Characterization and engineering of the biosynthesis gene cluster for antitumor macrolides PM100117 and PM100118 from a marine actinobacteria: generation of a novel improved derivative

Background PM100117 and PM100118 are glycosylated polyketides with remarkable antitumor activity, which derive from the marine symbiotic actinobacteria Streptomyces caniferus GUA-06-05-006A. Structurally, PM100117 and PM100118 are composed of a macrocyclic lactone, three deoxysugar units and a naphthoquinone (NQ) chromophore that shows a clear structural similarity to menaquinone. Results Whole-genome sequencing of S. caniferus GUA-06-05-006A has enabled the identification of PM100117 and PM100118 biosynthesis gene cluster, which has been characterized on the basis of bioinformatics and genetic engineering data. The product of four genes shows high identity to proteins involved in the biosynthesis of menaquinone via futalosine. Deletion of one of these genes led to a decay in PM100117 and PM100118 production, and to the accumulation of several derivatives lacking NQ. Likewise, five additional genes have been genetically characterized to be involved in the biosynthesis of this moiety. Moreover, the generation of a mutant in a gene coding for a putative cytochrome P450 has led to the production of PM100117 and PM100118 structural analogues showing an enhanced in vitro cytotoxic activity relative to the parental products. Conclusions Although a number of compounds structurally related to PM100117 and PM100118 has been discovered, this is, to our knowledge, the first insight reported into their biosynthesis. The structural resemblance of the NQ moiety to menaquinone, and the presence in the cluster of four putative menaquinone biosynthetic genes, suggests a connection between the biosynthesis pathways of both compounds. The availability of the PM100117 and PM100118 biosynthetic gene cluster will surely pave a way to the combinatorial engineering of more derivatives. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0443-5) contains supplementary material, which is available to authorized users.

This plasmid and fragment UP-MT (2,294 bp) were then digested with SpeI/NsiI and ligated to afford plasmid pMT-BESN. Insertion of UP-MT and DW-MT at both sides of the aac(3)IV gene in pMT-BESN was verified by PCR and sequencing with primers SpeI-MT, NsiI-MT, BglII-MT and EcoRV-MT. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pMT-BESN to produce the gene replacement plasmid pD-gonMT, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonMT deletion. Replacement of gonMT in the resulting Hyg s Amp R strain, ΔgonMT, was verified by PCR with primers cfMT (outside the deletion cassette) and ApraII (internal to the Apm R gene marker).

pD-gonSL
The upstream (UP-SL) and downstream (DW-SL) sequences flanking gene gonSL were amplified with the primer pairs SpeI-SL/NsiI-SL and NdeI-SL/EcoRV-SL (Table S1), respectively. UP-SL (2,138 bp) and plasmid pEFBA-oriT were digested with SpeI /NsiI and ligated to generate plasmid pSL-SN. This plasmid and fragment DW-SL were digested with NdeI/EcoRV. Digestion of DW-SL with NdeI generated two fragments, of 1.945 and 211 bp, respectively, the 211-bp fragment was cloned into pSL-SN (NdeI/EcoRV sites) to yield plasmid pSL-SN-200, which was then digested with NdeI and ligated to the 1.945-bp fragment to produce plasmid pSL-SNNE. The correct cloning orientation of the 1.945-bp DW-SL fragment was confirmed by PCR with primers NdeI-SL and EcoRV-SL. In addition, insertion of UP-SL and DW-SL at both sides of the aac(3)IV gene in pSL-SNNE was verified by sequencing with primers NdeI-SL, EcoRV-SL, SpeI-SL and NsiI-SL. A 1.6kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was then extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pSL-NESN to yield the gene replacement plasmid pD-gonSL, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonSL deletion.
Replacement of gonSL in the resulting Hyg s Amp R strain, ΔgonSL, was verified by PCR with primers cfSL (outside the deletion cassette) and Apra60 (internal to the Apm R gene marker).

pD-gonS1
The upstream (UP-S1) and downstream (DW-S1) sequences flanking gene gonS1 were amplified with the primer pairs fNsiI-S1/rNsiI-S1 and BglII-S1/EcoRV-S1 (Table S1), respectively. DW-S1 (2,131 bp) and plasmid pEFBA-oriT were digested with BglII/EcoRV and BamHI/EcoRV, respectively, and ligated to generate plasmid pS1-BE. This plasmid and fragment UP-S1 (2.096 bp) were then digested with NsiI and ligated to afford plasmid pS1-BENN. Correct cloning orientation of the gonS1 upstream fragment was confirmed by PCR with primers fNsiI-S1and apra60. In addition, insertion of UP-S1 and DW-S1 at both sides of the aac(3)IV gene in pS1-BENN was verified by sequencing with primers fNsiI-S1, rNsiI-S1, BglII-S1 and EcoRV-S1. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pS1-BENN to yield the gene replacement plasmid pD-gonS1, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonS1 deletion. Replacement of gonS1 in the resulting Hyg s Amp R strain, ΔgonS1, was verified by PCR with primers cfS1 (outside the deletion cassette) and Apra60 (internal to the Apm R gene marker).
Insertion of UP-S2 and DW-S2 at both sides of the aac(3)IV gene in pS2-NNNS was verified by sequencing with primers NsiI-S2, SpeI-S2, fNdeI-S2 and rNdeI-S2. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pS1-BENN to yield the gene replacement plasmid pD-gonS2, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonS2 deletion. Replacement of gonS2 in the resulting Hyg s Amp R strain, ΔgonS2, was verified by PCR with primers cfS2 (outside the deletion cassette) and ApraII (internal to the Apm R gene marker).

pD-gonCP
The upstream (UP-CP) and downstream (DW-CP) sequences flanking gene gonCP were amplified with the primer pairs SpeI-CP/NsiI-CP and BamHI-CP/EcoRV-CP (Table S1), respectively. DW-CP (2,548 bp) and plasmid pEFBA-oriT were digested with BamHI/EcoRV and ligated to generate plasmid pCP-BE. This plasmid and UP-CP (2,100 bp) were then digested with SpeI/NsiI and ligated to afford plasmid pCP-BESN. Insertion of UP-CP and DW-CP at both sides of the aac(3)IV gene in pCP-BESN was verified by PCR and sequencing with primers SpeI-CP, NsiI-CP, BamHI-CP and EcoRV-CP. A 1.6kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pCP-BESN to yield the gene replacement plasmid pD-gonCP, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonCP deletion.
Replacement of gonLCP in the resulting Hyg s Amp R strain, ΔgonCP, was verified by PCR with primers cfCP (outside the deletion cassette) and Apra60 (internal to the Apm R gene marker).
This plasmid and UP-MR (2,571 bp) were then digested with SpeI/NsiI and ligated to yield plasmid pMR-BESN. Insertion of UP-MR and DW-MR at both sides of the aac(3)IV gene in pMR-BESN was verified by PCR and sequencing with primers SpeI-MR, NsiI-MR, BglII-MR and EcoRV-MR. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pMR-BESN to yield the gene replacement plasmid pD-gonMR, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve gonMR deletion. Replacement of gonMR in the resulting Hyg s Amp R strain, ΔgonMR, was verified by PCR with primers cfMR (outside the deletion cassette) and ApraII (internal to the Apm R gene marker).
This plasmid and UP-orf9 (2,496 bp) were then digested with SpeI/NsiI and ligated to afford plasmid pGorf9-BESN. Insertion of UP-orf9 and DW-orf9 at both sides of the aac(3)IV gene in pGorf9-BESN was verified by PCR and sequencing with primers SpeI-orf9, NsiI-orf9, BglII-orf9 and EcoRV-orf9. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pGorf10-BESN to produce the gene replacement plasmid pD-orf9, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve orf9 deletion. Replacement of orf9 in the resulting Hyg s Amp R strain, Δ5201, was verified by PCR with primers cf09 (outside the deletion cassette) and Apra60 (internal to the Apm R gene marker).
Insertion of UP-orf11 and DW-orf11 at both sides of the aac(3)IV gene in pGorf11-SNNE was verified by PCR and sequencing with primers SpeI-orf11, NsiI-orf11, NdeI-orf11 and EcoRV-orf11. A 1.6-kbp fragment containing the hygromycin B resistance (Hyg R ) gene marker, hyg, was extracted from pLHyg by SpeI/NheI digestion and cloned into the XbaI site of pGorf11-SNNE to produce the gene replacement plasmid pD-orf11, which was transferred to Streptomyces caniferus GUA-06-05-006A by intergeneric conjugation to achieve orf11 deletion. Replacement of orf11 in the resulting Hyg s Amp R strain, Δ5259, was verified by PCR with primers cf11 (outside the deletion cassette) and ApraII (internal to the Apm R gene marker).

pC-gonP8
A DNA fragment of 7,326 bp containing the entire ORF of gonP8 (nt -20 to +7,306 from start codon) was amplified with the primer pair cpP8-Xb/cpP8-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonP8. Insertion of gonP8 into pSETHe was confirmed by PCR and sequencing with primers cpP8-Xb and cpP8-ERV. Plasmid pC-gonP8 was then transferred to the mutant strain gonP8by intergeneric conjugation to produce the strain CPgonP8 in which gonP8 inactivation was complemented.

pC-gonM4
A DNA fragment of 998 bp containing the entire ORF of gonM4 (nt -26 to +972 from start codon) was amplified with the primer pair cpM4-Xb/cpM4-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonM4. Insertion of gonM4 into pSETHe was confirmed by PCR and sequencing with primers cpM4-Xb and cpM4-ERV. Plasmid pC-gonM4 was transferred to the mutant strain ΔgonM4 by intergeneric conjugation to produce the strain CPgonM4 in which gonM4 deletion was complemented.

pC-gonMT
A DNA fragment of 1,211 bp containing the entire ORF of gonMT (nt -65 to +1,146 from start codon) was amplified with the primer pair cpMT-Xb/cpMT-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonMT. Insertion of gonMT into pSETHe was confirmed by PCR and sequencing with primers cpMT-Xb and cpMT-ERV. Plasmid pC-gonMT was transferred to the mutant strain ΔgonMT by intergeneric conjugation to produce the strain CPgonMT in which gonMT deletion was complemented.

pC-gonSL
A DNA fragment of 2,514 bp containing the entire ORF of gonSL (nt -23 to +2,491 from start codon) was amplified with the primer pair cpSL-Xb/cpSL-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonSL. Insertion of gonSL into pSETHe was confirmed by PCR and sequencing with primers cpSL-Xb and cpSL-ERV. Plasmid pC-gonSL was transferred to the mutant strain ΔgonSL by intergeneric conjugation to produce the strain CPgonSL in which gonSL deletion was complemented.

pC-gonS1
A DNA fragment of 1,135 bp containing the entire ORF of gonS1 (nt -24 to +1,111 from start codon) was amplified with the primer pair cpS1-Xb/cpS1-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonS1. Insertion of gonS1 into pSETHe was confirmed by PCR and sequencing with primers cpS1-Xb and cpS1-ERV. Plasmid pC-gonS1 was transferred to the mutant strain ΔgonS1 by intergeneric conjugation to produce the strain CPgonS1 in which gonS1 deletion was complemented.

pC-gonS2
A DNA fragment of 1,125 bp containing the entire ORF of gonS2 (nt -23 to +1,102 from start codon) was amplified with the primer pair cpS2-Xb/cpS2-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonS2. Insertion of gonS2 into pSETHe was confirmed by PCR and sequencing with primers cpS2-Xb and cpS2-ERV. Plasmid pC-gonS2 was transferred to the mutant strain ΔgonS2 by intergeneric conjugation to produce the strain CPgonS2 in which gonS2 deletion was complemented.

pC-gonCP
A DNA fragment of 1,361 bp containing the entire ORF of gonCP (nt -31 to +1,330 from start codon) was amplified with the primer pair cpCP-Xb/cpCP-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonCP. Insertion of gonCP into pSETHe was confirmed by PCR and sequencing with primers cpCP-Xb and cpCP-ERV. Plasmid pC-gonCP was transferred to the mutant strain ΔgonCP by intergeneric conjugation to produce the strain CPgonCP in which gonCP deletion was complemented.

pC-gonMR
A DNA fragment of 600 bp containing the entire ORF of gonMR (nt -30 to +570 from start codon) was amplified with the primer pair cpMR-Xb/cpMR-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonMR. Insertion of gonMR into pSETHe was confirmed by PCR and sequencing with primers cpMR-Xb and cpMR-ERV. Plasmid pC-gonMR was transferred to the mutant strain ΔgonMR by intergeneric conjugation to produce the strain CPgonMR in which gonMR deletion was complemented.

pC-gonL1
A DNA fragment of 3,005 bp containing the entire ORF of gonL1 (nt -24 to +2,981 from start codon) was amplified with the primer pair cpL1-Xb/cpL1-ERV (Table S1). The resulting PCR fragment was digested with XbaI/EcoRV and cloned into the same sites of plasmid pSETHe (see methods in the article), under the control of the ermE*p promoter, to yield the complementation plasmid pC-gonL1. Insertion of gonL1 into pSETHe was confirmed by PCR and sequencing with primers cpL1-Xb and cpL1-ERV. Plasmid pC-gonL1 was transferred to the mutant strain ΔgonL1 by intergeneric conjugation to produce the strain CPgonL1 in which gonL1 deletion was complemented.