acetate, and the solvent was removed under reduced pressure at 50 • C to dryness. The dry matter was dissolved in methanol and centrifuged to collect the supernatant containing the crude substances.
The crude substances generated using the method described above were processed using a semipreparative HPLC system (Waters 600, Milford, MA, USA) to collect fractions from the subfractions. One milliliter of crude extract solution was loaded and separated on a column (150 × 10 mm i.d., 5 µm, C18, Waters; flow rate: 6 mL/min; detection: 210 nm) from different subfractions using an isocratic program with acetonitrile−H 2 O (v/v) as the eluent. Finally, three novel compounds 1-3 were isolated, and ESI-HRMS analysis showed intense pseudo-molecular ions [M + H] + at m/z 329.2452, 315.2295, and 343.2604 for these three compounds.
To isolate the amicoumacins from B. subtilis fmb60, the acidified supernatant was adjusted to pH 7.0 with NaOH. The supernatant was then extracted with the same volume of ethyl acetate. The solvent was evaporated under reduced pressure at 50 • C to dryness. The dry matter was dissolved in methanol to collect the supernatant containing the crude substances. The crude extract was subjected to Sephadex LH-20 column chromatography (Φ2.6 × 100 cmi.d., Sigma, St. Louis, MO, USA) for further separation. The eluent was MeOH−H 2 O (4:1, v/v) and the flow rate was 12 mL/h. The eluent fraction was purified by Nexera X2 HPLC (SHIMADZU, Kyoto, Japan) with a gradient mobile phase (acetonitrile−H 2 O, from 50% to 60%; column: 150 × 10 mm i.d., 5 µm, C18, Waters; flow rate: 4 mL/min; detection: 250 nm) to yield compounds 4-16.

Determination of Amicoumacins by HPLC-MS
For liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis, an UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) equipped with a C18 column (5 µm, 4.6 × 250 mm i.d., Agilent Technologies, Palo Alto, CA, USA) was used. The spray voltage was set to 4.0 kV, and the heated transfer capillary temperature was set to 350 • C. High-resolution mass spectrometry analysis was performed with a Thermo Finnigan Surveyor-LCQ DECA XP Plus (Thermo Electron Corporation, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source.

NMR Spectroscopy Identification
The structures were identified by nuclear magnetic resonance (NMR) spectroscopy, and the samples were carried out in methanol-d 4 on Avance 600, 500, and 300 MHz Bruker NMR spectrometer (Bruker, Faellanden, Switzerland).

B. subtilis fmb60 Genomic DNA Isolation
Genomic DNA was isolated from lysed cells by phenol-chloroform extraction and ethanol precipitation [26]. B. subtilis fmb60 was cultured in 50 mL of medium LB at 30 • C overnight. After centrifugation at 8300× g for 5 min, the cells were washed twice with ddH 2 O, and then resuspended in 8 mL of SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0). After adding 10 mg lysozyme and incubating at 37 • C for 1-2 h in a water bath with occasional inverting, 500 µL of proteinase K (20 mg/mL) and 1 mL of 10% sodium dodecyl sulfate (SDS) were added, and the mixture was incubated at 50 • C with occasional inversion for 2 h until the solution became clear. Then, 3.5 mL of 5 M NaCl was added with inverting, after which 15 mL of phenol/chloroform/isoamyl alcohol (25:24:1) was added and mixed thoroughly by inversion to create an emulsion. After centrifugation at 8300× g for 30 min, 500 µL of the aqueous phase was transferred to a new 2 mL tube using an end-cut wide-bore 1 mL tip, then 35 µL of 3 M sodium acetate (pH 7.5) and 1.2 mL of absolute ethanol were added, and gently inverted to mix. The DNA was transferred to a 1.5 mL tube using a blue tip, rinsed with 75% ethanol, dried at room temperature (RT) for approximately 30 min, and dissolved with 200 µL ddH 2 O. Genomic DNA was prepared.

Direct Cloning of the NRPS/PKS and ace Gene Clusters
E. coli were cultivated and manipulated according to standard protocols. The strains and plasmids used in this study are listed in Table S1. Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Restriction enzymes were purchased from New England Biolabs Ltd. (Beijing, China), DNA polymerase (PrimerSTAR and T4), and DNA marker were purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China).
The genomic DNA was completely digested with the restriction enzymes PacI and SpeI to release the NRPS/PKS gene cluster. The linear cloning vector p15A-cm flanked with homology arms to target genes was amplified by PCR using p15A-cm-tet R -ccdBhyg as template, and primers (YM-ami-5 and YM-ami-3, Table S2) carrying the terminal homology arms to the target gene cluster. A direct cloning protocol was carried out according to our previous publication [26]. Recombinants carrying the entire gene cluster were selected by chloramphenicol resistance on the plate (the final concentration was 15 µg/mL) and subsequently verified by restriction enzyme analysis and sequencing, to obtain correct recombinants.
The ace gene cluster was amplified by PCR using primers ace-F and ace-R carrying the terminal homology arms to the pBR322 cloning vector. The linear cloning vector pBR322-apra flanked with homology arms to the ace gene cluster was amplified by PCR using pBR322-apra-OriT as template and primers (ace-pBR322-HAF and ace-pBR322-HAF, Table S2) carrying the terminal homology arms to the ace gene cluster. These two fragments were recombined using RecET recombinase in strain E. coli GB05-dir. The protocol was carried out according to our previous publication [14]. Recombinants carrying the entire gene cluster were selected by apramycin resistance on the plate (the final concentration was 50 µg/mL) and subsequently verified by restriction enzyme analysis and sequencing, to obtain correct recombinants.

Heterologous Expression of NRPS/PKS and ace Gene Clusters in E. coli
The recombinant plasmid p15A-cm-ami was transferred into E. coli GB05-MtaA by electroporation and verified by restriction enzyme analysis to obtain E. coli GB05-MtaAami [27]. The plasmid pBR322-apra-ace was transferred into E. coli GB05-MtaA-ami by electroporation to obtain recombinant E. coli GB05-MtaA-ami-ace containing the NRPS/PKS and ace gene clusters.
The correct transformants were subsequently fermented. The fermentation method for B. subtilis fmb60 is described above, while E. coli GB05-MtaA-ami and E. coli GB05-MtaA-ami-ace were cultured in beef peptone yeast medium at 37 • C overnight as seeds. One milliliter of seed culture was inoculated into a 250 mL shake-flask containing 50 mL of Landy medium with agitation at 200 rpm at 30 • C for 3 d. The extraction method is described above. The crude extract was obtained using HPLC-MS, which was carried out on a Thermo Scientific UltiMate 3000 HPLC system connected to a Bruker ESI-MS-MS Impact HD operating in positive ionization mode at a scan range of m/z 200-2000, auto MS 2 fragmentation. Reverse-phase chromatography of HPLC was carried out with 2.1 × 100 mm, 2.2 µm columns (PSLC 120 C18) in a solvent gradient, with solvents A (water and 0.1% formic acid) and B (CH3CN and 0.1% formic acid) for 25 min: 5% B from 0 to 3 min, 5% B-95% B within 15 min, followed by 4 min with 95% B and 3 min with 5% B at a flow rate of 0.3 mL/min. MS measurements were carried out using a standard ESI source.

Minimal Inhibitory Concentration (MIC) Assays and the Effects of Antimicrobials on Bacterial Morphology
The MICs of the amicoumacins were determined using a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [38]. The indicator bacteria in this assay were incubated in Mueller-Hinton broth (MHB) and normalized to an optical density of 10 6 CFU/mL. The amicoumacins were two-fold serially diluted in 96-well plates of MHB, with 50 µL in each well and concentrations ranging from 0.78 to 100 µg/mL. Then, 50 µL of bacterial suspension was added to each well, with a final inoculum density of approximately 5 × 10 5 CFU/mL, and the plates were incubated statically at 37 • C for 24 h [39]. Quality control and interpretations of results were performed according to CLSI guidelines [40]. The MICs of the amicoumacins were defined as the lowest concentration of amicoumacins, where no visible growth was observed in the wells of the microtiter plates. All assays were performed in triplicate.

Assay of ABTS + Radical Scavenging and Ferric-Reducing Activity
The ferric-reducing ability power (FRAP) and ABTS + radical scavenging activities of the amicoumacins were measured using commercial kits (Jiancheng Bio-engineering Institute, Nanjing, China). Trolox was used as a positive control.

Data Processing
SPSS 17.0 software was used for the experimental data processing. Antibacterial and antioxidant experiments were repeated three times, and the Duncan test was used for significance analysis (p < 0.05). All of the forms were made using Excel 2010.

Conclusions
In conclusion, five new metabolites, lipoamides D-F (1-3), amicoumacins D, E (4, 5), and known metabolites (6−16), have been identified from B. subtilis fmb60. Lipoamides D-F inhibited the growth of foodborne harmful bacteria, and amicoumacins D and E have antioxidant activities. The heterologous expression results demonstrated the function of NRPS/PKS in B. subtilis fmb60, and showed that the ace gene cluster can participate in the synthesis of amicoumacins.
Supplementary Materials: The following are available online. Table S1. Strains and plasmids in this study. Table S2. Primers used in this study. Table S3. Secondary metabolite biosynthetic gene clusters of B. subtilis fmb60. Table S4. NRPS/PKS gene clusters of B. subtilis fmb60. Figures S1-S18. 1D and 2D NMR spectra of lipoamide D, 1, amicoumacin D, 4, amicoumacin E, 5 in methanol-d 4 . Figure S19. Digestion map of NRPS/PKS and the ace gene cluster.
Author Contributions: J.Y. performed the experiments, analyzed the data, and wrote the manuscript; Q.Z., F.X., and M.Y. analyzed the data and wrote the manuscript; H.D. and X.B. analyzed and discussed the data; Z.L. provided samples and discussed the data; Y.L. and F.L. designed the research content, analyzed the data, and modified the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The authors declare that all data generated or analyzed during this study are included in this published article.