The Discovery of Cyclic Lipopeptide Olenamidonins in a Deepsea-Derived Streptomyces Strain by Knocking Out a DtxR Family Regulator

Three new cyclic lipopeptides, olenamidonins A-C (1–3), in addition to two previously reported metabolites (4 and 5), were accumulated in the ΔdtxRso deletion mutant of deepsea-derived Streptomyces olivaceus SCSIO 1071. The structures of these cyclic lipopeptides were determined by a combination of spectroscopic methods and circular dichroism (CD) measurement. The antibacterial assay results showed that compounds 1–5 displayed different degrees of growth inhibition against multidrug-resistant (MDR) bacterial strains Enterococcus faecalis CCARM 5172 and Enterococcus faecium CCARM 5203 with minimum inhibitory concentrations (MICs) of 1.56−6.25 μg/mL.


Introduction
Microbial genome sequencing has unearthed uncountable cryptic secondary metabolite biosynthetic gene clusters (BGCs) in Streptomyces.However, most of them are silent under normal laboratory conditions [1].Given that secondary metabolism is precisely controlled by a complex regulatory cascade network [2], the inactivation/overexpression of regulatory genes has been proven to be an effective way to unlock silent gene clusters [3].
DtxR (diphtheria toxin repressor) is a metal-dependent transcriptional regulator.DtxR family regulators, which were first identified in Corynebacterium diphtheriae [4], widely exist in high-GC content Gram-positive bacteria.They were found to be involved in the regulation of iron homeostasis, secondary metabolism, and morphological differentiation [5][6][7].For example, the deletion of a DtxR family regulator gene dmdR1 in Streptomyces coelicolor A3(2) led to a slow rate of spore formation and the loss of pigmented antibiotics undecylprodigiosin and actinorhodin production [8,9].The DtxR family regulator IdeR from Streptomyces avermitilis could regulate the production of avermectin and oligomycin in a positive and negative way, respectively; additionally, its deletion resulted in a bald phenotype and delayed morphological differentiation in S. avermitilis [5].
In our efforts to discover novel natural products from deepsea-derived Streptomyces strains, pleiotropic regulators were set as target genes for genetic manipulation to activate silent BGCs.A DtxR family regulatory gene dtxR so was identified from the genome of deepsea-derived Streptomyces olivaceus SCSIO 1071.The inactivation of dtxR so led to the activation of cyclic lipopeptide compound olenamidonins, including three new (1)(2)(3) and two known (4 and 5) compounds (Figure 1A).The related structural analogs of olenamidonins, enamidonins, were reported to have a variety of bioactivities, including macrophage foam two known (4 and 5) compounds (Figure 1A).The related structural analogs of ole namidonins, enamidonins, were reported to have a variety of bioactivities, including mac rophage foam cell formation inhibition [10], acyl-CoA/cholesterol acyltransferase (ACAT inhibition [11], and anti-Gram-positive bacteria activities [12].Herein, we describe the iso lation, structural identification, and antibacterial activity evaluation of these lipopeptides

Results
The deepsea-derived S. olivaceus SCSIO 1071 was taxonomically classified through the phylogenetic analysis of its 16S rRNA gene sequence (Table S1, Figure S1) against the EzTaxon-e server Database.DtxRso (Table S2) from S. olivaceus SCSIO 1071 showed 58.3% identity to DtxR (WP_010935052.1) from C. diphtheriae and is highly homologous to IdeR (BAC71567.1)from S. avermitilis (96.0%identity) and DmdR1 (CAC28070.1)from S. coeli color (98.0%identity).While the deletion of dtxRso had no obvious impact on the growth and morphological differentiation , a series of new peaks were accumulated in the fer mentation products of the ΔdtxRso mutant (Figure 1B, panel i) compared to those of the wild-type strain (Figure 1B, panel ii).

Results
The deepsea-derived S. olivaceus SCSIO 1071 was taxonomically classified through the phylogenetic analysis of its 16S rRNA gene sequence (Table S1, Figure S1) against the EzTaxon-e server Database.DtxR so (Table S2) from S. olivaceus SCSIO 1071 showed 58.3% identity to DtxR (WP_010935052.1) from C. diphtheriae and is highly homologous to IdeR (BAC71567.1)from S. avermitilis (96.0%identity) and DmdR1 (CAC28070.1)from S. coelicolor (98.0%identity).While the deletion of dtxR so had no obvious impact on the growth and morphological differentiation, a series of new peaks were accumulated in the fermentation products of the ∆dtxR so mutant (Figure 1B, panel i) compared to those of the wild-type strain (Figure 1B, panel ii).
To elucidate the chemical structures of the activated compounds, a total volume of 35 L fermentation cultures of S. olivaceus SCSIO 1071∆dtxR so was obtained, from which five compounds (1-5) were isolated.Their structures were identified via high-resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR) assignments.Compound 1 was isolated as a colorless oil.The molecular formula of 1 was determined to be C 34 H 51 N 7 O 6 according to the HRESIMS data ([M + H] + at m/z 654.3994, calcd 654.3979, Figure S6).The 1D and 2D NMR (HSQC, COSY, and HMBC) data showed the presence of amino acids phenylalanine (Phe), glycine (Gly), 2,3-diaminopropionic acid (Dpr), and a fatty acid moiety (Figures 2 and S7-S12) [8].HMBC correlations from H 2 -16 (∆ H 3.07, 2.78) to C6 (∆ C 171.1), C-17 (∆ C 137.3), and C-18/22 (∆ C 128.8), from H-20 (∆ H 7.17  The advanced Marfey's method [12] was used to determine the absolute configurations of the Phe and Dpr residues, and the result suggested the peptide backbone of compound 1 to be L-Phe-D-Dpr-L-Dpr-Gly (Figures S2 and S3).While the backbone was the same as those reported for autucedines and enamidonins [12,13], compound 1 contained a different 8 ′ -methyldecanoyl side-chain.Therefore, compound 1 was identified as a new cyclic lipopeptide, named olenamidonin A. The 1 H and 13 C chemical shifts of 1 are listed in Table 1. ).The advanced Marfey's method [12] was used to determine the absolute configurations of the Phe and Dpr residues, and the result suggested the peptide backbone of compound 1 to be L-Phe-D-Dpr-L- Dpr-Gly (Figures S2 and S3).While the backbone was the same as those reported for autucedines and enamidonins [12,13], compound 1 contained a different 8′-methyldecanoyl side-chain.Therefore, compound 1 was identified as a new cyclic lipopeptide, named olenamidonin A. The 1 H and 13 C chemical shifts of 1 are listed in Table 1.Compound 2 was isolated as a colorless oil.The molecular formula of 2 was determined to be C34H51N7O6 according to the HRESIMS data ([M + H] + at m/z 654.3991, calcd 654.3979, Figure S13), which was the same as 1.The NMR data showed that the backbone   S13), which was the same as 1.The NMR data showed that the backbone of 2 was the same as that of 1 (Figures 2 and S14-S17).While 2 also contained a C 11 fatty acyl chain, the COSY correlations of H 2 -8 ′ (∆ H 1.49)/H-9 ′ (∆ H 1.49)/H 3 -10 ′ /11 ′ (∆ H 0.84) combined with the HMBC correlations from H 2 -8 ′ (∆ H 1.13) to C-10 ′ ′ (∆ C 22.5) showed that the methyl group was attached to C-9 ′ (∆ C 27.3) instead of C-8 ′ (∆ C 33.7) compared with 1.The CD spectra of 2 and 1 in MeOH display similar Cotton effects, supporting that 2 shares the same absolute configurations as 1 (Figure S4).Thus, compound 2 was identified as a new olenamidonin analog, named olenamidonin B. The 1 H and 13 C chemical shifts of 2 are listed in Table 1.
Compound 3 was isolated as a colorless oil.The molecular formula of 3 was determined to be C 32 H 47 N 7 O 6 according to the HRESIMS data ([M + H] + at m/z 626.3675, calcd 626.3666, Figure S18), which had two less methyl groups than 1.The NMR data showed that the backbone of 3 was the same as 1 (Figures 2 and S19-S23).Eight carbons of the C 9 fatty acyl chain were identified by the COSY correlations, and two double-peak methyl signals with their HMBC correlations to C-7 ′ (∆ C 27.3) confirmed that the methyl group was attached to C-7 ′ (∆ C 27.3).The CD spectra of 3 and 1 in MeOH display similar Cotton effects, supporting that 3 shares the same absolute configurations as 1 (Figure S4).Compound 3 contained a different 7 ′ -methyloctanoyl side-chain.Thus, compound 3 was identified as a new olenamidonin analog, named olenamidonin C. The 1 H and 13   S31), respectively.By comparing the NMR data (Figures 2, S25-S30 and S32-S37) with those reported in the literature, 4 and 5 were respectively confirmed to be known compounds autucedine A and autucedine D previously isolated from Streptomyces olivaceus SCSIO T05 [13], which is the same species as that of 1071 but is a different strain.The 1 H and 13 C NMR data for 4 and 5 are listed in Table S3.
We further analyzed the olenamidonins' BGC (Table S4) from 1071 and found that it is identical to the aut BGC from T05 (GenBank ID: WP_194276129.1-WP_194276149.1)[13] except a couple of nucleotides in between open reading frames.Based on the previously proposed biosynthetic pathway of autucedines [13], the 3-oxoacyl-ACP synthase OleH, the homolog of AutS, may display flexible substrate specificity, ligating different acyl-CoAs to DHA-ACP to finally generate lipopeptides with different lipid chains.While a molecular network analysis of autucedine analogs showed that AutS was able to use saturated/unsaturated lipochains with a length of C7 to C13, these compounds were not structurally characterized except autucedines A-E [13].
The antibacterial activities of compounds 1-5 were evaluated against three Grampositive (Micrococcus luteus ML01, Enterococcus faecalis CCARM 5172, and Enterococcus faecium CCARM 5203) and two Gram-negative (Escherichia coli CCARM 1009 and Pseudomonas aeruginosa 15690) multidrug-resistant (MDR) bacterial strains.As shown in Table 2, while no antibacterial activities toward M. luteus ML01, E. coli CCARM 1009, or P. aeruginosa 15690 were detected for all the compounds, considerable growth inhibitions against E. faecalis CCARM 5172 and E. faecium CCARM 5203 were observed.Noticeably, compounds 1-2 and 5 exhibited stronger anti-E.faecalis CCARM 5172 activities (MICs = 1.56−3.12µg/mL) than the positive control ciprofloxacin (MIC = 6.25 µg/mL), while 3-4 displayed comparable inhibition activities to that of ciprofloxacin.Compounds 1-2 and 5 also showed similar anti-E.faecium CCARM 5203 activities to that of the positive control tetracycline (MIC = 1.56 µg/mL), which were stronger than those of 3-4 (MIC = 3.12-6.25µg/mL).The comparison of the structures and antibacterial activities of these compounds showed lipopeptides with C 11 -and C 10 -fatty acyl chains (1-2, 5) have better inhibition potentials against E. faecalis CCARM 5172 and E. faecium CCARM 5203 than C 9 -and C 8 -fatty acyl chain analogs (3)(4), suggesting that the length of the fatty acyl chain probably plays an important role in antibacterial activities.We further analyzed the distribution of the DtxR so homologs across diverse bacteria.A sequence similarity network (SSN) analysis of 2375 DtxR so homologs from the UniProt database was performed.As shown in Figure 3, these homologs mostly exist in Actinomycetes (in red), including families of Microbacteriaceae, Streptosporangiaceae, Nocardiaceae, Micrococcaceae, and Corynebacteriaceae.Noticeably, the Acidimicrobiia clade (in green) mainly has three paralogous groups, which are respectively associated with homolog(s) belonging to the Actinomycetes clade, suggesting their close relationship during evolution.Additionally, DtxR family regulators from Magnoliopsida (in orange) and Myxococcia (in dark green) are clustered with DtxR so /IdeR in the Actinomycetes clade, suggesting a possible horizontal transfer of the DtxR so homologs across the bacterial world.Considering the massive cryptic secondary metabolite BGCs and the wide distribution of DtxR family regulators in Actinomycetes, the genetic manipulation of these regulators would be an alternative approach to unlock silent or cryptic gene clusters.We further analyzed the distribution of the DtxRso homologs across diverse bacteria.A sequence similarity network (SSN) analysis of 2375 DtxRso homologs from the UniProt database was performed.As shown in Figure 3, these homologs mostly exist in Actinomycetes (in red), including families of Microbacteriaceae, Streptosporangiaceae, Nocardiaceae, Micrococcaceae, and Corynebacteriaceae.Noticeably, the Acidimicrobiia clade (in green) mainly has three paralogous groups, which are respectively associated with homolog(s) belonging to the Actinomycetes clade, suggesting their close relationship during evolution.Additionally, DtxR family regulators from Magnoliopsida (in orange) and Myxococcia (in dark green) are clustered with DtxRso/IdeR in the Actinomycetes clade, suggesting a possible horizontal transfer of the DtxRso homologs across the bacterial world.Considering the massive cryptic secondary metabolite BGCs and the wide distribution of DtxR family regulators in Actinomycetes, the genetic manipulation of these regulators would be an alternative approach to unlock silent or cryptic gene clusters.

Conclusions
In this study, three new compounds, olenamidonins A-C (1-3), and two known compounds (4 and 5) were obtained from the ∆dtxR so mutant of deepsea-derived S. olivaceus SCSIO 1071.Compounds 1-5 showed different degrees of growth inhibitory activities against E. faecalis CCARM 5172 and E. faecium CCARM 5203.The comparison of their structures and antibacterial activities suggested that the length of the fatty acyl chain probably plays an important role in antibacterial activities.Feeding lipids with longer chains may contribute to the generation of olenamidonin derivatives with better activities.The activation of olenamidonin production by knocking out dtxR so is very intriguing, and the underlying regulatory mechanism is worthy of further investigation.Changing the fermentation conditions of ∆dtxR so could serve to investigate the impacts of the dtxR so deletion on the productions of other metabolites.Given the wide distribution of DtxR so homologs, they could serve as alternative targets for the activation of silent BGCs in actinomycetes strains.

General Materials and Methods
Bacterial strains and plasmids used in this study are listed in Table S1; primers are listed in Table S2.Streptomyces olivaceus SCSIO 1071 was isolated from a deepsea mud sample from the South China Sea.The NMR spectra were recorded on a Bruker Avance III 600.Chemical shifts were referenced to the residual DMSO-d 6 signal (∆ H 2.50 and ∆ C 39.5 ppm for DMSO-d 6 ).HRESIMS data were obtained on a Q-TOF Ultima Global GAA076 MS spectrometer.DNA isolation was carried out according to established protocols [15].Plasmid was extracted using commercial kits (OMEGA).
The EFI-Enzyme Similarity Tool (EFI-EST) was used to generate sequence similarity networks (SSNs) [16].For the distribution of DtxR so homologs, the DtxR so sequence was used as the query for searching a nonredundant protein sequence database using PSI-BLAST (position-specific iterated BLAST).A total of 2316 DtxR so homologs were used to build the SSNs.The 10 −80 SSN was generated by applying an E value cutoff of 10 −80 to the full network.Each node in the network represents a single sequence, and each edge represents the pairwise connection between two sequences for which the BLASTP E value was lower than the cutoff value.SSNs were visualized and colored by Cytoscape (v3.7.2) [14].

The Construction of the dtxR so Mutant Strain
A PCR-targeting strategy was adopted to obtain the dtxR so mutant strain [17,18].Briefly, the amplified aac(3)IV-oriT resistance gene from pIJ773 was transformed into E. coli BW25113/pIJ790/pWLI615 to replace the dtxR so gene, resulting in the mutant cosmid pWLI1002 (∆dtxR so ).The mutant cosmid was passed through E. coli ET12567/pUZ8002 and then introduced into S. olivaceus SCSIO 1071 via conjugation [19].The dtxR so mutant strain was selected through apramycin-resistant and neomycin-sensitive phenotype screening, followed by PCR confirmation.

Production and Purification of Olenamidonins
The fermentation of S. olivaceus SCSIO 1071/pWLI1002 in a total volume of 35 L was performed by growing cultures in 250 mL baffled Erlenmeyer flasks each containing 50 mL of AF/MS medium (glucose 20 g, yeast extract 2 g, soya flour 8 g, CaCO 3 4 g, NaCl 1 g, distilled H 2 O 1000 mL, pH = 7.3), incubated at 30 • C on a rotary shaker at 220 rpm for 7 days.The cells were extracted with acetone by sonication.The combined organic extracts were concentrated and then partitioned between 90% MeOH and n-hexane to yield two residues.The aqueous MeOH layer (1.05 g) was subjected to a reversed-phase (C18) open column chromatography with 20−100% MeOH to afford 6 fractions.Compounds 1-5 were obtained by a further purification of fraction 4 on reversed-phase HPLC (YMC-Pack ODS-A column 250 mm × 10 mm, i.d. 5 µm; wavelength at 260 nm) eluting with 49% CH 3 CN + 0.1% HCOOH (v/v) (1.5 mL/min). Olenamidonin

Antibacterial Assay
The MDR bacterial strains M. luteus ML01, E. faecalis CCARM 5172, E. faecium CCARM 5203, E. coli CCARM 1009, and P. aeruginosa 15690 were grown overnight at 37 • C in liquid LB medium, then diluted with the LB broth to 10 6 CFU/mL.The sample solutions were diluted with MeOH to make a series of concentrations.After that, 20 µL of the sample solutions with different concentrations was dispensed into 180 µL of the bacterial suspension in 96-well plates and incubated at 37 • C for 18 h.The growth of MDR strains was measured on a microplate reader at a wavelength of 620 nm.Tetracycline (for M. luteus ML01, E. faecium CCARM 5203, E. coli CCARM 1009, and P. aeruginosa 15690) or ciprofloxacin (for E. faecalis CCARM 5172) was used as a positive control, methanol was used as a negative control, and LB broth was used as a blank.

Figure 3 .
Figure 3. Distribution of DtxRso homologs in bacteria.SSN analysis of DtxRso homologs in diverse bacteria is based on cutoff E value of 10 −85 and visualized by Cytoscape [14].

Figure 3 .
Figure 3. Distribution of DtxR so homologs in bacteria.SSN analysis of DtxR so homologs in diverse bacteria is based on cutoff E value of 10 −85 and visualized by Cytoscape [14].

Author
Contributions: Q.S. performed chemical and bioactive experiments, analyzed the data, and wrote the manuscript.D.Y. measured the NMR spectra.X.Z.formal analysis.F.X. and W.L. designed the experiments and revised the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by grants from the Shandong Provincial Natural Science Foundation (ZR2021ZD28), the National Natural Science Foundation of China (32070054, U22A20582, 81991525 and 32100051), and the Fundamental Research Funds for the Central Universities (202172002).
C chemical shifts of 3 are listed in Table 1.Compounds 4 and 5 were isolated as a colorless oil.The molecular formulas of 4 and 5 were determined to be C 31 H 45 N 7 O 6 ([M + H] + at m/z 612.3522, calcd 612.3510, Figure S24) and C 33 H 49 N 7 O 6 ([M + H] + at m/z 640.3840, calcd 640.3823, Figure