In vitro characterization of nonribosomal peptide synthetase-dependent O-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine

Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene (azs) cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the Streptomyces fragilis genome. Azaserine was produced by the heterologous expression of the azs cluster in Streptomyces albus. In vitro enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes O-(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.

. Primers used for cloning of the azs cluster. Table S2. Primers used for the construction of plasmids for the preparation of Azs proteins. Table S3. The final concentration of IPTG for gene expression. Table S4. Primers used for site-directed mutagenesis of AzsO. Figure S1. The azs cluster homologs discovered from the genome database. Figure S2. Amino acid sequence alignment of the Cy domains of AzsO homologs and representative Cy domains (EpoB and BmdB). Figure S3. The structure models of AzsO from Streptomyces fragilis constructed by AlphaFold2. Figure S4. MS/MS spectra of authentic azaserine and the compound produced by Streptomyces albus-azs. Figure S5. SDS-PAGE analysis of recombinant Azs proteins. Figure S6. In vitro analysis of AzsS. Figure S7. Phosphopantetheine ejection assay for the reaction products of AzsP, AzsT, and AzsF. Figure S8. In vitro analysis of AzsD using HDA-NAC (4′) as a substrate. Figure S9. Phosphopantetheine ejection assay for the reaction product of AzsO. Figure S10. LC-HRMS analysis of HDA-AzsO-CP (9) in the reaction catalyzed by AzsD, AzsN, and AzsO. Figure S11. LC-MS analysis of HDA-Ser (8) release catalyzed by AzsB. Figure S12. Synthetic standard of HDA-Ser. Figure S13. Site-directed mutagenesis of the Cy domain of AzsO. Figure S14. Sequence logo around conserved motifs generated from 1,165 Cy domains. Figure S15. Mechanism of the reactions catalyzed by Cy domains. Figure S16. 1 H NMR data of Fmoc-HDA-NAC.

Materials
Streptomyces fragilis NBRC 12682 was obtained from the National Institute of Technology and Evaluation (NITE). Streptomyces niger JCM3158 was obtained from Japan Collection of Microorganisms (JCM). Streptomyces albus J1074 (Streptomyces albidoflavus J1074) used for the heterologous expression of azs genes was kindly provided from Prof. T. Kuzuyama. Escherichia coli DH5α (TaKaRa Bio Inc, Shiga, Japan) and E. coli HST08 (TaKaRa Bio Inc) were used for DNA manipulation, and E. coli BL21(DE3) (Merck KGaA, Darmstadt, Germany) was used for expressing recombinant proteins. In addition, E. coli S17-1 was used for conjugation. Enzymes used for DNA manipulation, including DNA polymerase and restriction enzymes, were purchased from TaKaRa Bio Inc. Primers were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All chemicals used in this work were purchased from Sigma-Aldrich (St. Louis, MO, USA), New England BioLabs (Ipswich, MA, USA), and Tokyo Chemical Industry (Tokyo, Japan). Azaserine was purchased from Sigma-Aldrich.

Conjugational transfer
E. coli S17-1 harboring a plasmid for heterologous expression was inoculated into 5 mL of Luria-Bertani (LB) medium and incubated at 37°C overnight. The culture was transferred to 100 mL LB medium and incubated at 37°C until OD600 reached 0.6. The cells were harvested and washed twice with fresh LB medium and resuspended in 10 mL LB medium. Spores of S. albus J1074 in 100 µL of 10% glycerol were suspended in 0.5 mL TSB medium (30 g/L tryptic soy broth) and incubated at 50°C for 10 min. The spores and 500 µl of E. coli cells were mixed and inoculated on an MS agar plate (20 g/L soya flour, 20 g/L mannitol, and 20 g/L agar) containing 20 mM MgCl2. After incubation at 30°C for 18-20 h, the plate was overlaid with an antibiotic solution. The antibiotic solution contained nalidixic acid (0.75 g/L), thiostrepton (0.75 g/L), and/or kanamycin (0.75 g/L). After incubation at 30°C for one week, several antibioticresistant colonies were obtained.

Construction of plasmids for heterologous expression
The primers used for plasmid construction are listed in Table S1. The whole azs cluster was divided into three fragments and amplified by PCR using appropriate primers (Table S1) and S. fragilis genomic DNA as a template. In addition, linearized pTYM19 with the homologous region for gene cloning was amplified by PCR using appropriate primers (Table S1) and pTYM19 as a template. [1] The amplified DNA fragments were assembled by using In-Fusion (TaKaRa Bio Inc.), resulting in pTYM19-azs.
For the construction of pHKO4-azsR, azsR was amplified by PCR using appropriate primers (Table S1) and S. fragilis genomic DNA as a template and cloned into the NdeI and SphI sites of pHKO4 by using In-Fusion (TaKaRa Bio Inc.).

Heterologous expression of azs cluster in S. albus J1074-azs
S. albus J1074-azs was constructed by sequential transformation of S. albus J1074 using pTYM19-azs and pHKO4-azsR. S. albus J1074-azs was inoculated into 50 mL preculture medium (10 g/L glucose, 20 g/L dextrin, 5 g/L yeast extract, 5 g/L NZ-amine A, and 1 g/L CaCO3) and cultured while shaking (150 rpm) at 30°C for 2 days. A portion (5 mL) of the preculture was inoculated into 100 mL ISP2 medium (4 g/L yeast extract, 10 g/L malt extract, and 4 g/L glucose) and cultured while shaking (150 rpm) at 30°C for 2 days. Then, 10 mg/L of thiostrepton was added to induce the expression of azsR and cultured while shaking (150 rpm) at 30°C for 1 day. A portion (10 mL) of the culture was harvested and stirred with 0.5 g of activated carbon powder (Nacalai Tesque, Kyoto, Japan) for 1 h. The mixture of cells and activated carbon powder were harvested by centrifugation at 13,100 g for 10 min. After the supernatant was removed, an equal volume of distilled water was added to the mixture, which was then gently resuspended. The sample was centrifuged at 13,100 g for 10 min, and the supernatant was removed. The compounds were extracted from the mixture using 10 mL of 60% methanol. After centrifugation, the supernatant was transferred to a new tube and evaporated to dryness under reduced pressure. The residual material was dissolved in 100 µL of 60% methanol.
The obtained samples were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESIMS) using the LC-2040C 3D Plus system (Shimadzu Corp., Kyoto, Japan) equipped with a COSMOSIL 2.5HILIC packed column (2.0 mm ID × 50 mm, Nacalai Tesque) coupled with a model LCMS-8040 liquid chromatograph tandem mass spectrometer (Shimadzu Corp.). The compounds were eluted with a linear gradient of acetonitrile/20 mM ammonium acetate. Then the UV chromatogram was obtained by photodiode array spectrophotometer in the LC-2040C 3D Plus system.
To obtain high resolution (HR) MS spectra, the samples were analyzed by LC-HRESIMS using the UFLC Nexera system (Shimadzu, Kyoto, Japan) equipped with a COSMOSIL 2.5HILIC packed column (2.0 mm ID × 50 mm, Nacalai Tesque) coupled with the SCIEX Triple TOF 5600 system (SCIEX, Framingham, MA). The compounds were eluted with a linear gradient of acetonitrile/20 mM ammonium acetate.
Production and purification of recombinant AzsS, AzsP, AzsQ, AzsF, AzsD, AzsN, AzsN-CP, AzsO, AzsO-CP, AzsB, and Sn_AzsT pColdI-azsS, pColdI-azsP, pColdI-azsQ, pColdI-azsF, pColdI-azsD, pColdI-azsN, pColdI-azsN-CP, pColdI-azsO, pColdI-azsO-CP, and pColdI-azsB were constructed by amplification of the genes with PCR using appropriate primers (Table S2) and genomic DNA of S. fragilis as a template followed by gene cloning using In-Fusion (TaKaRa Bio Inc.). pColdI-Sn_azsT was constructed by using the azsT gene amplified from the genomic DNA of Streptomyces niger. Each plasmid was introduced into E. coli BL21(DE3). To obtain holo-AzsQ, holo-AzsN, holo-AzsN-CP, holo-AzsO-CP, and holo-AzsO, corresponding plasmids were introduced to E. coli BL21(DE3) harboring pACYC-sfp. [2] To obtain Sn_AzsT, pColdI-Sn_azsT was introduced into E. coli with pTf-16 (TaKaRa Bio Inc.). Each obtained strain was cultured in 100 mL LB medium with ampicillin (1 mg/mL arabinose was added to LB medium for Sn_AzsT) while shaking (150 rpm) at 37°C until the OD600 reached 0.6. After cooling the culture on ice, isopropyl-D-thiogalactopyranoside (IPTG) was added to the culture to induce gene expression, and the culture was incubated while shaking (150 rpm) at 15°C for 24 h. The final concentration of IPTG is listed in Table S3. The cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, 10% glycerol, and 200 mM NaCl, pH 8.0). After lysis by sonication, the cell debris was removed by centrifugation. The recombinant protein was purified using His60 Ni Superflow Resin (TaKaRa Bio Inc.). The resin was added to the clear lysate and incubated with gentle shaking at 4°C for 1 h. After incubation, the resin was loaded onto a column and washed with 50 mL lysis buffer. The protein was eluted by lysis buffer containing different concentrations of imidazole. The buffer was desalted and concentrated with lysis buffer using an Amicon Ultra centrifugal filter with a suitable molecular mass cutoff (Merck Millipore, Burlington, MA, USA).

Chemical synthesis of hydrazine acetic acid (HAA)
HAA was synthesized according to the previously described procedure. [3] Bromoacetic acid (276 mg, 2 mmol) dissolved in ethanol was added to a three-necked flask equipped with a Dimroth condenser. The flask was substituted with nitrogen gas. Then, hydrazine (500 mg, 15 mmol) dissolved in ethanol was added dropwise. The reaction mixture was refluxed at 80°C for 5 h. The reaction solution was then allowed to stand at 4°C overnight. The supernatant was obtained after centrifugation at 13,000 rpm for 30 min. The solution was evaporated to dryness under reduced pressure, resulting in HAA.
Chemical synthesis of N-succinyl-hydrazinoacetic acid (succinyl-HAA, 1) 9-Fluorenylmethyl carbazate (1 mmol) and succinic anhydride (1 mmol) were dissolved in 10 mL of methanol and the solution was stirred at room temperature for overnight. The reaction solution was evaporated to dryness under reduced pressure. The residual material was dissolved in 2 mL of dimethylformamide (DMF) with 500 µL of piperidine and the solution was stirred at room temperature for 2 h. Then, water and ethyl acetate were added to the solution. The aqueous layer was harvested and evaporated to dryness under reduced pressure. The residual material was dissolved in a mixture of 5 mL of water and 5 mL of acetonitrile. Bromoacetic acid (1 mmol) in acetonitrile was added dropwise to the solution and refluxed at 80°C for 4 h. The solution was evaporated to dryness, resulting in N-succinyl-hydrazinoacetic acid (succinyl-HAA) (1).

Chemical synthesis of N-acetylcysteamine thioester of 2-hydrazineylideneacetic acid (HDA-NAC)
Fmoc-HDA (1 mmol), WSC (1.1 mmol), DMAP (0.1 mmol), and NAC (1.1 mmol), were dissolved in dichloromethane and the solution was stirred at room temperature overnight under nitrogen atmosphere to obtain N-acetylcysteamine thioester of Fmoc-HDA (Fmoc-HDA-NAC). The liquid-liquid extraction was carried out with water and chloroform, and the chloroform layer was collected. The solution was evaporated to dryness under reduced pressure. Then, the residual material was applied to medium pressure liquid chromatography (MPLC) Purifi-Comp (Shoko Scientific) equipped with a silica gel column (Purif-Pack, Shoko Scientific). A linear gradient of hexane/ethyl acetate was used for the elution. Fmoc-HDA-NAC was dissolved in DMSO-d6, and the structure was determined by the JNM-ECA500II NMR system (JEOL, Tokyo, Japan, Figure S16). Since HDA-NAC was unstable, HDA-NAC was used for in vitro assay without further purification after deprotection of Fmoc-HDA-NAC as described below. Four milligram of Fmoc-HDA-NAC was dissolved in 100 µL of DMF with 5 μL of piperidine and the solution was stirred for 30 min at room temperature. After that, water (400 µL) and chloroform (400 µL) were added, and liquid-liquid extraction was then carried out. The aqueous layer was collected and used as 20 mM HDA-NAC.
Authentic HDA-Ser (8) was synthesized by reducing azaserine immediately before use. Azaserine (2 mg) and NaBH4 (1.8 mg) were dissolved in 500 µL EtOH and incubated for 3 h. The synthesis of HDA-Ser was confirmed by HRMS and MS/MS ( Figure S12) because it was highly unstable. The synthesized HDA-Ser (8) was purified by using MPLC (Purif-RP2, Shoko Scientific) equipped with a HILIC column (20 ID × 250 mm, Nacalai Tesque). The compound was eluted using linear gradient of acetonitrile/20 mM ammonium acetate. The fractions containing HDA-Ser were evaporated to dryness and dissolved in the reaction mixture without Azs proteins for analysis, because the retention time was affected and ionization efficiency decreased, by the compounds dissolved in the buffer.

Construction and analysis of AzsO variants
A DNA fragment with each mutation was amplified by PCR using an appropriate pair of primers listed in Table S4 and pColdI-AzsO as a template. The obtained DNA fragment was cyclized using In-fusion and introduced into E. coli HST08. The mutations were confirmed by DNA sequencing. AzsO variants were produced and purified as the same method used for wild-type AzsO.
In vitro reaction of the AzsO variants was performed using the method which was used for the detection of the intermediates binding to the CP domain of AzsO. The compounds were analyzed by LC-ESIMS using the LC-2040C 3D Plus system (Shimadzu Corp.) equipped with a BioResolve RP mAb packed column (2.1 mm ID × 100 mm, Waters) coupled with a model LCMS-8040 liquid chromatograph tandem mass spectrometer (Shimadzu Corp.). The compounds were eluted with a linear gradient of water/acetonitrile containing 0.1% formic acid.    Figure S1. The azs cluster homologs discovered in the genome database. The clusters were analyzed by antiSMASH [4] and BiGSCAPE [5] .             The mutation did not result in cyclization ability. These results showed that these residues are important in catalysis. At least three independent replicates were performed for each assay. All the results showed same trends. Representative results are shown. WT, wild type.