New insights into paulomycin biosynthesis pathway in Streptomyces albus J1074 and generation of novel derivatives by combinatorial biosynthesis
© González et al. 2016
Received: 18 January 2016
Accepted: 9 March 2016
Published: 21 March 2016
Streptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.
Inactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.
The paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure–activity relationships.
Paulomycins A and B antibiotics containing an isothiocyanate group (paulic acid) and mainly active against Gram-positive bacteria, were initially isolated from Streptomyces paulus strain 273 [17, 18]. Later on, the absolute stereochemistry of these compounds was reported [19, 20] and they were found to be closely related to antibacterial compounds senfolomycins A and B, only differing in the stereochemistry of methoxy group present in their respective deoxysugar moieties . Streptomyces paulus produces paulomycins as a family of compounds that include paulomycin A, A2, B, C, D, E and F , O-demethylpaulomycins A and B, paulomenol A and B, hydrogen sulfide adducts of paulomycin A and B , paldimycin A and B, and 273a2α and 273a2β, derivatives of paulomycin A and paulomycin B containing one or two N-acetyl-l-cysteine groups, respectively [24, 25]. Antibiotic activity of paldimycins was assessed in vitro against 215 Gram-positive bacteria and found comparable to that of vancomycin . In addition, paulomycins and paldimycins were found to be able of killing Staphylococcus aureus intracellular cells surviving into polymorphonuclear leukocytes [27, 28]. On the other hand, paulomenol A and B lack antibacterial activity pointing to paulic acid as determinant of the antibiotic properties of paulomycins and paldimycins . Paulomycin A and B were also found to be produced by S. albus G . Regarding the biosynthetic origin of paulomycins, addition of some precursors was found to increase production yields and they included valine, isoleucine, isobutyric acid, 2-methylburytic acid , l-methionine, l-threonine and α-ketoburyric acid [31, 32]. The gene cluster involved in paulomycins biosynthesis has been recently reported in S. albus J1074 , S. paulus NRRL8115 and Streptomyces sp. YN86  sharing between them an overall identity of 99 %. A pathway for paulomycin biosynthesis in S. paulus NRRL8115, based on in silico gene analysis, has been recently proposed .
In this work, we characterize several steps of paulomycins biosynthesis in S. albus J1074 including the glycosylation and acylation steps and the biosynthesis of the paulic acid moiety. Inactivation experiments allowed to determine genes (and enzymes) involved in these steps and to identify several biosynthetic intermediates and some products accumulated by the corresponding mutants which provided insights on the biosynthesis pathway. One of the genes studied in this work, and shown to be involved in paulomycin biosynthesis, has not been previously annotated at S. paulus NRRL8115 and Streptomyces sp. YN86 paulomycin biosynthesis gene clusters. We propose a refined version of the paulomycin biosynthesis pathway based on experimental evidence. In addition, we have generated by combinatorial biosynthesis several novel bioactive derivatives of paulomycin A and B carrying modifications in the L-paulomycose moiety using different plasmids that direct the biosynthesis of different deoxyhexoses.
Delimiting the boundaries of paulomycin biosynthesis cluster
Proposed functions of paulomycin biosynthesis cluster deduced proteins from S. albus J1074
S. albus J1074a
S. paulus NRRL8115b
Streptomyces sp. YN86c
RNA polymerase sigma factor
M23 family peptidase
TetR-family transcriptional regulator
LuxR-family transcriptional regulator
Conserved hypothetical protein
EmrB/QacA subfamily transporter
Elongation factor G 1
Dehydrogenase E1 alpha subunit
Dehydrogenase E1 beta subunit
SARP-family transcriptional regulator
Predicted GCN5-related N-acetyltransferase
Hypothetical protein, Reductase
3-oxoacyl-ACP synthase III
LuxR-family transcriptional regulator
dTDP-6-deoxy-L-hexose 3-O- methyltransferase
Regulation of paulomycin biosynthesis gene cluster
Glycosyltransferases involved in paulomycin biosynthesis
Two glycosyltransferases must be involved in the incorporation of D-allose and L-paulomycose during paulomycin biosynthesis. Plm12 and Plm23 show similarities to O- and C-glycosyltransferases from different streptomycetes including Pau15/PauY15 and Pau25/PauY25, respectively, of paulomycin biosynthesis clusters in S. paulus (KJ721164.1) and Streptomyces sp.YN86 (KJ721165.1). In addition, Plm11 shows similarity to cytochrome P450-like enzymes that lack the conserved Cys necessary to bind heme prosthetic group. These enzymes have been shown to participate in glycosylation by activating their counterpart glycosyltransferase .
SAM5335 mutant strain, which was generated by deletion of plm23, produces four compounds (Fig. 4) with UPLC retention times of 2.0, 2.7, 2.9 and 3.0 min, respectively. Compound 8 with UPLC retention time of 2.0 min, showed maxima of absorbance at 223 and 329 nm, and a mass of m/z 138 [M + H]+. It was identified as 2-aminobenzoic acid (anthranilic acid) (Fig. 5) by NMR (Additional file 3: Figures S20–S23, Table S4). Compounds 9 and 10 with UPLC retention times of 2.7 and 2.9 min, respectively, shared a similar absorption spectrum with maxima at 223, 251 and 302 nm. Compound 10, revealed by mass analysis an ion of m/z 180 [M + H]+, and was identified by NMR (Additional file 3: Figures S24–S26) as N-acetyl-orto-benzoic acid (Fig. 5). Compound 11 with UPLC retention times of 3.0 min, a mass of m/z 209 [M + H]+ and maxima of absorption at 207 and 288 nm, was characterized by NMR (Additional file 3: Figures S27–S29) as deoxydehydrochorismic acid (Fig. 5). These compounds, 2-aminobenzoic acid (8), N-acetyl-orto-benzoic acid (10) and deoxydehydrochorismic acid (11), are not paulomycin intermediates since they failed restoring paulomycins biosynthesis in bioconversion experiments using S. albus B29 mutant strain. Complementation of SAM5324 and SAM5335 using pEM4HT5324 and pEM4HT5335 respectively, partially restored (20 % and 10 %, respectively) paulomycins and paulomenols production (Additional file 1: Figure S5).
Acyltransferases involved in paulomycin biosynthesis
Three acyltransferases might be involved in the incorporation of paulyl-CoA, acyl-CoA and 2-methylbutyryl-CoA or isobutyryl-CoA during paulomycin A and B biosynthesis, respectively. Plm3 and Plm22 show similarity to O-acyltransferases MegY (AAG13909.1) and TcaM (ACB37733.1) involved in megalomicin and tetrocarcin A biosynthesis, respectively. In addition, they show similarity to isovaleryltransferases from different origins including Pau6/PauY6 and Pau24/PauY24, respectively, in S. paulus (KJ721164.1) and Streptomyces sp.YN86 (KJ721165.1) paulomycin biosynthesis clusters. On the other hand, Plm19 show similarity to GCN5-related N-acetyltransferase bthur0008_41520 (ZP_04104064.1). However, Plm19 has no orthologues annotated in paulomycin biosynthesis clusters in S. paulus (KJ721164.1) and Streptomyces sp.YN86 (KJ721165.1) , but the corresponding gene is present in both sequences between pau21 and pau22 in S. paulus NRRL8115 and pauY21 and pauY22 in Streptomyces sp.YN86.
Enzymes involved in paulic acid biosynthesis
Several activities might be involved in the biosynthesis of the paulic acid moiety. Paulic acid contains an isothiocyanate residue (−N = C = S) that determines the paulomycin characteristic absorbance peak at 275 nm and confers antibiotic activity to paulomycins . Since the isothiocyanate moiety presents nitrogen and sulfur atoms there must be in the cluster, genes encoding an aminotransferase and a sulfotransferase involved in the introduction of these components. In the biosynthesis cluster, plm28 encodes a protein containing an UBA/THIF-type NAD/FAD binding fold (IPR000594) that might act as a sulfotransferase based on the similarity to MoeZ-like enzymes, which transfer sulfur during molybdopterin biosynthesis . On the other hand, plm29 encodes a putative aminotransferase. For both enzymes, Plm28 and Plm29, there are orthologues (Pau30/PauY30 and Pau31/PauY31) annotated in paulomycin biosynthesis clusters of S. paulus (KJ721164.1) and Streptomyces sp.YN86 (KJ721165.1), respectively.
Biosynthesis of L-paulomycose and generation of novel paulomycin derivatives
The paulomycin biosynthesis gene cluster contains all genes necessary for the biosynthesis of L-paulomycose. An intermediate in the biosynthesis of L-olivose, NDP-4-keto-L-olivose , might be generated by the activity of d-glucose-1-phosphate synthase Plm21, dTDP-glucose 4,6-dehydratase Plm20, NDP-hexose 2,3-dehydratase Plm13, dTDP-4-keto-6-deoxy-L-hexose 2,3-reductase Plm40 and dTDP-4-keto-6-deoxyhexose 3,5-epimerase Plm42. The intermediate NDP-4-keto-L-olivose might then suffer an O-methylation, most probably by dTDP-6-deoxy-L-hexose 3-O-methyltransferase Plm41, and the incorporation of a two-carbon side chain at C4 to generate L-paulomycose (Fig. 7). Two enzymes from the pathway with high similarity to pyruvate dehydrogenase E1 α and β subunits, Plm8 and Plm9, might participate in the incorporation of such side chain. A similar mechanism has been shown to participate in the biosynthesis of kosinostatin , it has been biochemically characterized in yersinose A biosynthesis , and it has been genetically elucidated in avilamycin A pathway . In all those cases the mechanism involves the transfer of a two-carbon side chain into the deoxysugar from pyruvate. All enzymes involved in L-paulomycose biosynthesis mentioned in this section present orthologues into paulomycin biosynthesis clusters in S. paulus and Streptomyces sp.YN86 (Table 1).
∆SUG mutant strain was used as recipient to host plasmid pFL844T  that contains all genes required for the biosynthesis of L-amicetose and L-olivose. Plasmid pFL844T contains, in addition, a 3-O-methyltransferase coding gene (oleY). OleY is able to introduce an O-methyl group into L-olivose and other deoxysugar moieties . The resultant strain ∆SUG/844 showed the appearance of five new UPLC peaks (Fig. 10a), with retention times of 4.0 min (17), 4.7 min (18), 4.9 min (19), 5.3 min (20) and 5.7 min (21). These compounds showed the characteristic paulomycin absorption spectrum with maxima at 236, 275 and 323 nm, and possessed masses of m/z, 687 [M + H]+ (17), 759 [M + H]+ (18), 773 [M + H]+ (19), 757 [M + H]+ (20) and 743 [M + H]+ (21). NMR characterization of these paulomycin derivatives (Additional file 3: Figures S62–S95, Tables S9–S10) showed they correspond to: 3′-O-demethyl-paulomycin E (17), 3′-O-demethyl-paulomycin B (18), 3′-O-demethyl-paulomycin A (19), 3′-demethoxyl-paulomycin A (20) and 3′-demethoxyl-paulomycin B (21) (Fig. 10b). All these compounds are novel derivatives of paulomycin A, B and E with modifications at the C3′ position of the deoxysugar, lacking the methoxy group (compounds 20 and 21) or the O-methyl group (compounds 17, 18 and 19). Curiously, no compounds that present a 3′-O-methylation, showing the effect of OleY, have been identified. O-methyltransferase OleY has been previously reported to methylate the C3′ hydroxyl group of different deoxysugars, including L-olivose, when attached to macrolide or anthracycline type compounds [41, 42].
Biological activity of novel paulomycins
The antibacterial activity of paulomycin A (1), paulomycin B (2), paulomycin E (3), paulomycin F (12), 13-O-deacetyl-13-O-paulyl-paulomycin E (14′), 13-O-deacetyl-13-O-paulyl-paulomycin B (15′), 13-O-deacetyl-13-O-paulyl-paulomycin A (16′), 3′-O-demethyl-paulomycin B (18), 3′-O-demethyl-paulomycin A (19), 3′-demethoxyl-paulomycin A (20) and 3′-demethoxyl-paulomycin B (21) was monitored against Gram-positives Micrococcus luteus, Staphylococcus aureus, S. epidermidis, and Streptococcus agalactiae, and Gram-negatives Escherichia coli, Pseudomonas aeruginosa, Serratia marcenscens and Klebsiella pneumonia.
Paulomycins A (1), B (2), E (3) and F (12) showed a good antibacterial activity against all Gram-positive bacteria tested, being more active against S. agalactiae with inhibition halos of 21, 21, 19 and 18 mm, respectively (Additional file 4: Figure S96). Paulomycin derivatives carrying modifications in the L-paulomicose moiety (compounds 18, 19, 20 and 21) retain antibacterial activity but were less active than paulomycins A and B. The best activity observed was also against S. agalactiae, being the most active 3′-O-demethyl-paulomycin A (19) with an inhibition halo of 20 mm. Paulomycin derivatives carrying an acyl migration of paulic acid to the C13 hydroxyl group (compounds 14′, 15′ and 16′) showed no antibacterial activity against the Gram-positive bacteria tested (Additional file 4: Figure S96). All paulomycins and their derivatives were found inactive against the all Gram-negative bacteria tested.
Genome mining of S. albus J1074 chromosome sequence predicted the presence of 27 gene clusters putatively involved in secondary metabolites biosynthesis . Five of these clusters were found to direct the biosynthesis of different metabolites: blue pigment indigoidine, polycyclic tetramate macrolactam 6-epi-alteramides, polyene candicidins, non-ribosomal peptide antimycins and glycosylated antibiotic paulomycins . Paulomycin biosynthesis gene cluster was initially reported to comprise a region of approximately 60 kb from sshg_05313 (plm1) to sshg_05354 (plm42) based on in silico analysis . These limits have been confirmed in this work by analysis of gene expression and gene inactivation experiments. Production of paulomycins by S. albus J1074 was previously reported to be very variable and a reproducible pattern for production of these compounds was not achieved using different batches of media or changing the culture conditions . Furthermore, paulomenols, initially proposed to be paulomycins intermediates , were found to be paulomycins degradation products . More recently, additional biosynthesis gene clusters involved in paulomycins biosynthesis have been reported at S. paulus NRRL8115 and Streptomyces sp. YN86, and a putative biosynthesis pathway has been proposed base on in silico analysis .
In addition to a clear instability of paulomycins, which turn into paulomenols by loss of the paulic acid moiety , paulomycins intermediates also showed structural instability since products expected to be accumulated by mutant strains generated in this work were mostly degraded or modified into shunt products. According to their respective accumulated products, these mutant strains can be divided in two groups: (i) one group including mutant strains affected in steps occurring before paulic acid incorporation (SAM5331, SAM5335, SAM5340 and SAM5341) and (ii) a second group including mutant strains defective in enzymes acting in late steps once paulic acid has been incorporated into the corresponding intermediate (SAM5315, SAM5324, SAM5334 and ∆SUG). On the basis of all these mutants a pathway for paulomycins biosynthesis is proposed (Fig. 7).
The first set of mutant strains are affected in genes coding for: acyltransferase Plm19, which we propose to be involved in the incorporation of paulic acid while Li and coworkers  proposed to be performed by 3-oxoacyl-ACP synthase III Pau29 (Plm27); C-glycosyltrasferase Plm23; sulfotransferase Plm28; and aminotransferase Plm29, the last two enzymes likely involved in paulic acid biosynthesis. These mutants accumulate shunt products that do not contain paulic acid: 2-aminobenzoic acid (8), N-acetyl-orto-aminobenzoic (10) and deoxydehydrochorismic acid (11). 2-aminobenzoic acid (anthranilic acid, 8), might derive from DHHA dehydration (Fig. 5). Anthranilic acid should also be the intermediate leading to N-acetyl-orto-aminobenzoic (10) by N-acetylation. Aminotransferase Plm29 might be involved in paulic acid biosynthesis and acyltransferase Plm19 should transfer the paulic acid moiety to an early paulomycin intermediate in form of C-glycosylated quinone (Fig. 7). This putative glycosylated intermediate has not been identified in the aminotransferase and acyltransferase mutants. Perhaps it is unstable and therefore leads to the accumulation of early precursors that are then transformed into anthranilic acid (8) and N-acetyl-orto-aminobenzoic (10). The fact that inactivation of plm23 led also to accumulate N-acetyl-orto-aminobenzoic (10) points to Plm23 C-glycosyltransferase acting at early stages of paulomycin biosynthesis on a quinone intermediate that has not been detected in SAM5322 mutant strain. Thus, this unidentified quinone intermediate might, in addition, be unstable (Fig. 5). The same interpretation can be applied to the accumulation of deoxydehydrochorismic acid (11), probably generated by chorismate dehydration, by SAM5335 mutant strain. Other compounds not characterized, such as 9, produced by SAM5331, SAM5341 and SAM5335 strains, shared similar absorption spectrum to N-acetyl-orto-aminobenzoic (10), 2-aminobenzoic acid (anthranilic acid) (8) and deoxydehydrochorismic acid (11) and possess masses lower than 150 Daltons pointing to possible modification of other paulomycin intermediates such as 2-amino-2-deoxyisochorismate (ADIC) or 3-hydroxyanthranilic acid (3-HAA) (Figs. 5, 7).
The second set of mutant strains mentioned above are defective at: acyltransferase Plm3, responsible for conversion of paulomycin F (12) into paulomycin A (1) and B (2) by incorporation of 2-methylbutyrate and isobutyrate, respectively; glycosyltransferase Plm12, involved in incorporation of paulomycose; acyltransferase Plm22 (Pau24), which incorporates acetate into 13-O-deacetyl-paulomycin E (14) to generate paulomycin E (3) in a different way as it was proposed previously  involving the incorporation of an acetyl group into TDP-D-allose prior to its attachment to the quinone moiety; and enzymes involved in L-paulomycose biosynthesis: dTDP-4-keto-6-deoxy-L-hexose 2,3-reductase Plm40, dTDP-6-deoxy-L-hexose 3-O-methyltransferase Plm41, and dTDP-4-keto-6-deoxyhexose 3,5-epimerase Plm42 (Fig. 7). These mutants accumulated compounds containing paulic acid. SAM5324 and ∆SUG mutant strains produce 6-hydroxyl-paulinone (6). SAM5315 mutant strain produces intermediate paulomycin F that might derive of paulomycin E by reduction of paulomycose keto group leading to a hydroxyl group (Fig. 5). On the other hand, SAM5334 produces 13-O-deacetyl-paulomycin A (16) and 13-O-deacetyl-paulomycin B (15) that might originate from 13-O-deacetyl-paulomycin E (14) (paulomycin intermediate also identified at SAM5334) by incorporation of 2-methylbutyrate and isobutyrate, respectively (Fig. 5). These results point acyltranferase Plm3 is a flexible enzyme, capable of introducing 2-methylbutyrate and isobutyrate either on paulomycin F (12) (Fig. 7) or on13-O-deacetyl-paulomycin F (Fig. 8). Furthermore, since 13-O-deacetyl-paulomycin E (14), 13-O-deacetyl-paulomycin A (16) and 13-O-deacetyl-paulomycin B (15) can be converted into paulomycin E, A and B, respectively, acyltransferase Plm22 must also be flexible enough to introduce acetate into those compounds (14, 15 and 16), acting in an alternative pathway (Route B) for the transformation of 13-O-deacetyl-paulomycin E (14) into paulomycin A (1) and B (2) (Fig. 8). Compounds 6, 14, 15 and 16, lacking the acetate moiety at C13 hydroxyl group, suffer a paulic acid migration to that position, thus becoming shunt products: 6-hydroxyl-13-O-paulyl-paulinone (6′), 13-O-deacetyl-13-O-paulyl-paulomycin E (14′), 13-O-deacetyl-13-O-paulyl-paulomycin B (15′) and 13-O-deacetyl-13-O-paulyl-paulomycin A (16′). Migration of acyl groups in aqueous solutions has been demonstrated to occur in other compounds such as betacyanins where glucose 6′-O-position is always favored , thuggacins that suffer acyl migrations of their lactone group , and chloramphenicol that undergoes an intra molecular rearrangement of an acetyl group from 3-hydroxyl to 1-hydroxyl group . It has been demonstrated that acyl migration occurs to primary hydroxyl groups, which is the most stable position for acyl moieties [46, 47]. Stabilization of paulic acid to its natural position at C11 might be determined by transferring acetate at C13 hydroxyl group by acyltransferase Plm22.
Regarding paulic acid biosynthesis, in addition to sulfotransferase Plm28 and aminotransferase Plm29, some other activities are necessary for its biosynthesis, (Fig. 7). However, the lack of compounds containing incomplete versions of paulic acid being produced by SAM5340 or SAM5341 mutants suggest a high specificity of acyltransferase Plm19 for paulic acid and makes characterization of this subpathway a complex issue that might be addressed in a different work. A related issue is paulic acid biosynthetic origin. Based on structural similarities, paulic acid might derive from butyrate or crotonate (Fig. 7). If butyrate corresponds to the real precursor then feeding experiments using valine or isoburyrate should increase the production of all paulomycins (A, B, C and E), but only enhanced production of paulomycin B has been reported .
The expression of pFL844T, containing a set of deoxysugar biosynthesis genes for the biosynthesis of L-amicetose and L-olivose, into ∆SUG mutant strain, affected in the biosynthesis of L-paulomycose, led to the generation of new glycosylated forms of paulomycins. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. On the other hand, O-methyltransferase OleY, which has been previously shown to be flexible for modification of deoxysugar moieties attached into macrolide or anthracycline type aglyca [41, 42], is not apparently able to methylate 3′-O-demethyl-paulomycin E (17), 3′-O-demethyl-paulomycin B (18) or 3′-O-demethyl-paulomycin A (19), which will lead to recover the production of paulomycin A, B and E. Bioactivity testing of this paulomycin derivaties showed that the removal of either the L-paulomycose moiety C3′ methoxy group (compounds 20 and 21) or the O-methyl group (compounds 18 and 19) clearly decreases the antibacterial activity of the compounds with respect to paulomycin A and B. In contrast, the acyl migration of paulic acid to the C13 hydroxyl group due to the absence of the characteristic acetate moiety (compounds 14′, 15′ and 16′) render these paulomycin derivatives inactive.
Paulomycin biosynthesis has been shown to be repressed by afsA-y, γ-butyrolactone synthase gene in Streptomyces sp. YN8 . However, there is not an afsA homologue present in S. albus J1074 or S. paulus NRRL 8115 , indicating that the upper (pleiotropic) level of paulomycin regulation is strain-specific. Regulation of paulomycins biosynthesis is controlled in S. albus J1074 by at least three pathway-specific regulatory systems: LuxR-family Plm2, SARP-family Plm10 and LuxR-family Plm30. Inactivation of genes encoding the last two transcriptional regulators abrogates paulomycin production, while inactivation of plm2 led to a considerable reduction of paulomycins yields (20 %). On the other hand, plm1 encoding a TetR-family transcriptional regulator acts as a repressor of the pathway since its inactivation lead to an increased production of paulomycin B and paulomenol B.
We have unraveled paulomycins biosynthesis pathway by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A, B and E containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. Furthermore, the pyruvate dehydrogenase system formed by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. Bioactivity testing of paulomycin derivatives showed that the L-paulomycose moiety C3′ methoxy group is important for the bacterial activity since those compounds lacking this group are less active than their corresponding counterparts. In addition, the paulic acid moiety is not only essential for paulomycins antibacterial activity but also its location is important. The lack of the D-allose acetate moiety and its substitution by a paulic acid moiety renders the corresponding paulomycin derivatives inactive as antibacterial agents.
Strains and culture conditions
Bacterial strains used in this work were S. albus J1074  and S. albus B29 . Escherichia coli DH10B (Invitrogen) and ET12567 (pUB307)  were used for subcloning and intergeneric conjugation, respectively. Growth medium for S. albus was tryptone soya broth (TSB), MA medium was used for sporulation and R5A as regular production medium . MFE medium (Glucose (10 g/L), Soy bean flour (5 g/L), MOPS (21 g/L), Yeast extract (0.2 g/L), MgSO4·7H2O (0.6 g/L), K2HPO4 (1.75 g/L), CaCl2 (5 mg/L), MnCl2 (1 mg/L), ZnSO4 (1 mg/L), FeSO4 (5 mg/L), pH 6.8) was used for production and purification of compounds 6, 12, 14, 15, 16, 17, 18, 19, 20 and 21. E. coli media were those described in the literature (LB and TB) . When plasmid-containing clones were grown, media were supplemented with appropriate antibiotics: ampicillin (100 µg/mL), tobramycin (20 µg/mL), apramycin (25 µg/mL), thiostrepton (50 µg/mL), tetracycline (10 µg/mL), chloramphenicol (25 µg/mL) and nalidixic acid (50 µg/mL).
DNA manipulation and plasmids
DNA manipulations were performed according to standard procedures for E. coli  and Streptomyces . PCR conditions used for all amplifications were 99.9 °C for 4 min; 20 cycles of 99.9 °C for 20 s, 65–45 °C touchdown for 20 s and 72 °C for 45 s followed by 10 cycles of 99.9 °C for 20 s, 60 °C for 20 s and 72 °C for 45 s. Final extension was performed at 68 °C for 10 min. Pfx DNA polymerase (Invitrogen) and 2.5 % dimethylsulphoxide (DMSO) were used for all amplifications. PCR products of the expected sizes were initially cloned into pCR-BLUNT for sequencing verification. All oligoprimers used for PCR amplifications are shown in Additional file 1: Table S1. Plasmids used in this work were: pOJ260  for gene disruption; pEFBAoriT  and pHZ1358  for gene replacement; pEM4T  was used for gene expression; pLHyg  was the source of the hygromycin resistance gene hyg; and pCR-BLUNT (Invitrogen) was used for cloning PCR products. Plasmid pFL844T  was used for the generation of novel paulomycin derivatives by combinatorial biosynthesis.
Methods regarding the construction of plasmids for gene inactivation and ectopic expression and the generation of S. albus J1074 mutant strains are provided in Additional file 1. Methods for co-culture experiment are provided in Additional file 2.
Isolation of total RNA and gene expression analysis
Mycelium from R5A liquid cultures of S. albus J1074 was obtained at 48 h following a previously described procedure . Transcript detection analysis was carried out by using the SuperScript one-step RT-PCR with Platinum® Taq DNA polymerase (Invitrogen) with 100 ng of total RNA as a template. Dimethyl sulphoxide (5 % v/v, final) was added to all reactions along with RNAguard RNase inhibitor (32.2 U per reaction) (Amersham Pharmacia Biotech, Europe GmbH, Barcelona, Spain). Conditions were those described before  but using specific amplification temperatures depending of each set of primers. Primers listed in Additional file 1: Table S1 were used to generate PCR products of different lengths around 500 bp. Negative controls for each pair of primers were carried out with Platinum® Taq DNA polymerase (Invitrogen) in the absence of reverse transcriptase to confirm that amplified products were not due to the presence of contaminating chromosomal DNA in RNA preparations. Oligonucleotides HRDB-GB1-F and HRDB-GB2-R for hrdB [57, 58], encoding a constitutively expressed housekeeping sigma factor, were used as an internal control to normalize RNA samples. RT-PCR analysis were carried out at least three times for each pair of primers and the RT-PCR products were separated in agarose gels and visualized by ethidium bromide staining. Identity of PCR products was verified by direct sequencing with one of the amplification primers.
Analysis of metabolites by UPLC and HPLC–MS and isolation of compounds
Whole cultures of S. albus J1074 and mutants generated in this work were extracted with ethyl acetate containing formic acid (1 %), to enhance the extraction of compounds containing ionizing groups, and analyzed by UPLC and LC–MS for the production of paulomycins, following previously described methods [14, 59]. Reversed phase chromatography was performed in an Acquity UPLC instrument fitted with a BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters). Samples were eluted with 10 % acetonitrile for 1 min, followed by a linear gradient from 10 to 100 % acetonitrile over 7 min, at a flow rate of 0.5 mL/min and a column temperature of 35 °C. For HPLC–MS analysis, an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 µm, 2.1 × 150 mm, Waters) was used. Solvents were the same as above and elution was performed with an initial isocratic hold with 10 % acetonitrile during 4 min followed by a linear gradient from 10 to 88 % acetonitrile over 26 min, at 0.25 mL/min. MS analysis were done by electrospray ionization in the positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 V. Detection and spectral characterization of peaks was performed in both cases by photodiode array detection in the range from 200 to 500 nm, using Empower software (Waters) to extract bidimensional chromatograms at different wavelengths, depending on the spectral characteristics of the desired compound.
Isolation of compounds accumulated by S. albus J1074 mutants SAM5315, SAM5324, SAM5331, SAM5334, SAM5335 and ∆SUG, affected in the production of paulomycins was performed following the procedure previously described for isolation of paulomycins .
Structural characterization of compounds
Compounds 6′, 7, 8, 10, 11, 12, 14′, 15′, 16′, 17, 18, 19, 20 and 21 corresponding to 6-hydroxyl-13-O-paulyl-paulinone (6′), (2E)-17-(4′-aminophenyl)-3,11,15-trihydroxy-10,12,14-trimethyl-17-oxo-heptadeca-4,6,8-trienoic acid (7), 2-aminobenzoic acid (anthranilic acid) (8), N-acetyl-orto-aminobenzoic acid (10), deoxydehydrochorismic acid (11), paulomycin F (12), 13-O-deacetyl-13-O-paulyl-paulomycin E (14′), 13-O-deacetyl-13-O-paulyl-paulomycin B (15′), 13-O-deacetyl-13-O-paulyl-paulomycin A (16′), 3′-O-demethyl-paulomycin E (17), 3′-O-demethyl-paulomycin B (18), 3′-O-demethyl-paulomycin A (19), 3′-demethoxyl-paulomycin A (20) and 3′-demethoxyl-paulomycin B (21) were subjected to LC/ESI-TOF analysis in order to determine their molecular formula. The structural elucidation of compounds 6′, 7, 8, 10, 11, 12, 14′, 15′, 16′, 17, 18, 19, 20 and 21 was carried out by analysis of a combination of 1D (1H and 13C), and 2D (1H-1H COSY, TOCSY, 1H-13C heteronuclear single-quantum correlation (HSQC)-edited and 1H-13C heteronuclear multiple-bond correlation (HMBC) NMR experiments and comparison of the spectra obtained with those described in the literature (supporting information). Solvents used in the NMR analyses were deuterated methanol (CD3OD) for compounds 8, 10 and 11, or deuterated DMSO (DMSO-d 6 ) for compounds 6′, 7, 12, 14′, 15′,16′, 17, 18, 19, 20 and 21 (Additional file 3).
Methods regarding analysis of the antibacterial activity of paulomycins are provided in Additional file 4.
CO, JAS and CM conceived and designed the project; AG and MR conducted experiments; AG, MR, CM, JAS and CO analyzed the data; AFB carried out compound purifications; CO drafted the manuscript and CO, JAS and CM contributed to preparing the final version of the paper. All authors read and approved the final manuscript.
This research was supported by the Spanish Ministry of Economy and Competitiveness Grants, MINECO (BIO2012-33596 to J. A. S. and PIM2010EEI-00752 to C. M.) and “Apoyo a grupos de excelencia”, Principado de Asturias-FEDER (FC-15-GRUPIN14-014). A. G. was the recipient of a fellowship of FICYT (Asturias, Spain). We thank Fundación Bancaria Caja de Ahorros de Asturias for financial support to C. O. We also like to thank Dr. Fernando Reyes from Fundación Medina for technical support in the structural elucidation of compounds.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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