Mutational Analysis Reveals That All Tailoring Region Genes Are Required for Production of Polyketide Antibiotic Mupirocin by Pseudomonas fluorescens

Abstract

The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with ΔmupC and ΔmupO, ΔmupU, ΔmupV, or ΔmacpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V. The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with ΔmupC and ΔmupO, ΔmupU, ΔmupV, or ΔmacpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V. Mupirocin is a polyketide antibiotic active against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. It is a competitive inhibitor of isoleucyl-tRNA synthetase (IleRS), blocking binding of isoleucine and ATP to IleRS (1Hughes J. Mellows G. Biochem. J. 1978; 176: 305-318Crossref PubMed Scopus (171) Google Scholar). Mupirocin is a mixture of pseudomonic acids (PA) 5The abbreviations used are: PA, pseudomonic acid; ER, enoyl reductases; WT, wild type; ORF, open reading frame; PKS, polyketide synthase; KS, ketosynthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; TE, thioesterase; MeT, methyltransferase; HMG, 3-hydroxy-3-methylglutaric acid; PPTase, phosphopantetheinyl transferase; N-AHL, N-acyl homoserine lactone; IPTG, isopropyl-1-thio-β-d-galactopyranoside; HPLC, high pressure liquid chromatography.5The abbreviations used are: PA, pseudomonic acid; ER, enoyl reductases; WT, wild type; ORF, open reading frame; PKS, polyketide synthase; KS, ketosynthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; TE, thioesterase; MeT, methyltransferase; HMG, 3-hydroxy-3-methylglutaric acid; PPTase, phosphopantetheinyl transferase; N-AHL, N-acyl homoserine lactone; IPTG, isopropyl-1-thio-β-d-galactopyranoside; HPLC, high pressure liquid chromatography. produced by Pseudomonas fluorescens NCIMB 10586, with PA-A accounting for 90%. PA-A consists of a C9 saturated fatty acid (9-hydroxynonanoic acid, 9HN) joined via an ester linkage to a C17 unsaturated polyketide moiety containing a tetrahydropyran ring (monic acid, MA, Fig. 1A) (2Chain E.B. Mellows G. J. Chem. Soc. Chem. Commun. 1974; : 847-848Crossref Google Scholar, 3Chain E.B. Mellows G. J. Chem. Soc. Perkin Trans. 1977; 1: 294-309Crossref Google Scholar). PA-B has an additional hydroxyl group at C-8 (4Chain E.B. Mellows G. J. Chem. Soc. Perkin Trans. 1977; 1: 318-322Crossref Scopus (47) Google Scholar), in PA-C the C-10,11 epoxide group is replaced with a double bond (5Clayton J.P. O'Hanlon P.J. Rogers N.H. Tetrahedron Lett. 1980; 21: 881-884Crossref Scopus (43) Google Scholar), and PA-D has an additional double bond at C-4′,5′ in the 9-HN moiety (6O'Hanlon P.J. Rogers N.H. Tyler J.W. J. Chem. Soc. Perkin Trans. 1983; 1: 2655-2665Crossref Scopus (35) Google Scholar). Polyketides are biosynthesized by condensation of small carboxylic acids catalyzed by ketosynthases (KS) generating β-carbonyl groups that may then be left unreduced or reduced partially or fully by the combined activities of ketoreductases (KR), dehydratases (DH), and enoyl reductases (ER). In archetypal Type I polyketide synthase (PKS) systems the starter and extender carboxylic acids are loaded onto KS and acyl carrier proteins (ACP), respectively, via acyl-CoA-activated intermediates, catalyzed by acyltransferases (AT). The growing polyketide chain remains attached to an ACP as a thiolester until its release, typically by hydrolysis catalyzed by a terminal thioesterase (TE). The polyketide backbone may then require further modification to produce the final metabolite. Post-PKS tailoring includes such processes as further oxidative-reductive steps, addition of moieties such as sugar units, methylation, halogenation, and cyclization. It is this variety in reduction, addition of functional groups, and choice of carboxylic units that make the polyketides a very diverse class of metabolites in both structure and activity (7Staunton J. Weissmann K.J. Nat. Prod. Rep. 2001; 18: 380-416Crossref PubMed Scopus (1253) Google Scholar). There are several examples of PKS combinatorial biosyntheses resulting in novel polyketide metabolites (for a review see Ref. 8Weissman K.J. Leadlay P.F. Nat. Rev. Microbiol. 2005; 3: 925-936Crossref PubMed Scopus (344) Google Scholar), and such an approach is also being applied to tailoring enzymes (for a review see Ref. 9Rix U. Fischer C. Remsing L.L. Rohr J. Nat. Prod. Rep. 2002; 19: 542-580Crossref PubMed Scopus (223) Google Scholar). The 74-kb mupirocin biosynthetic gene cluster has been sequenced and many of the open reading frames (ORFs) assigned putative functions (Table 1 and Fig. 2) (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The first half of the cluster contains 3 large Type I multifunctional PKS genes plus associated trans-acyltransferase as well as two single ORFs mupA and mupB, while the second half, referred to as the tailoring region, contains 26 single ORFs (mupC-X, and macpA-E), plus two PKS-like genes mmpE and mmpF. A model for biosynthesis of the mupirocin backbone was proposed based on the cluster sequence, feeding experiments (11Feline T.C. Jones R.B. Mellows G. Phillips L. J. Chem. Soc. Perkin Trans. 1977; 1: 309-318Crossref Scopus (43) Google Scholar, 12Martin F.M. Simpson T.J. J. Chem. Soc. Perkin Trans. 1989; 1: 207-209Crossref Scopus (25) Google Scholar) and comparisons with other polyketide biosynthetic pathways (Fig. 2B) (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). MmpD and MmpA together contain 6 modules for condensation and reduction of acetate-derived units, which with two methyl transferase domains and assuming that the DH domain in module 1 is inactive (because it contains aspartic acid and alanine residues instead of glycine and proline in the DH motif HXXXGXXXXP) could generate the backbone of a The of 9-HN is to A starter may be by catalyzed by a fatty acid synthase or with additional activity by The resulting backbone further to produce the PA-A in Fig. to or after of and oxidation of the C-10,11 double bond to the C-10,11 epoxide double bond reduction and oxidation to produce the tetrahydropyran and of the methyl group a that of a putative a novel mupirocin the tetrahydropyran ring S.M. J. Hothersall J. Simpson T.J. Thomas C.M. Chem. Commun. 2005; : PubMed Scopus Google Scholar). to be for of the methyl group required for the of the tetrahydropyran while of or an immediate with 9-HN and other tailoring tetrahydropyran ring ring after or is of the enzymes involved in In the of of other ORFs mupO, and macpE in a to PA-B as the and that are for PA-A but not PA-B S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). that PA-B is not as been by of but is instead a to PA-A or a function of mupirocin biosynthesis KS, KS, ACP, KS, KR, ACP, KS, ACP, KS, DH, KR, ACP, ACP, ACP, KS, DH, KR, MeT, ACP, KS, DH, KR, ACP, KS, DH, KR, MeT, ACP, KS, KR, synthase in a work also of and of which and I could be assigned a in of of the cluster (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the of all ORFs in the tailoring region that all are required for production. of the of mutants with NCIMB 10586, a of novel by mupirocin and mupirocin in Fig. B and C. The of proteins in mupirocin biosynthesis is while of of and other novel metabolites be in and fluorescens NCIMB Thomas C.M. Scopus (25) Google Scholar) was used as the mupirocin J. Biol. 1983; PubMed Scopus Google Scholar) was used for and U. 1983; 1: Scopus Google Scholar) was used to and E. J. Microbiol. PubMed Scopus Google Scholar) was used in to mupirocin production. for and used or in this are in and of used for gene and experiments in a of gene and acid to of of of as of WT, not double mutants of with mupO, U, or macpE as for single mutants of mupO, U, and macpE as S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). in a a acid to of of of as of WT, not double mutants of with mupO, U, or macpE as for single mutants of mupO, U, and macpE as S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). and at in Thomas C.M. E. PubMed Scopus Google Scholar) and with with fluorescens at in and and of was with antibiotic as and and was by the of and J. PubMed Scopus Google Scholar), or with of was J. A Scholar). a E. with the of U. PubMed Scopus Google Scholar). was the which is based on the chain U. 1977; Scopus Google Scholar). The on an of was to the and E. to fluorescens as A mixture of of E. containing the and fluorescens on to a The was on at The mixture was in and on or with the antibiotic to for the of the and not E. in which the the the was in at antibiotic on with and onto that and for Mupirocin of fluorescens to the onto and at for The then with with a and at for and for the of around the fluorescens of at and for at at for and the at to was or at and acid at 1 of and as for was at for The was with to then with The was The then by The was in of and by as with to of the of a the and the and by The was in 1 of by on a of of Pseudomonas at for was to 1 onto containing mupirocin and at for or of was was or 1 of fluorescens with two of at for and at with in 1 was with both an during and with a a and was on an was generated a and with at for then first and and at for The was at for of the first was then used as in the second two to generate and The was of at for at for for mupF, and or for for and at for by a single final at for ORFs of the for Mupirocin ORFs of the tailoring region and to as S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The is by of of was by two and which the region to be The generated and includes the first of while the generated and includes the final (for see two via a the to an of which but not to was to fluorescens NCIMB by with E. the by in or and was by the on the of the to of bacteria, of of in which the the by in or or by on the the gene whose in the to with identified by with and A of ΔmupC mutants a reduction in activity against to of (Table 3 and Fig. The was then by and the promoter (Table for see The resulting was the ΔmupC and (Table the in must be to of function and not to a on for ΔmupC activity to of In the further tailoring region ORFs and and and mmpE and by the of mutants a with a reduction in activity against to of 3 and Fig. mutants reduced of and was as a in to be in to the (Table of and whose to be in with is in the of the other activity in the with containing the to this and whose the as the (Table is an isoleucyl-tRNA synthetase to mupirocin on E. (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). as that NCIMB is to mupirocin in of while the a between and on with not while the in may which mupirocin its to mupirocin is The other deletions that not that are as pairs of with and with to the that gene pairs must be the to be polypeptides may to to functional this and together and (for see was both and and by for its to mupirocin production. was in the and together which mupirocin to but not for the of in the or the single mutants was that the in the a that with the functional of the two ORFs double deletions both and or both and of or respectively, double mutants antibiotic activity to with the also required with a to be functional in was together with the in both and and to and mupirocin production. of of ORFs based on the of the region indicated that all the ORFs in the and it is to that the region mupA to a single transcriptional two of the genes of the tailoring region, and on and the of binding to in the (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), for both are ORFs on the not which is the the with a while the with CoA in the or on the and genes as well as the genes of and to which could be as to generate in all several and for both for and which being used as the of the whose was by and The for and of the in both The was and sequenced and the region been of in both was for the which was in the and to be the this it to that could mupA to further the of mupF, with ORFs in both to the promoter and for to The been for the of mupF, but in the the the be in the by a second of the first of the of the the of the and by together as a single including of the putative of the control of the promoter in this was to the in it could partially a that includes the of the it runs in the was and as an together with and such that or and respectively, Fig. be the to the and deletions implying that and has its promoter (Fig. was also by the in on its the to that the of of by is to a of and to be a the active of that it not be in the a such as reduction of in of mupirocin as indicated by both and of but could be by further the of with ORFs in both and for of the which was for both of and reduced activity to of The with as by while the not (Table 3 and Fig. was by The of PA-A was to WT, while the in the to the in not the PA-A which at MupC and for PA-A was on of the mutants to produce novel metabolites that the function of to be all mutants reduced PA-A and produced the of and by as a with which has been identified by to be PA-A S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). PA-A has a of A second with is also to be to of a reduction in the PA-A with WT, and the of a with (Fig. The of that PA-A was at with it also a of novel with PA-A (Fig. and of be of the other mutants in the of PA-A and PA-B (Table and of and reduced of PA-A at and to WT, of PA-B with the of PA-B are not at the high of other for mupO, and which a of to PA-B S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). also reduced mupirocin which could be functions as an isoleucyl-tRNA synthetase that mupirocin on is proposed to be a phosphopantetheinyl transferase and a be to on or Type I PKS and and be to produce a mupirocin backbone the novel of and as and by indicated a of with for PA-A for mupirocin and for the novel mupirocin The (Fig. B and by of the of and of metabolites other be in The of mupirocin and mupirocin are with of novel with and two double bond PA-A as required by the indicated by further the that MupC is required double mutants in which both and mupO, or macpE by (Fig. and of the double mutants the mupO, U, V, and macpE mutants, which all PA-B instead of PA-A S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) indicating that MupC after the of In with work S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) the of the cluster tailoring region that all 26 tailoring ORFs are required for normal mupirocin production. activity as a isoleucyl-tRNA synthetase (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) not be involved in biosynthesis the reduction in activity was it was of that mupirocin was not to normal by in of its to mupirocin was The reduction in mupirocin of may be to on genes of the other deletions could be that the of that do not be that of a involved in could as a to antibiotic in the of the The of is also by the on deletions in mupD/E and The that deletions in of ORFs could be by single genes in but double deletions could be by double that genes to be The of of single gene deletions that or gene in the that the of genes not a state but also the with further It be in further work to for for genes to and which functions many pairs of that a functional the of MupJ and are and of which as ORFs a polyketide biosynthesis cluster of function C. PubMed Scopus Google Scholar). A cluster in has been assigned to J. J. G. G. J. PubMed Scopus Google Scholar), and the structure of has been an of the biosynthetic gene cluster to be proposed J. U. PubMed Scopus Google Scholar). the and K gene pair is also associated with an ACP, KS, and as it is in the cluster and in the A and A of J. Chem. Soc. PubMed Scopus Google Scholar, Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). gene is proposed to a methyl onto a polyketide The pair and the A cluster been to of to and to J. Chem. Soc. PubMed Scopus Google Scholar). in work has been U. PubMed Scopus Google Scholar) to the of genes the ACP has been to be to the by a transferase to by the KS which then with an to which is then and as in the It is that MupJ and K with synthase function in a to of an at with the and required to generate the methyl in a to the model for biosynthesis (10El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) (Fig. that in of in the of a novel mupirocin whose structure be as being by of the polyketide of Cooper S.M. J. Hothersall J. Simpson T.J. Thomas C.M. Chem. Commun. Scholar). and a for the also been proposed to be in other systems to and activity of of the large E. enzyme is to be via a Microbiol. PubMed Scopus Google Scholar). to of and of and by or but could be by a all or several of the J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. 1989; PubMed Google Scholar). that of genes may be required for and the the to for of and J. PubMed Scopus Google Scholar). of to the of is its is to J. 2002; PubMed Scopus Google Scholar). It is proposed that such that a at a gene is for and the is may also function to of the is associated with the enzyme in the C. L. Biochem. Scopus Google Scholar). on the cluster that two ORFs and counter to the of the cluster (10El-Sayed A.K. Hothersall J. 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Thomas C.M. 2001; PubMed Scopus Google Scholar). was also by and the region the control of the must also its of is an which may be a putative is to the of The that a in the active of be by the promoter in that the in macpC, which could not be that are for a second the that the in required for but this the then the of in to the the to that of a and this not to be the that may be other function in this region such as a small or additional that remains to be approach of tailoring ORFs and of novel has also in other polyketide systems to the of of in the B biosynthetic generated an that the was for of the bond in the moiety of B J. E. 2005; PubMed Scopus Google Scholar). a putative enzyme of the biosynthetic was in for of in tailoring steps, in and reduction L. U. C. V. Rohr J. J. Chem. 2005; PubMed Scopus (47) Google Scholar). of and of the also generated two as and G. G. U. Chem. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of a in the gene cluster in in the of Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Deletion of in a that could be as a to both a double bond and a C-7 during polyketide chain of in to a C-7 comparisons are with as a ketoreductase, MupC to a dienoyl CoA and other which be with it after a by reduction of the double bond in the module 3 Fig. this the of which also to be implicated in reduction of the double bond S.M. Hothersall J. A.K. C. J. Simpson T.J. Thomas C.M. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). this double mutants of of with and the that also PA-B indicated that MupC after is a putative acyl CoA it is to that it may an onto MupC may on an also attached to proposed that work in the biosynthetic after the of the with but to ring the structure of mupirocin the that and MupC may work during the of the between putative modules and is that and MupC after and proteins that it may work the putative The proposed in Fig. 6 is with this and with the metabolites as a of of and to this PA-B be a on the to is as biosynthetic that PA-B is produced by of that the of the MmpD module is the as in Fig. but with an bond between C-8 and the dienoyl could be by double bond between the module and with of the module of the KS a of to a domain which was not not but it may be that this region between the KS and a DH which are to that this could be an for the proposed double bond then as proposed to produce the 1 (Fig. catalyzed of the double bond and of the hydroxyl group on the resulting epoxide to that to the which could be to PA-B after with 9-HN and C-10,11 the of which to be by to of the C-7 to the 3 catalyzed by the the for to the fully The be reduced by MupC to which on further catalyzed which be to PA-A as for be fully with the of small of PA-B with the the of to not being this the to PA-B as this a for to a of The the for further double and that further for the that the the of the cluster the of between the gene and the proposed biosynthetic gene must with the of genes and could that may to multifunctional the of an bidirectional promoter region the of gene the in the tailoring genes to be involved in tetrahydropyran ring the tailoring region to been by of a then it is that and at been to mupO, U, V, and with which work to the cluster to functions together and polypeptides may the of PA-A production. for of the putative was by the in the of was by of of with