Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586

The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU + macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A. MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.


Construction of mutagenesis plasmids
All plasmid constructs reported in this paper were checked by suitable restriction digests and Sanger sequencing of inserted DNA.

pAW1 -mupV domain 1 Y167F
Primer pairs AW1 plus AW2 and AW3 plus AW4 were used to amplify 555 bp and 502 bp segments of the NCIMB10586 chromosome, respectively. Primers AW2 and AW3 were designed to generate a 23 bp overlap, which included the mupV Y167F mutation (DNA codon TAC to TTC). The products were spliced by overlap PCR using primers AW1 and AW4, and cloned using MfeI/BamHI digestion and ligation with pAKE604 digested with EcoRI/BamHI to yield mutagenesis vector pAW1.

pHHMZC25 -mupZ C25X (TERM)
Primers HHMZ1F plus HHMZ1R and HHMZ2F plus HHMZ2R were used to amplify 546 bp and 582 bp fragments from the NCIMB10586 chromosome. Primers HHMZ1R and HHMZ2F were designed to generate a 21 bp overlap which contained the base to be mutated from C to A, generating a TGA stop codon. The products were spliced by overlap PCR using primers HHMZ1F and HHMZ2R, and cloned using BamHI/XbaI digestion and ligation with pAKE604 digested with EcoRI/BamHI to yield mutagenesis vector pHHMXC25.

R
To allow easy selection of transconjugants after transfer of suicide mutagenesis plasmids from E. coli to P. fluorescens (which is naturally Ap R ) the bla gene was deleted. Primers F∆bla and R∆bla were used to amplify the 6574 bp segment of pAKE604 that excludes the bla gene, recircularised by ligation after cutting with SacI and introduced into E. coli DH5 selecting Km R . A correct clone not conferring Ap R was designated pJC70.

pJC102 -mupV domain 2 V581A, H631A
Primer pairs JC1 plus JC2 and JC3 plus JC4 were used to amplify 546 bp and 500 bp segments of the NCIMB10586 chromosome, respectively. Primers JC2 and JC3 were designed to generate a 21 bp overlap, which included the mupV H631A mutation (DNA codon CAT to GCA). The products were spliced by overlap PCR using primers JC1 and JC4, and then A-tailed and ligated into pGEM-T Easy (Promega). Sequencing revealed a secondary, unintended point mutation (V581A), which was deemed acceptable as it was also in domain 2 of mupV. The DNA fragment was cloned using SalI/EcoRI digestion and ligation into pJC70 to yield mutagenesis vector pJC102.

pMY21 -deletion of mupQ mupS macpD and mmpF
Primers QDA1F plus QDA1R and QDA2F plus QDA2R were used to amplify 547 bp and 504 bp fragments from the NCIMB10586 chromosome. Primers QDA1R and QDA2F were designed to incorporate an XbaI site allowing the two arms to be joined to an in-frame deletion starting in mupQ and ending in mmpF and cloned using EcoRI/BamHI digestion and ligation with pAKE604 digested with EcoRI/BamHI to yield mutagenesis vector pMY21.

Construction of expression plasmids
Initial constructions were designed when we predicted mupO, macpE, mupU, mupV, mupC and mupF to be sufficient for the PAB to PAA conversion. A series of intermediate plasmids were constructed using AT-cloning by the protocol in the pGEM-T Easy manual prior to generation of combinations of genes and then cloning into the broad host-range expression vector pJH10. Primers were designed to include the native NCIMB10586 ribosome binding site (RBS) for each gene. The template DNA for all initial PCRs was the P. fluorescens NCIMB10586 chromosome.

pMMG1 -intermediate carrying mupO
Primers FmupO and RmupO were used to amplify a 1.4 kb fragment, which was AT-cloned into pGEM-T Easy.

pMMG2 -intermediate carrying macpE
Primers FmacpE and RmacpE were used to amplify a 257 bp fragment, which was AT-cloned into pGEM-T Easy.

pMMG34 -intermediate carrying mupU and mupV
Primers FmupUV and RmupUV were used to amplify a 3.6 kb fragment encoding both mupU and mupV, which was AT-cloned into pGEM-T Easy.

pMMG5 -intermediate carrying mupC
Primers FmupC and RmupC were used to amplify a 1.3 kb fragment, which was AT-cloned into pGEM-T Easy.

pMMG6 -intermediate carrying mupF
Primers FmupF and RmupF were used to amplify a 1.0 kb fragment, which was AT-cloned into pGEM-T Easy.

pMMH6 -expression plasmid carrying mupO, macpE, mupU, mupV, mupC and mupF
The 7.6 kb fragment from pMMG66 containing all six genes was released by KpnI/XbaI digestion and cloned into broad host-range expression vector pJH10 cut with KpnI/XbaI.

pJC124 -expression plasmid carrying mupO, mupP, macpE, mupU, mupV, mupC and mupF
Primers FmupOP and RmupOP were used to amplify a 2.4 kb fragment including genes mupO and mupP. Primers FmacpE2 and RmacpE2 were used to amplify a 278 bp fragment carrying macpE. Primers RmupOP and FmacpE2 were designed to overlap. The products were spliced by overlap PCR with primers FmupOP and RmacpE2, to give a 2.6 kb fragment that was digested with KpnI/AflII and inserted into pMMH6 to replace the mupO-macpE segment, yielding pJC124.

pJC134 -expression plasmid pJC133 with point mutation H256A in MupL
Primers FmupLMN and RmupL-H256A were used to amplify an 809 bp N-terminal segment of mupL while primers FmupL-H256A and RmupMN were used to amplify a 4.3 kb fragment with the Cterminal part part of mupL plus mupM and mupN. The overlap in primers FmupL-H256A and RmupL-H256A included the mupL H256A mutation (DNA codon CAC to GCC) allowing overlap PCR using outer primers FmupLMN and RmupMN, to generate a 5.1 kb fragment including mupLH256A, mupM and mupN. This product was AT-cloned into pGEM-T Easy and then released by XbaI/BsaI digest before insertion into XbaI/NotI-digested pJC124 yielding pJC134. Figure