An Amidinohydrolase Provides the Missing Link in the Biosynthesis of Amino Marginolactone Antibiotics

Abstract Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with γ‐aminobutanoyl‐CoA but with 4‐guanidinylbutanoyl‐CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final‐stage de‐amidination of the corresponding guanidino‐substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective‐group chemistry.


Materials, DNA isolation and manipulation.
Bacterial strains, plasmids and oligonucleotides (Eurofins) used in this work are summarized in Tables S1, S2 and S3 respectively. Restriction endonucleases were purchased from New England Biolabs (NEB). T4 DNA ligase and alkaline phosphatase were purchased from Fermentas. All chemicals were from Sigma-Aldrich. Liquid cultures for isolation of genomic DNA were grown in tryptone soya broth (Difco). DNA isolation and manipulation in Streptomyces, and E. coli were carried out using standard protocols. [1,2] PCR amplifications were carried out using Phusion ® High-Fidelity DNA Polymerase (NEB). E. coli BL21(DE3) (Novagen) was used for protein expression.
Streptomyces olivaceus Tü4018 was grown in GYM medium (0.4% glucose, 0.4% yeast extract, 1% malt extract, pH 7.2) for 2-3 days. 1 mL samples of culture broth were centrifuged at 20,000 x g for 15 min. The mycelia pellets were then extracted with 1 mL of methanol at 60°C for 2 hours. The mixture was spun down and the clear methanol extract was evaporated to dryness and dissolved in 200 µL of methanol. 10 µL of the extract was analyzed by LC-MS. LC-MS analyses were performed on a HPLC (Agilent Technologies 1200 series) coupled to a Thermo Fisher LTQ mass spectrometer fitted with an electrospray ionization (ESI) source. For extracts from Streptomyces macronensis and from Streptomyces olivaceus Tü4018, a Luna 5µ C18 column (2.0 x 250 mm, Phenomenex) was used, and the samples were eluted using MQ containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.2 ml min -1 . The linear elution gradient for extracts from Streptomyces macronensis was 25% to 50% B over 20 min, 50% to 100% B over 9 min. The elution gradient for extracts from Streptomyces olivaceus Tü4018 was 25% to 50% B over 15 min, 50% to 75% B over 30 min, 75% to 100% B over 4 min. For extracts from Saccharomonospora azurea and from Streptomyces violaceusniger DSM 4137, a Prodigy 5µ C18 column (4.6 x 250 mm, Phenomenex) was used, and the samples were eluted using MQ containing 20mM ammonium acetate (A) and methanol (B) at a flow rate of 0.7 ml min -1 . The elution gradient for both extracts was 60% to 95% B over 30 min. The mass spectrometer was run in positive ionization mode, scanning from m/z 200 to 2000 in full scan mode.
MS/MS analysis were performed on [M+H] + ions with a normalized collision energy of 30%. Highresolution mass analysis was carried out on Thermo Fisher Orbitrap mass spectrometer with resolution set up at 60 K.
For desertomycin B production and isolation, six 250 ml Erlenmeyer flasks with spirals, containing 50 ml TSBY medium, were inoculated with 1 ml 2-day TSBY seed culture of S. macronensis dstH-deletion mutant, and incubated at 30 °C, 200 rpm. After 3 days, the broth was centrifuged at 9,000 rpm for 20 min. The pellet was resuspended in methanol and incubated at 60 °C for 2 h. The methanol extract of mycelia pellets was evaporated to dryness under reduced pressure with the rotary evaporator.
The residue was dissolved in methanol, and desertomycin B was purified from a preparative HPLC (Agilent 1200) fitted with a Luna C18 column (100Å, 21.20 x 250 mm, Phenomenex). Compounds were eluted with MQ containing 0.1% formic acid (A) and MeCN containing 0.1% formic acid (B) with a linear gradient of 5% to 35% B over 10 min, 35% to 65% B over 15 min, 65% to 100% B over 10 min at a flow rate of 20 ml/min. Fractions were collected, and checked by MS analysis. Fractions containing desertomycin B were combined. Acetonitrile was removed under reduced pressure, and sample was lyophilized.
For kanchanamycin C production and isolation, six 1 L Erlenmeyer flasks with spirals, containing 250 ml GYM medium, were inoculated with 2.5 ml 2-day GYM seed culture of Streptomyces olivaceus Tü4018 and incubated at 30 °C, 200 rpm. After 6 days, the broth was centrifuged at 9,000 rpm for 20 min. The pellet was resuspended in methanol and incubated at 60 °C for 2 h. The suspension was centrifuged in Falcon tubes at 2,500 rpm for 15 min. The supernatants were combined and filtered into a 1 L round flask. The methanol was removed under reduced pressure with the rotary evaporator to give a yellowish residue. The residue was extracted two times with diethyl ether/water. The diethyl ether was removed with the rotary evaporator. After lyophilisation the residues were dissolved in methanol for purification by preparative HPLC. Compounds were eluted with 5 mM ammonium acetate (A) and methanol (B) with a linear gradient of 60% B to 95 % B over 30 min, at a flow rate of 20 ml/min. Fractions were collected, and checked by MS analysis. Fractions containing kanchanamycin C were combined. After removing the methanol under reduced pressure, sample was lyophilized.
For azalomycin F4a production and isolation, six 1 L Erlenmeyer flasks with spirals, containing 250 ml TSBY medium, were inoculated with 2.5 ml 2-day TSBY seed culture of Streptomyces violaceusniger DSM 4137 and incubated at 30 °C, 200 rpm. After 3 days, the broth was centrifuged at 9,000 rpm for 20 min. The pellet was resuspended in methanol and incubated at 60 °C for 2 h. The methanol extract of mycelia pellets was evaporated to dryness under reduced pressure with the rotary evaporator. The residue was dissolved in MeOH, and separated on a sephadex LH20 column with MeOH/chloroform (1:1). The fractions were checked by MS. Fractions containing azalomycin F4a were combined, and solvents were removed under reduced pressure. The residue was dissolved in MeOH, and further purified by semi-preparative HPLC on a Prodigy C18 column (10 x 250 mm, Phenomenex) with a linear gradient of 45% MeCN, 55% 5 mM ammonium acetate to 56% MeCN, 44% 5 mM ammonium acetate over 35 minutes with a flow rate of 10 ml/min. Fractions containing azalomycin F4a were combined. Acetonitrile was removed under reduced pressure, sample was lyophilized.

Gene knock-out in S. macronensis
The amidinohydrolase gene dstH in S. macronensis was knocked out by in-frame deletion. To construct the deletion plasmid pYH7-dstH, dstH upstream and downstream fragments (about 2 kb) were amplified from S. macronensis genomic DNA by PCR with primers dstH-up F, dstH-up R and dstH-dn F, dstH-dn R, respectively. The cloning vector pYH7 [4] was digested with NdeI, treated with shrimp alkaline phosphatase (SAP) and gel purified. To ligate the two fragments into pYH7, the isothermal assembly method was used as described. [5] The mixture was incubated at 50°C for 60 min, and then was used to transform E. coli DH10B. The integrity of the plasmid was checked by restriction digestion and sequencing.
The construct was then introduced by conjugation into S. macronensis. The donor strain was E.
coli ET12657/pUZ8002, and conjugation was carried out on 20 ml of SFM plates (2% mannitol, 2% soya flour, 2% agar). After incubating at 30°C for 20 hours, exconjugants were selected with 50 µg ml -1 apramycin and 25 µg ml -1 nalidixic acid. Single colonies from this plate were transferred to a SFM plate containing 50 µg ml -1 apramycin to double check for antibiotic resistance. Mutant screening was carried out by streaking transformants on SFM agar medium for non-selective growth, then patching single colonies onto both SFM agar and SFM agar containing apramycin (50 µg ml -1 ) in parallel. Candidate colonies with the correct phenotype (Apr S ) were selected for further screening by PCR with a pair of primers dstH-CP1 and dstH-CP2 to identify double cross-over mutants. The PCR fragments from the double cross-over mutants were further verified by sequencing.

Protein expression and purification
The dstH gene was amplified by PCR, using genomic DNA of Streptomyces olivaceus Tü4018 as template, and inserted into vector pET28a via NdeI and HindIII restriction sites to yield pET28a-dstH. The identity of the plasmid was confirmed by DNA sequencing. Gel (Novex) analysis and the concentration of the protein was measured by Bradford assay using bovine serum albumin as a standard.

In vitro activity assays of DstH
Each reaction mixture (25 µl) contained 5 µM purified DstH, 1 mM CoCl 2 (or NiCl 2 , MnCl 2 , ZnCl 2 , MgCl 2 , MQ as no-metal control), in 50 mM Tris-HCl buffer pH 9.0. After incubation at 37°C for 30 min, 0.5 µl of purified desertomycin B (or primycin A1, kanchanamycin C, azalomycin F4a) stock solution (in DMSO) was added to a final concentration of 0.3 mM, and the reaction was allowed to continue at 37°C for 3 hr. 10 µl of the reaction mixture was taken, mixed with 50 µl methanol, and analyzed by HPLC-MS with a Luna 5µ C18 column (2.0 x 250 mm, Phenomenex) eluting with MQ containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.2 ml min -1 . The linear elution gradient for assays when desertomycin B or primycin A1 was used as substrate was 25% to 50% B over 20 min, 50% to 100% B over 9 min. The elution gradient for assays when kanchanamycin C was used as substrate was 25% to 50% B over 9 min, 50% to 72% B over 26 min, 72% to 100% B over 5 min. The elution gradient for assays when azalomycin F4a was used as substrate was 25% to 50% B over 5 min, 50% to 75% B over 20 min, 75% to 100% B over 5 min.

Streptomyces olivaceus Tü4018), primycin in Saccharomonospora azurea DSM 43044, kanchanamycin in Streptomyces olivaceus Tü4018 and azalomycin in Streptomyces violaceusniger
HAAGMAQSTALVDCSVEEFAEVVAGKVAGAVNLHELTEDL--DAFIVFSSIAATWGSGGQCGYAAGNAFLD A1 *  Kishi et al., [7] all the other 20 stereocenters have the same configurations as those established by Kishi and colleagues.   The two peaks for deguanidino-amino-kanchanamycin C at 18.0 min and 20.2 min are probably isomers, the same is true for kanchanamycin C at 19.1 min and 21.4 min. The two isomers, by analogy with azalomycin, are likely due to a different site of attachment of the malonyl group, either at C23-OH or at C25-OH. [8]

Figure S11. HPLC-ESI-MS total ion current traces of in vitro amidinohydrolysis of desertomycin B catalysed by DstH in the presence of various divalent ions.
Desertomycin B is efficiently converted to its amino form 1a in the presence of either Co 2+ or Ni 2+ .

Figure S12. HPLC-ESI-MS total ion current traces of in vitro convertion of primycin A1 catalysed by DstH with various divalent ions.
Primycin A1 can be efficiently converted to its amino form under the assay conditions used.

Figure S13. HPLC-ESI-MS total ion current traces of in vitro convertion of kanchanamycin C catalysed by DstH with various divalent ions.
Kanchanamycin can be almost completely converted to its amino form under the assay conditions used.

Figure S14. HPLC-ESI-MS total ion current traces of in vitro convertion of azalomycin F4a
catalysed by DstH with various divalent ions. Azalomycin F4a can not be converted to its amino form under the assay conditions used. The two peaks are isomers of The two peaks are azalomycin F4a isomers, differing in the attachment site of the malonyl group, either at C23-OH or at C25-OH. [8] [9] Streptomyces olivaceus Tü4018

Supplementary Tables
Desertomycin A-and kanchanamycin-producing strain [10] Streptomyces macronensis Desertomycin A-producing strain [11] Streptomyces spectabilis Desertomycin A-producing strain [12] Saccharomonospora azurea (syn. S. caesia) Primycin-producing strain [13] S. violaceusniger DSM4137 Azalomycin-producing strain [14]   Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. The sequence has been deposited under the accession number XXXXX. Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. The sequence has been deposited under the accession number XXXXX.  Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. The sequence has been deposited under the accession number XXXXX. Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. The sequence has been deposited under the accession number XXXXX.