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Isolation, Structure Elucidation, and Biosynthesis of a CysteateContaining Nonribosomal Peptide in Streptomyces lincolnensis Min Wang,† Dandan Chen,†,§ Qunfei Zhao,†,‡ and Wen Liu*,†,‡,§ †

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State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Sciences, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China ‡ State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China § Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China S Supporting Information *

ABSTRACT: The species Streptomyces lincolnensis is known as a producer of lincomycin A, a clinically important lincosamide antibiotic with activity against Gram-positive bacteria. Here, we report that S. lincolnensis produces a new cysteate-containing lactone product, cysteoamide (1), which arises from nonribosomal peptide synthetase-programmed sequential assembly of the monomers phenylacetic acid, valine, cysteate, threonine, β-hydroxyleucine, and β-alanine and subsequent intramolecular cyclization to form a lactone ring. The structure of 1 was determined by combined analysis of NMR and MS spectra, while the amino acid absolute configurations in 1 were assigned by Marfey’s analysis following acid hydrolysis. The biosynthetic gene cluster of 1 was defined in the genome of S. lincolnensis by bioinformatics analysis and in vivo genetic study. In addition, in vitro assay revealed that OrfA, a pyridoxal 5′-phosphate-dependent protein, is responsible for the formation of the unusual cysteate unit. Cysteate-containing nonribosomal peptides appear to be widely present in various Streptomyces strains, and this study generates interest in their intrinsic functions that remain poorly understood.

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INTRODUCTION Natural products have been well-known as a valuable source of new drugs. They have played a particularly vital role in battling infectious and tumor-related diseases, as the majority of related small molecule based chemotherapeutics in clinical use are natural products, their derivatives, or chemically synthesized mimics inspired by pharmaceutically important moieties arising from natural products.1 The strain Streptomyces lincolnensis is known as a producer of lincomycins, a group of lincosamide antibiotics among which the A component has been widely used to treat Gram-positive bacterial infections.2 In addition to an investigation of the biosynthesis of these antibiotics,3−5 we are interested in the chemical profile of S. lincolnensis for the following reasons: (1) traditionally, the Streptomyces genus has been a rich source of various bioactive natural products;6 (2) although the strain S. lincolnensis was discovered more than half a century ago, no small molecules other than lincomycin-related products have been isolated from this bacteria;7 and (3) a preliminary bioinformatics analysis of its genome using antiSMASH8 showed ∼30 biosynthetic gene clusters (BGCs) coding for a wide variety of small molecules, including terpenes, ribosomally encoded and posttranslationally modified peptides (RiPPs), nonribosomal peptides (NRPs), polyketides (PKs), and their hybrids thereof (Figure S1). Here we report that S. lincolnensis © 2018 American Chemical Society

produces a new cysteate-containing product, cysteoamide (1, Figure 1), which arises from NRP synthetase (NRPS)programmed assembly using six different amino acid monomers.

Figure 1. Structure of compound 1. The unusual monomer D-cysteate in this NRP was highlighted in red. Special Issue: Synthesis of Antibiotics and Related Molecules Received: January 8, 2018 Published: March 20, 2018 7102

DOI: 10.1021/acs.joc.8b00044 J. Org. Chem. 2018, 83, 7102−7108

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The Journal of Organic Chemistry

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RESULTS AND DISCUSSION Isolation and Structural Elucidation of 1. S. lincolnensis was fermented under published conditions for lincomycin production.3 HPLC−MS analysis of the resulting fermentation broth revealed the production of 1 as a minor product, which was then scaled-up for structural elucidation. The molecular formula of 1 was determined by HR-ESI-MS as C29H43N5O11S ([M + H]+, m/z 670.2761, calcd for C29H44N5O11S+ 670.2578) (Figure S2A). The 1H NMR spectrum of 1 in DMSO-d6 displayed featured signals of a peptide backbone, which included four α-proton signals at δ 3.97 (1H, t, J = 6.8 Hz), 4.36 (1H, dd, J = 2.1, 9.6 Hz), 4.44 (1H, q, J = 5.5 Hz), and 4.11 (1H, dd, J = 6.2, 7.9 Hz) as well as five methyl signals at δ 0.83 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J = 6.4 Hz), 1.06 (3H, d, J = 6.6 Hz), 0.89 (3H, d, J = 6.4 Hz), and 0.86 (3H, d, J = 6.8 Hz) (Figure S3 and Table 1). Furthermore, in the downfield 1H NMR spectrum we observed a pair of multiplets at δ 7.19 (1H,

m) and 7.25 (4H, m), suggesting the presence of a monosubstituted phenyl group. The HSQC spectrum showed the signals of two methines (δC‑21 29.4, δH‑21 1.73 and δC‑27 30.1, δH‑27 2.13), two oxygenated methines (δC‑20 73.2, δH‑20 3.52 and δC‑24 71.3, δH‑24 5.50), and four methylene groups (δC‑2 33.7, δH‑2 2.38/2.16; δC‑3 36.1, δH‑3 3.41/2.88; δC‑13 42.1, δH‑13 3.57/3.53 and δC‑26 50.2, δH‑26 3.05/2.93) (Figures S4 and S5 and Table 1). Further analysis of the HSQC, HMBC, and COSY spectra revealed that the structure of 1 was composed of a phenylacetic acid (Paa) residue, a valine (Val) residue, a cysteate (Cya) residue, a threonine (Thr) residue, a βhydroxyleucine (β-OHleu) residue, and a β-alanine (β-Ala) residue (Figures S6−S9). The complete residue sequence was established as Paa-Val-Cya-Thr-β-OHleu-β-Ala through the HMBC couplings from α-protons and/or α-NH to carbonyl carbons of adjacent residues (Figure 2). Moreover, the Thr and

Table 1. NMR Spectroscopic Data for 1a position

δC, typeb

1 2

169.9, C 33.7, CH2

3

36.1, CH2

3-NH 4 5 5-NH 6 7 7-NH 8 9 9-NH 10 11 11-NH 12 13 14 15, 19 16, 18 17 20 20-OH 21 22 23 24 25 26 27 28 29

170.5, C 60.8, CH 170.7, C 56.8, CH 170.7, C 51.5, CH 171.1, C 58.4, CH 171.1, C 42.1, CH2 136.3, 129.0, 128.1, 126.2, 73.2,

C CH CH CH CH

29.4, 20.0, 16.5, 71.3, 16.1, 50.2,

CH CH3 CH3 CH CH3 CH2

30.1, CH 19.6, CH3 18.1, CH3

δH (J, Hz) 2.38, 2.16, 3.41, 2.88, 7.35,

dt (5.3, 12.9) m m dd (3.2, 12.8) d (8.0)

3.97, t (6.8) 8.00, d (7.0) 4.36, dd (2.1, 9.6) 8.09, d (9.6) 4.44, q (5.5) 8.77, d (5.5) 4.11, dd (6.2, 7.9) 8.41, d (8.1) 3.57, d (14.2) 3.53, d (14.2) 7.25, 7.25, 7.19, 3.52, 4.91, 1.73, 0.86, 0.89, 5.50, 1.06, 3.05, 2.93, 2.13, 0.89, 0.83,

m m m m d (6.7) m d (6.8) d (6.4) dq (2.1, 6.7) d (6.6) dd (5.8, 14.0) dd (5.4, 14.0) m d (6.4) d (6.8)

HMBCc 3, 3-NH 2, 3-NH

Figure 2. Key correlations observed in COSY (bold lines) and HMBC (arrows) spectra of 1. 3-NH, 5 5-NH, 20, 21

β-Ala residues were linked by an ester bond to form a lactone ring, which was demonstrated by the long-range correlation signal between the oxygenated methine proton H-24 and the carbonyl carbon C-1 observed in HMBC, as well as the acylated shift at H-24 shown in 1H NMR spectrum (Figure 2). In addition, 1 was subjected to partial hydrolysis in LiOH to produce compound 2, the structural composition of which was supported by ESI-MS/MS analysis (Figure S2B). The MS2 spectrum of the parent ion peak at m/z 688 (compound 2, C29H45N5O12S) showed daughter ions at m/z 670.45 [M + H − H2O]+, m/z 599.33 [M + H − β-Ala]+, m/z 471.39 [M + H − Paa − Val]+, m/z 470.27 [M + H − β-OHleu − β-Ala]+, m/z 369.31 [M + H − Thr − β-OHleu − β-Ala]+, m/z 320.39 [M + H − Paa − Val − Cya]+, m/z 219.22 [M + H − Paa − Val − Cya − Thr]+, and m/z 218.20 [M + H − Cya − Thr − βOHleu − β-Ala]+ (Figure S2B). The above MS2 fragmentation mode was consistent with the NMR analysis results. Taken together, the planar structure of 1, as shown in Figure 2, was fully established by extensive NMR and MS analysis. The absolute configuration of the Val, Cya, and Thr residues of 1 was determined by Marfey’s method.9 Briefly, 1 was completely hydrolyzed after treatment with 6 N HCl in 120 °C for 14 h. The resulting residues were then derivatized with Nα(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (L-FDAA) to produce the respective L-FDAA derivatives, which were further subjected to HPLC−DAD (340 nm) and HPLC−MS analysis. By comparing the retention times, mass data, and UV spectra of the L-FDAA derivatives of the acid hydrolysate with those of the L-FDAA derivatives of standard amino acids, it was concluded that the amino acid residues Val and Thr possess the L configuration, whereas the Cya residue is in the D configuration (Figure 3). However, the absolute configuration of the β-hydroxyleucine residue could not be determined due to the lack of standard amino acids. Further efforts to determine

5-NH, 7 25 7, 7-NH, 9, 26 9-NH, 26 9-NH, 11, 11-NH 11-NH, 27, 28, 29 11, 11-NH, 13 15, 19 15, 19, 16, 18, 13 16, 18, 13 15, 19 15, 19 5-NH, 5, 20-OH, 21, 22, 23 5, 20, 20-OH, 22, 23 20, 21 20, 21 7, 25 24 9, 9-NH 11, 28, 29 11, 27 11, 27

a

500 MHz for 1H, 100 MHz for 13C, DMSO-d6. Chemical shifts are reported in ppm. bMultiplicity and assignment from HSQC experiment. cDetermined from HMBC experiment, nJCH = 8 Hz, recycle time 1 s, from carbon(s) stated to the indicated proton(s). 7103

DOI: 10.1021/acs.joc.8b00044 J. Org. Chem. 2018, 83, 7102−7108

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Figure 3. Determination of the absolute configurations of amino acids in 1. (A) HPLC-DAD (340 nm) chromatogram of L-FDAA amino acid derivatives, which were conducted by incubating L-FDAA with the complete acid hydrolysate of 1 (i), L-valine standard (24.90 min (ii), D-valine standard (26.96 min (iii), L-cysteate standard (14.14 min (iv), D-cysteate standard (13.52 min (v), L-threonine standard (19.31 min (vi, vii) L-allothreonine standard (19.42 min, vii), D-threonine standard (20.86 min, viii), and D-allo-threonine standard (20.05 min (ix). The chromatogram of (i) displayed peaks corresponding to D-cysteate (13.47 min), L-threonine (19.32 min), and L-valine (24.90 min). (B) The ESI-MS spectra of D-cysteate, L-threonine, β-alanine, L-valine, and β-hydroxyleucine−FDAA derivatives, which were extracted from the HPLC−ESI-MS chromatogram of (i).

its absolute configuration using Mosher’s method failed due to the insufficient amount of compound 1. Nevertheless, HPLC− MS analysis of the L-FDAA derivatives confirmed the presence of a β-OHleu in addition to a β-Ala in compound 1 (Figure 3). Thus, the absolute structure of 1 (except the residue β-OHleu) was conclusively established, as shown in Figure 1. Identification and Analysis of the Biosynthetic Gene Cluster of 1. The peptidyl nature of 1, particularly the presence of four nonproteinogenic amino acid monomers in its structure, suggests that compound 1 is a product of NRPSs, which are often giant enzymes organized into modules.10 Each NRPS module typically consists of an adenylation (A) domain responsible for amino acid recognition and activation, a peptidyl carrier protein (PCP) domain for activated monomer thioesterification, and a condensation (C) domain for trans-

peptidation between the aligned peptidyl and aminoacyl thioesters to accomplish one-round peptide chain growth. Therefore, we analyzed the genome of S. lincolnensis (GenBank accession no. NZ_CP016438), with a focus on the gene clusters coding for NRPSs that contain at least five C-A-PCP modules for elongation. This analysis resulted in the identification of one candidate gene cluster, which is composed of eight orfs (orfA ∼ H) with an overall GC content of 74.4% (Figure 4 and Table S3). In this gene cluster, three orfs (orf B, orf C, and orf D) code for NRPSs. OrfB and OrfC share the same domain/module organization, i.e., C-A-PCP-C-A-PCPepimerization (E) domains, while OrfD comprise C-A-PCPthioesterase (TE) domains. The presence of two E domains indicates that the mature product might have two D-amino acid residues. Additionally, the substrate specificities of each A 7104

DOI: 10.1021/acs.joc.8b00044 J. Org. Chem. 2018, 83, 7102−7108

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Figure 4. Biosynthetic gene cluster and proposed biosynthetic pathway of 1. This gene cluster encodes three NRPSs: OrfB, OrfC, and OrfD (blue). Their domain architectures are shown, and they activated and condensed, in order, L-valine, L-cysteate, L-threonine, L-leucine, and β-alanine. Nomenclature for catalytic domains: A, adenylation domain; C, condensation domain; E, epimerization domain; PCP, peptidyl carrier protein domain; TE, thioesterase domain.

tailoring enzyme and functions after the assembly cannot be excluded at this time). Taken together, the above analyses suggest that these gene products coordinate and constitute a biosynthetic pathway to produce 1 by programming the preparation of the unusual starter and extender units as well as the sequential assembly of the Paa, Val, Cya, Thr, β-OHleu, and β-Ala residues (Figure 4). Correlation of the Biosynthetic Gene Cluster with Production of 1. To validate that the gene cluster identified above was involved in 1 biosynthesis, orf B, one of the NRPSencoding genes, was inactivated by in-frame deletion according to a previously described method.3 Briefly, two fragments respectively containing the partial N- and C-terminal sequences of orf B were amplified from the genome of S. lincolnensis and then cloned into the plasmid pKC1139 to yield a recombinant plasmid that lacked an in-frame coding region of orf B. After conjugation of the resulting construct into S. lincolnensis and double-crossover homologous recombination, an apramycinsensitive mutant with the correct disruption of orf B was obtained (Figure S10). Compared with the wild type, the resulting Δorf B mutant failed to produce 1, as shown in the HPLC−MS profile (Figure 5). This result confirmed that the identified biosynthetic gene cluster is indeed responsible for the production of 1 and its NRP biosynthetic origin. The identification of this gene cluster inspired us to further analyze the absolute configuration of the β-OHleu residue in the structure of 1. The R configuration of C5 was deduced from the presence of an E domain in module 4 of the related NRPS OrfC. The absolute configuration of C20 was determined using J-based configuration analysis.14 A large coupling constant of 3 JH−H = 6.8 Hz indicated an antiorientation between H5 and H20. Thus, the absolute configuration at C20 was accordingly deduced to be R, and this β-OHleu residue is (3R)-3-hydroxyD-leucine (Figure S11).

domain analyzed by NRPSpredictor2 are in good agreement with the amino acid sequence of 1 (Table S4). Upstream of this gene cluster exists two closely linked orfs (orf H and orf G), which encode enzymes belonging to the three-component pyruvate dehydrogenase complex. OrfH is a dual functional protein comprising pyruvate dehydrogenase (E1 component β subunit) and dihydrolipoyl transacetylase (E2 component) activities, while OrfG shows a high homology to the pyruvate dehydrogenase (E1 component α subunit). Although none of the genes in this cluster encode for dihydrolipoyl dehydrogenase (E3 component), we could find one homologue, orf I (GenBank accession no. ANS62246.1), in other region of the genome of S. lincolnensis. A recent in vitro reconstitution assay has demonstrated that a pyruvate dehydrogenase-like protein complex could convert phenylpyruvate into phenylacetyl-S acyl carrier protein, which is supplied to the subsequent biosynthetic assembly line to finally produce ripostatin.11 We thus propose that OrfH, OrfG, and OrfI could act together to perform the decarboxylation of phenylpyruvate and produce phenylacetyl-CoA, which would be utilized as the starter unit to initiate the NRPS-programmed assembly (Figure 4). OrfA is highly homologous to MA3297, a pyridoxal 5′-phosphate (PLP) dependent enzyme capable of catalyzing the reaction between L-phosphoserine and sulfite with the production of L-cysteate and phosphate.12 Presumably, OrfA might be responsible for the biosynthesis of Cya, which serves as the second extender unit (following Val) to be incorporated into the NRPS machinery (Figure 4). OrfE shares high sequence similarity to MbtH, a group of relatively small proteins often found embedded in NRPSs, which is likely to participate in the amino acid activation.13 Additionally, OrfF shows high homology to cytochrome P450 and could catalyze the formation of β-OHleu by hydroxylation of Leu during the assembly process (although the possibility that OrfF acts as a 7105

DOI: 10.1021/acs.joc.8b00044 J. Org. Chem. 2018, 83, 7102−7108

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formation of the Cya unit by in vitro assay. The structure of 1 features the residue Cya, an unnatural amino acid. Currently, a dozen Cya-containing peptides have been reported thus far, but most are produced by marine sponges, including polydiscamides,15 halicylindramide,16 discodermins,17 Oriamide,18 corticiamide,19 and microspinosamide.20 These structurally similar cyclic peptides have been reported to possess various biological properties, including antimicrobial, antifungal, and cytotoxic activities. In contrast, there are only three Cya-containing peptides isolated from the Gram-positive bacteria Actinobacteria, i.e., JBIR-96 from S. sp. RI051-SDHV6,21 JBIR-95 from Kibdelosporangium sp. AK-AA56,22 and stenothricin D from S. roseosporus.23 The structure of 1 is very similar to that of JBIR96, with only one different residue (Thr replaced by β− OHleu). Recently, Takeda et al. reported the biosynthetic gene cluster of JBIR-95 and proved the biochemical activity of cysteate synthase in vitro.24 A further sequence survey revealed that a number of NRPS-encoding biosynthetic gene clusters from the genus Streptomyces contain genes homologues of orfA (Figure S13). This finding indicates that Cya-containing NRPs that are structurally related to 1 are widely present, and an investigation into their biological functions in this genus would be interesting.

Figure 5. Examination of the production of 1 in S. lincolnensis wild type (i, WT) and Δorf B mutant strain (ii) by HPLC−-MS analysis. Extracted ion chromatogram (EIC) for [M + H]+ signal of 1 is shown.

Biochemical Characterization of OrfA. OrfA exhibits high sequence homology with the PLP-dependent L-cysteate synthase. To confirm its expected activity to supply the extender unit Cya, we performed an in vitro biochemical assay following the overexpression and purification of OrfA from Escherichia coli. The resulting recombinant protein was light yellow and exhibited a characteristic λmax at 417 nm, suggesting the presence of a PLP cofactor bound in the form of a Schiff base (internal aldimine) (Figure S12). The assay was performed by adding enzyme OrfA into substrates mixture (L-phosphoserine and sodium sulfite), and the reactions without OrfA or sodium sulfite served as negative controls. HPLC analysis of the product profile after derivation with LFDAA revealed effective transformation of phosphoserine and production of Cya (Figure 6). Therefore, OrfA, a likely PLPdependent protein, was indeed capable of catalyzing the conversion of L-phosphoserine and sulfite into Cya and phosphate.

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EXPERIMENTAL SECTION

General Materials and Methods. Materials, Bacterial Strains and Plasmids. Biochemicals and media were purchased from Sinopharm Chemical Reagent Co., Ltd. (China), Oxoid Ltd. (U.K.), TCI (Shanghai) Development Co., Ltd. (China), or Sigma-Aldrich Corp. (USA) unless otherwise stated. Restriction endonucleases were obtained from Thermo Fisher Scientific Co. Ltd. (USA). The bacterial strains, plasmids, and primers used in this study are summarized in Tables S1 and S2. DNA Isolation, Manipulation, and Sequencing. DNA isolation and manipulation in Escherichia coli or Streptomyces strains were carried out according to standard methods. PCR amplifications were performed on an Applied Biosystems Veriti Thermal Cycler using PrimeSTAR HS DNA polymerase (Takara Bio Inc., USA). Primers were synthesized at Shanghai Sangon Biotech Co. Ltd. (China), and DNA sequencing was carried out at Shanghai Majorbio Biotech Co. Ltd. (China). Analysis of Metabolites and Enzymatic Products. Optical rotations ([α]D) were measured on a JASCO P-1030 polarimeter in a 100 × 2 mm cell at room temperature. UV spectra were analyzed on a NanoDrop 2000C UV/vis spectrophotometer (Thermo Fisher Scientific Inc., USA). IR spectra were recorded on a Nicolet 380 FT-IR Spectrometer (Thermo Fisher Scientific Inc., USA). High performance liquid chromatography (HPLC) analysis was carried out on an Agilent 1200 HPLC system (Agilent Technologies Inc., USA). HPLC Electrospray ionization MS (HPLC-ESI-MS) analysis was performed on a Thermo Fisher LTQ Fleet ESI-MS spectrometer (Thermo Fisher Scientific Inc., USA), and the data were analyzed using Thermo Xcalibur software. HPLC ESI-high resolution MS (HPLC-ESI-HRMS) analysis was carried out on a 6530 Accurate-Mass Q-TOF LC/MS System (Agilent Technologies Inc., USA), and the data were analyzed using Agilent MassHunter Qualitative Analysis software. NMR data were recorded on a Bruker AV500 spectrometers (Bruker Co. Ltd., Germany) and referenced to residual signal in DMSO-d6. Production and Analysis of Cysteoamide (1). The growth, fermentation and HPLC−MS analysis of the S. lincolnensis wild type strain and its mutant were performed in the same method as reported previously.3 Isolation, Purification and Characterization of Cysteoamide (1). The S. lincolnensis wild-type fermentation culture broth (about 1 L) was treated with Amberlite XAD-2 resin (Rohm and Haas Co., USA) and Sephadex LH20 column (3.5 × 200 cm, GE Healthcare, USA)

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CONCLUSION We have isolated and characterized a new nonribosomal peptide (cysteoamide, 1), identified its biosynthetic gene cluster by bioinformatics analysis and in vivo genetic study in S. lincolnensis, and revealed the enzyme responsible for the

Figure 6. HPLC-DAD (340 nm) analysis of in vitro OrfA assays followed by derivatization with L-FDAA. Reactions were performed as follows: (i) L-phosphoserine + Na2SO3 + OrfA, (ii) L-phosphoserine + Na2SO3, (iii) L-phosphoserine + OrfA, (iv) L-phosphoserine standard, and (v) L-cysteate standard. 7106

DOI: 10.1021/acs.joc.8b00044 J. Org. Chem. 2018, 83, 7102−7108

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heated at 50 °C for 30 min and cooled down to rt, and the reaction was quenched by addition of 2 N HCl (20 μL). Standard amino acids (L-phosphoserine and L-cysteate) were reacted with L-FDAA in the same way. HPLC analysis of the resulting mixtures was carried out on the analytical Aglient Zorbax column by gradient elution of solvent A (H2O + 0.1% TFA) and solvent B (CH3CN + 0.1% TFA) with a flow rate of 1 mL/min over a 30 min period as follows: T = 0 min, 10% B; T = 5 min, 10% B; T = 20 min, 30% B; T = 23 min, 80% B; T = 26 min, 80% B; T = 28 min, 10% B; T = 30 min, 10% B (mAU at 340 nm).

according to a method described before.3 Final purification of compound 1 was carried out on an Aglient Zorbax column (SBC18, 5 μm, 9.4 × 250 mm, Agilent Technologies Inc., USA) by gradient elution of solvent A (10 mM NH4Ac) and solvent B (CH3CN) with a flow rate of 2 mL/min over a 30 min period as follows: T = 0 min, 10% B; T = 5 min, 10% B; T = 25 min, 40% B; T = 26 min, 50% B; T = 27 min, 10% B; and T = 30 min, 10% B (mAU at 210 nm). The targeted compound was eluted at 23 min with a yield of 6.8 mg. Cysteoamide (1): colorless amorphous solid; [α]28D +3.3 (c 0.55, MeOH); UV (MeOH) λmax(ε) 258 (315) nm; IR (KBr) νmax 1737, 1657, 1535, 1240, 1176, 1042 cm−1; 1H and 13C NMR (500 and 125 MHz, respectively, DMSO-d6) data see Table 1; ESI-HR-MS calcd for C29H44N5O11S+ 670.2578 [M + H]+, found 670.2761 (Figure S2) Determination of the Absolute Configurations of Amino Acids in Cysteoamide (1). Compound 1 (1.0 mg) was treated with 6 N HCl (0.2 mL) at 110 °C for 14 h. After concentration in vacuo, the reaction mixture was dissolved in H2O (100 μL). The above 50 μL aliquot or 50 mM standard amino acids aqueous solution was added to 1 M NaHCO3 (20 μL) and 1% of L-FDAA solution in acetone (100 μL). The solutions were heated at 50 °C for 30 min and cooled down to room temperature, followed by quenching with 2 N HCl (20 μL). Solvents were evaporated under nitrogen, and residues were redissolved in CH3CN (1 mL). The resulting mixtures were analyzed by HPLC or HPLC−MS on the analytical Aglient Zorbax column by gradient elution of solvent A (H2O + 0.1% FA) and solvent B (CH3CN + 0.1% FA) with a flow rate of 1 mL/min over a 40 min period as follows: T = 0 min, 10% B; T = 5 min, 10% B; T = 25 min, 40% B; T = 26 min, 50% B; T = 27 min, 10% B; and T = 30 min, 10% B (mAU at 340 nm). Gene Inactivation of orf B by In-Frame Deletion. The genomic DNA of the S. lincolnensis wild-type strain was used as the PCR template. The 1.83 kb fragment obtained using primers orfB-L-for and orfB-L-rev was digested by EcoRI and XbaI and cloned into the same sites of pKC1139 to yield plasmid pLL1201. The 1.91-kb fragment obtained using primers orfB-R-for and orfB-R-rev was digested by XbaI and HindIII and cloned into the same sites of pLL1201 to yield the recombinant plasmid pLL1202, in which a 6.89-kb in-frame coding region of orf B was deleted. To transfer pLL1202 into the S. lincolnensis wild type, conjugation between E. coli ET12567-Streptomyces was carried out following the standard procedure as previously described.3 The colonies with apramycin resistance were identified as integrating mutants, which were further subjected to single and double homologous recombination events according to a previously described method.3 The resulting apramycin-sensitive isolates were subjected to PCR amplification using primers orfB-gt-for and orfB-gt-rev to identify a desired 1.65-kb product (Figure S10). DNA sequencing of this 1.65kb product confirmed the genotype of LL1201, in which orf B was inframe deleted. Expression and Purification of Protein OrfA. The gene orfA was amplified using primers orfA-for and orfA-rev with engineered restriction sites. The target gene fragment was cloned into a pET28a(+) vector (Novagen). The resultant plasmid pLL1204 was transformed into E. coli BL21 (DE3) for protein overexpression. The purification of OrfA from E. coli transformant LL1202 were carried out following a method described previously.3 The resulting protein was concentrated and stored at −80 °C for in vitro assays. The purity of OrfA was examined by 12% SDS-PAGE analysis (Figure S12A), and the concentration was determined by Bradford assay. The UV−vis spectra of the recombinant protein OrfA was recorded at the concentration of 1 mg/mL from 250 to 600 nm (Figure S12B). Characterization of OrfA-Catalyzed Conversions in Vitro. The assays were carried out in a 50 μL reaction mixture containing 50 mM Tris−HCl (pH 7.5), 2 mM L-phosphoserine, 2 mM Na2SO3, 0.2 mM PLP, and 20 μM OrfA. The reactions proceeding in the absence of the enzyme or Na2SO3 were utilized as the negative controls. After incubation at 30 °C for 20 min, the reactions were quenched by adding an equal volume of acetonitrile. After centrifugation (12000 rpm, 10 min), 20 μL of the supernatants was added to 1 M NaHCO3 (15 μL) and 1% of L-FDAA solution in acetone (5 μL). The solutions were

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b00044. Figures S1−S13, and Tables S1−S4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-21-54925111. Fax: 8621-64166128. ORCID

Min Wang: 0000-0002-4060-1375 Wen Liu: 0000-0001-8835-8012 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by grants from the NSFC (21472231, 21520102004, 31430005, 21750004, and 21621002), CAS (SQYZDJ-SSW-SLH1037 and XDB20020200), STCM (17JC1405100 and 15JC1400400), the National Mega-project for Innovative Drugs (2018ZX09711001-006-010), K. C. Wong Education Foundation, Chang-Jiang Scholars Program, and Youth Innovation Promotion Association CAS (2017303) of China.

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