Fig 1.
Examples of polyketide antibiotics with stereospecific centers and proposed biosynthetic pathways of medermycin and actinorhodin.
A: three members of BIQ family shares opposite conFig uration at C3 and C15 (either 3S15R, or 3R15S). Two well-known antibiotics, daunorubicin and avermectin, have more than one stereospecific center and genes for stereochemical control at C13 and C5 for these two antibiotics respectively will be discussed in this study. B: Biosynthetic pathways of MED 2 and ACT 1 were proposed to share common earlier stages to produce a bicyclic intermediate 4. The formation of this intermediate was catalyzed by polyketide synthases (minimal PKS: KS, CLF and ACP) and related enzymes (KR, ARO and CYC). It could be converted into two shunt products (DMAC 5 and aloesaponarin II 6) in the actVI-ORF1-deficient mutant strain. The keto-reduction at C-3 of this bicyclic intermediate 4 in ACT 1 pathway was performed by ActVI-ORF1, probably by Med-ORF12 in MED 2 pathway, followed by spontaneous cyclization and dehydration, leading to the production of DNPA 7 with 3S configuration.
Fig 2.
Comparison between two biosynthetic gene clusters for medermycin and actinorhodin and phylogenetic analysis of Med-ORF12 and its homologies.
A: Predicted function of genes in the clusters for ACT 1 and MED 2 respectively is indicated as bars with different filling styles. B: Phylogenetic tree was established using the amino acid sequences of Med-ORF12 and Med-ORF6 (accession number: BAC79036 and BAC79042, for MED 2) from Streptomyces sp AM-7161, ActVI-ORF1 and ActIII (NP_629223 and NP_629236, for ACT 1) from S. coelicolor A3(2), DauB (AAA87616 for aklaviketone) from Streptomyces sp., Gra-ORF5 and Gra-ORF6 (P16542 and P16543 for GRA 3) from Streptomyces violaceoruber, AveF (NP_822111 predicated as ketoreductase for C5 of avermectin) from Streptomyces avermitilis, DnrU(Q_9ZAU1, marked as daunorubicin C-13 ketoreductase) from Streptomyces peucetius, Sa10 (ACK77759 for indigoidine/auricin) from Streptomyces aureofaciens, 3HAD (3HAD_B) from human heart and a hypothetical protein (WP_019763579) from Streptomyces sp Wigar10. The bar indicated the evolutionary distance. The numbers on branch nodes were percentages of 1000 sets of bootstrap supports. 3HAD: 3-hydroxyacyl-CoA dehydrogenase protein family; SDR: short-chain alcohol dehydrogenases family. C: Comparison between the reactions catalyzed by 3HAD and Med-ORF12 respectively.
Table 1.
Primers used in this study.
Fig 3.
Structure-based sequence alignment between Med-ORF12 and its homologies and the modeled structure of Med-ORF12
. A: The primary sequence of Med-ORF12 is aligned with the ActVI-ORF1, 3HAD, Sa10 and WP_019763579. The sequences are annotated with corresponding secondary structures in 3HAD. Arrows represent β -sheets and helices indicate α-helices. The similar residues are shaded and identical residues are highlighted in black. The key catalytic residues are marked by ☆. The numbers indicating amino acid positions of each entry refer to the nucleotide sequence deposited in the databases. Sequences retrieved from GenBank were aligned using Clustal X. B: Homology model of Med-ORF12 using one chain of 3HAD from human heart as template. Left: The homology modeled structure of Med-ORF12 obtained using PyMOL. β-strands and α-helices are shown in yellow and red, respectively. Right: The NADH molecule is modeled in active sites of Med-ORF12 by the docking program Autodock. The NADPH is shown in cyans sticks, the catalytic dyad (His141 and Glu153) shown in magenta sticks, the residues (Pro142, Phe143, Asn144, Ser229 and Thr230) interacted by hydrogen bonding with NADH shown as sky-blue sticks and the hydrogen bonds shown in blue dots.
Fig 4.
Inactivation and complementation of med-ORF12.
A: Schematic representation of knock-out med-ORF12. A 2.8 kb genomic fragment is composed of left arm (L-arm) and right arm (R-arm) referring to the upstream and downstream regions flanking med-ORF12 respectively. These two DNA regions are linked together and then inserted into a suicide vector pYH7, giving a suicide plasmid pHSL124. After double- crossover between chromosome of the wild type strain and homologous regions on pHSL124, med-ORF12-deficient mutant strain was acquired. On the chromosome of the mutant, an artificially-designed sequence (ATGTCTAGATGA) containing XbaI site in underline and start codon/stop codon in bold to replace in-frame med-ORF12 in the full-length. B: PCR confirmation of med-ORF12-deficient mutant strain (MS) and complementary strain (CS). The amplification using wild-type strain genomic DNA (lane 6) as template and a primer set (med12-qc1/ med12-qc1, showed in arrow in A) produced a 1 488 bp PCR product, while the genomic DNA of the med-ORF12- deficient mutant strains (lane 1–5) gave a 513 bp band as expected. The 975 bp genomic fragment of med-ORF12 was amplified by PCR (lane 8) and inserted into an integrative vector pIJ8600 (8.1 kb, lane 9: digestion with NdeI and BamHI), generating an expression plasmid pHSAYT19 (lane 7: double digestion with NdeI and BamHI). Genomic DNA isolated from the complementary strains (lane 10) and a primer set (med 12-A/med 12-B shown in arrow in A) were used for PCR amplification, giving a 975 bp band indicating the presence of med-ORF12, as same as for wild type strain genomic DNA as template (lane 11). On the contrary, the med-ORF12-deficient mutant strain (lane 12) could not produce an expected a 975 bp PCR product. M is 1 kb ladder as DNA marker.
Fig 5.
Metabolite analysis of Streptomyces strains.
A: Comparison of the pigmentation in liquid cultures between medermycin-producing wild type strain (WT) and its derived strains. Red-brown pigmentation acts as indicator for MED 2 production. The mutant strain (MS) was a med-ORF12 deficient strain. The complementary strain was obtained by transforming the expression plasmid pHSAY19, derived from pIJ8600, into med-ORF12 deficient mutant strain. Expression of med-ORF12 on pHSAY19 in the complementary strain was induced by thiostrepton (12.5 mg/ml). The wild type and mutant strains were transformed with the vector pIJ8600. B: HPLC spectra of metabolites in wild type, mutant and complementary strains, indicated as UV absorption at 434 nm of crude extracts of Streptomyces strains in A. In a contrast to wild type strain (WT/pIJ8600), the mutant strain (MS/pIJ8600) could not produce MED 2 due to the deficiency of med-ORF12. The reintroduction of med-ORF12 could restore the production of MED 2 in the complementary strain in a comparable yield to that in the wild type strain.
Fig 6.
Detection of two shunt products in the mutant strain.
A: HPLC spectra of metabolites by the wild type strain (WT/pIJ8600), mutant strain (MS/pIJ8600) and control strain (CH999/pRM5), indicated as UV absorption at 434 nm. Control: CH999/pRM5 was a recombinant strain of S. coeliololar able to accumulate two shunt products DMAC 5 (Mr: 298, 16.7 min) and aloesaponarin II 6 (Mr. 254, 22.6 min), due to the presence of 6 genes on pRM5, encoding KS, CLF, ACP, KR, ARO and CYC (Fig 1B) from the earlier stages of ACT 1 pathway. These 6 genes control the formation of the bicyclic intermediate 4. Then the bicyclic intermediate was spontaneously converted into two shunt products in CH999/pRM5 (Fig 1B). B: Mass spectra of peaks for authentic DMAC 5 and aloesaponarin II 6 by CH999/pRM5 and for the compounds at 16.9 min and 22.6 min by the med-ORF12- deficient mutant strain.