Annabel C.
Murphy‡§
a,
Shu-Shan
Gao‡
a,
Li-Chen
Han
a,
Simon
Carobene
a,
Daisuke
Fukuda
bc,
Zhongshu
Song
a,
Joanne
Hothersall
b,
Russell J.
Cox
a,
John
Crosby
a,
Matthew P.
Crump
a,
Christopher M.
Thomas
b,
Christine L.
Willis
*a and
Thomas J.
Simpson
*a
aSchool of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK. E-mail: chris.willis@bristol.ac.uk; tom.simpson@bristol.ac.uk; Fax: +44 (0)117 925 1295; Tel: +44 (0)117 928 7660
bSchool of Biosiences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
cDiscovery Science and Technology Department, Daiichi Sankyo RD Novare co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-9630, Japan
First published on 1st November 2013
The biosynthesis of the mixed PKS-FAS-NRPS hybrid antibiotic thiomarinol A was investigated using feeding studies to both wild type and mutant strains of the marine bacterium Pseudoalteromonas. Particularly interesting features of the pathway include assembly of the 8-hydroxyoctanoic acid side-chain via chain extension of a C4-precursor (4-hydroxybutyrate), and construction of the pyrrothine unit from cysteine via (HolA-D, F-H) prior to intact incorporation into thiomarinol (catalysed by TmlU). A series of thiomarinol-related and other minor metabolites have been isolated from wild-type and mutant strains. The results of these investigations are rationalised in terms of the overall biosynthetic pathway.
We have recently identified the thiomarinol (tml) biosynthetic gene cluster via full genome sequencing of SANK 73390. It is contained on a 97 kb plasmid consisting almost entirely of the thiomarinol biosynthetic genes.17 These consist of trans-AT PKSs (tmpA, C and D), a putative FAS (tmpB) and associated tailoring and resistance genes (tmlA-Z) with high homology to the mupirocin (mup) cluster (Fig. S1, ESI†). A non-ribosomal peptide synthetase (NRPS) linked to a set of tailoring enzymes (holA-H) is also present similar to that recently shown to control holomycin biosynthesis in Streptomyces clavuligerus.18,19 Analysis of the WT culture and mutant strains in which the PKS and NRPS parts of the cluster had been deactivated by in-frame deletions, resulted in isolation of a number of new thiomarinol related compounds.20 Analysis of extracts of the WT strain by reversed-phase HPLC-ESIMS led to the isolation of two additional polar metabolites which lacked the pyrrothine moiety. These were shown to be marinolic acid A 15 and the corresponding amide, marinolic amide 16. Similar analysis of the ΔNRPS mutant strain showed, as expected, that no pyrrothine containing compounds were produced, the major thiomarinol metabolite being marinolic acid A, but no amide 16. Two minor metabolites were isolated: marinolic acid A617 and A418 in which the octanoate moiety on marinolic acid A has been replaced by hexanoate and butanoate respectively. Re-examination of the WT extracts revealed trace amounts of 17. A number of new acyl-pyrrothine containing compounds were also isolated. On the basis of their similarity to metabolites previously isolated21–23 from Xenorhabdus spp, they were designated as xenorhabdins 8–13 (19–25) respectively (Fig. 2).
We now report stable isotope labelling and other feeding experiments with WT and mutant strains of Pseudoalteromonas which confirm the origins of all of the atoms in thiomarinol, provide a rationalisation for the occurrence of the minor related metabolites, and for the timing of pyrrothine formation and its linkage to marinolic acid.
Scheme 1 Incorporation of [13C]- and [18O]-labelled acetates and [methyl-13C]-methionine into thiomarinol A. |
The incorporation pattern confirms the similarity of the pseudomonic acid A11,12 and thiomarinol A biosynthetic pathways. A regular acetate incorporation pattern is observed in the C1–C14 fragment of thiomarinol. A high level of 13C incorporation (5–6%) is observed at all seven expected positions in the polyketide-derived moiety with high levels of 18O retention (ca. 80%) at carbons 1, 5, 7 and 13 (Table 1). The 4- and 6-hydroxyl groups are not labelled from 18O-acetate and are at positions consistent with their introduction being via tailoring enzymes using molecular oxygen. The 16-methylene (55% enriched from [methyl-13C]methionine) and 17-methyl carbons (40% enriched) are methionine derived consistent with the presence of C-MeT domains in the first and third extension modules, and the 15-methyl is derived from the methyl of a cleaved acetate unit consistent with it being derived via the HMGS β-branch pathway as in pseudomonic acid A.
Carbon | δ C | [1,2-13C2]-acetatea % (JC–C Hz) | [2-13C]-acetatea % | [1-13C, 18O2]-acetatea % ΔδCb (ppm) |
---|---|---|---|---|
a Percentage enrichment (>1 to nearest integer); significant differences from average values indicated in bold. b Average ratio of enriched (13C) to enriched + isotopically shifted (13C18O) signals is 1:4 except C-1′ which is ca. 1:1, all values to nearest integer. c Spectra determined in d6-DMSO, and d4-MeOH in which the 15- and 17-methyls appear respectively at 14.9 and 15.2 ppm. | ||||
1 | 166.1 | 2 (76) | 5 (0.03) | |
2 | 114.3 | 2 (76) | 7 | |
3 | 160.8 | 2 (43) | 5 | |
4 | 72.4 | 2 (43) | 7 | |
5 | 76.2 | 2 (43) | 6 (0.02) | |
6 | 63.8 | 2 (43) | 7 | |
7 | 69.6 | 2 (35) | 6 (0.02) | |
8 | 42.2 | 2 (35) | 7 | |
9 | 31.8 | 2 (44) | 6 | |
10 | 127.7 | 2 (44) | 7 | |
11 | 134.1 | 2 (44) | 6 | |
12 | 43.1 | 2 (44) | 7 | |
13 | 69.2 | 2 (39) | 6 (0.02) | |
14 | 20.0 | 2 (39) | 7 | |
15 | 15.7 | 2 | 7 | |
16 | 64.2 | |||
17 | 15.7 | |||
1′ | 171.8 | 2 (50) | 6 (0.03) | |
2′ | 34.6 | 2 (50) | 7 | |
3′ | 24.9 | 2 (35) | 6 | |
4′ | 28.4 | 2 (35) | 7 | |
5′ | 28.3 | 1 (34) | 3 | |
6′ | 25.3 | 1 (34) | 4 | |
7′ | 28.2 | 0.3 (38) | 2 | |
8′ | 63.0 | 0.3 (38) | 2 | 1 |
1′′ | 167.9 | |||
2′′ | 115.3 | |||
3′′ | 133.9 | |||
4′′ | 133.6 | |||
5′′ | 110.3 |
Feeding [2,3-13C2]succinate to cultures of wild-type Pseudoalteromonas SANK 73390 revealed an intact incorporation confirmed by observation of a 13C–13C coupling of 34 Hz between C6′ and C7′ (ca. 0.5% enrichment, Scheme 2, see insert expansion in Fig. S10†) in accord with the proposed C4 precursor. A lower level of incorporation (0.15%) was also observed elsewhere within the molecule resulting from degradation of [2,3-13C2]succinate to [1,2-13C2]acetate.
Scheme 2 Incorporation of [13C]-labelled succinate, 4-hydroxybutyrate and cystine into thiomarinol A. |
It is known that 4-hydroxybutyrate can be formed via reduction of succinyl CoA.24 The overall results of acetate incorporation are also consistent with this: acetate is incorporated intact into only carbons 3 and 4 of succinyl CoA (Scheme S2, ESI†) which explains the significant but lower incorporation into C5′/C6′ of thiomarinol, presumably due to dilution by a pool of endogenously derived Krebs Cycle intermediate. Further cycling of labelled succinate would give single labels at carbons 1 and 2 of succinyl CoA which explains the lower levels of labelling from [2-13C]acetate into C7′ and C8′. To explain the low level of intact acetate labelling of C7′/C8′, the conversion of succinyl CoA to symmetrical succinate must be partially reversible so that ca. 30% of the succinate is incorporated via this route. The low level of labelling at C8′ from [1-13C]acetate is completely consistent with this hypothesis.
To further investigate the proposed C4 side-chain precursor, [2,3-13C2]-4-hydroxybutyrate was prepared from [2,3-13C2]succinate via cyclisation to succinic anhydride, reduction to the lactone with LiAlH4 followed by hydrolytic ring opening with aqueous NaOH (Scheme S1†). Feeding [2,3-13C2]-4-hydroxybutyrate to cultures of the wild-type Pseudoalteromonas SANK 73390 showed a site-specific labelling (0.2%) of thiomarinol A at C6′, C7′ confirming intact incorporation (Scheme 2, see insert expansion in Fig. S12†).
Thus the biosynthetic origin of C5′ to C8′ is somewhat similar to the situation presumed for 9-hydroxynonanoic in mupirocin biosynthesis, where labelling studies are consistent with a 3-hydroxypropionate starter unit, which then undergoes three reductive chain elongations. 3-Hydroxypropionate can be formed in bacteria by reduction of the thiolester of malonyl CoA25 and both the mup and tml clusters show the presence of genes encoding a malonyl/succinyl CoA dehydrogenase clustered with an acyl CoA synthetase and an ACP.
Interestingly, the biosynthetic gene cluster for difficidin,26 another trans-AT metabolite of Bacillus amyloliquefaciens, also contains an oxo-acyl CoA reductase encoded by difE which as in mupirocin/thiomarinol is grouped with an acyl CoA synthetase and an ACP (difD and difC). In addition there is an adjacent kinase (difB) which would be suitable for catalysing dehydration of 3-hydroxypropionate to produce the acryl thiol ester required to prime difficidin chain elongation.
The retention of 18O label at C1′ of thiomarinol A is slightly less than half that at the other acetate-oxygen bearing positions. This is consistent with C1′ existing as the free carboxylate, resulting in randomisation of the label between both carboxylate oxygens, before ligation via an amide bond to the pyrrothine. Analysis of the structure of the pyrrothine moiety of thiomarinol suggest that it is formed via two molecules of cysteine. To gain evidence for this proposal, [2-13C]cystine was prepared from ethyl [2-13C]pyruvate, (Scheme S2†) and fed to WT Pseudoalteromonas. A high level of incorporation of 13C into the C2′′ and C4′′ positions (16 and 20% respectively) confirmed that the pyrrothine is indeed cysteine, and therefore NRPS, derived (Scheme 2).
The biosynthetic gene cluster for holomycin biosynthesis in Streptomyces clavuligerus has been isolated and a pathway from cysteine proposed. This pathway involves initial dipeptide formation followed by a series of oxidative cyclisations, the final step being acetylation of the free pyrrothine.18 To prove that the pyrrothine moiety in thiomarinol is assembled and then incorporated as an intact unit rather than being assembled on marinolic acid, pyrrothine was synthesised as previously described27 and fed to the ΔNRPS mutant strain of SANK 73390. The mutant was prepared by a deletion in the holA gene encoding the amino acid activation domain which, from sequence analysis of the active site, is predicted to selectively activate cysteine. As indicated above, the ΔNRPS mutant normally produces mainly marinolic acid. When cultures were supplemented with pyrrothine, the normal WT phenotype was restored indicating that Pseudoalteromonas takes the intact pyrrothine as substrate (Scheme 3). Interestingly, the concentration of the feeds was critical, with maximum restoration of thiomarinol A production being observed at a pyrrothine concentration of 20 mg ml−1. At concentrations of >80 mg ml−1, no thiomarinol A was observed and production of marinolic acid was also completely inhibited (Fig. S3, ESI†).
To test if HolE can also transfer a range of non-endogenous substrates, various short, medium and long chain fatty acids and others with ω-functionalized side chains, e.g. hydroxyl, amino, carboxyl and phenyl were fed to the ΔPKS mutant (Scheme 4). Some of these were incorporated with varying efficiencies, presumably after conversion to their respective CoA esters, sometimes after catabolic chain shortening to give 19 and the novel acyl-pyrrothines 26–30.
The isolation of truncated metabolites marinolic acids A4 and A6, is consistent with the isotope labelling studies which suggest that a C4 intermediate is added to the product of the PKS, e.g. by trans-esterification. This would then be elongated to give an enzyme bound hexanoate analogue which would be further elongated to give the completed octanoate side chain. In the absence of the pyrrothine in the ΔNRPS mutant they would be released either by enzymatic or spontaneous hydrolysis as marinolic acids. We have previously shown that release of assembly intermediates occurs when flux of intermediates along the normal mupirocin pathway is impaired by a range of targeted mutations of trans-acting and other tailoring genes.28 Similar mechanisms would allow leakage of marinolic acids A4 and A6 into the medium. It is significant that this appears to be more pronounced in the mutant than the WT. Truncated forms of mupirocin and related metabolites with C7 and C5 hydroxy acids have also been observed in both mutant strains and when the quorum sensing regulation mechanism has been manipulated to increase mupirocin production.29
Footnotes |
† Electronic supplementary information (ESI) available: General experimental details, feeding experiments, synthesis of labelled precursors, spectroscopic data and NMR spectra of labelled thiomarinol A. See DOI: 10.1039/c3sc52281d |
‡ These authors contributed equally to the work. |
§ Current address: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA. |
This journal is © The Royal Society of Chemistry 2014 |