Genome mining and heterologous expression 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 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 by non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid The transfer of BGCs from the original host into a feasible heterologous host, resulting in heterologous expression, has become a valid alternative approach for identifying the gene clusters required for the biosynthesis of compounds. In 2012, we described a method termed linear plus linear homologous recombination (LLHR) to directly clone large BGCs from digested genomic DNA in Escherichia coli. LLHR is mediated by the prophage recombinase RecET in E. coli [14]. In 2016 an improved method, exonuclease combined with RecET recombination, was described [15]. 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 -CH2 (C 30.4, 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 as lipoamide E.
The structure of compound 3 was investigated using NMR spectroscopy. The 1DNMR 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 (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 as 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   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 twenty-six 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 -COCH3 [24].
This acetyl group was identified by a distinct singlet peak observed at H 2.05 in the 1 H NMR spectrum and a carbonyl carbon (C 172.1) in the 13  Compound 6 was identified as a new natural amicoumacin, which is referred to herein as N-butanonyl-amicoumacin C.

Direct Cloning and Heterologous Expression of NRPS/PKS Gene Cluster
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 [29,30]. 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 [31]. 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 ExoCET 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.
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 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 and ace Gene Cluster
Early reports indicated that this may in part be due to the possibility that many of the amicoumacin analogues discussed here may be isolation artifacts. 4 The formation mechanism of the 2-hydroxymorpholine moiety, an unusual cyclic structure in bacilosarcin, cannot be elucidated by bioinformatics analysis alone [32]. Earlier 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 [24, 33]. 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 bacilosarcins. 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)

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

Genome Sequencing, Annotation, and Bioinformatics Analysis
The genome of B. subtilis fmb60 was sequenced using the third-generation single-

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) to collect fractions from the subfractions.   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. 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