A new peucemycin derivative and impacts of peuR and bldA on peucemycin biosynthesis in Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is known to produce a variety of secondary metabolites, including two important antitumor anthracyclines: daunorubicin and doxorubicin. Identification of peucemycin and 25-hydroxy peucemycin (peucemycin A), as well as their biosynthetic pathway, has expanded its biosynthetic potential. In this study, we isolated a new peucemycin derivative and identified it as 19-hydroxy peucemycin (peucemycin B). Its antibacterial activity was lower than those of peucemycin and peucemycin A. On the other hand, this newly identified peucemycin derivative had higher anticancer activity than the other two compounds for MKN45, NCI-H1650, and MDA-MB-231 cancer cell lines with IC50 values of 76.97 µM, 99.68 µM, and 135.2 µM, respectively. Peucemycin biosynthetic gene cluster revealed the presence of a SARP regulator named PeuR whose role was unknown. The presence of the TTA codon in the peuR and the absence of global regulator BldA in S. peucetius reduced its ability to regulate the peucemycin biosynthetic gene cluster. Hence, different mutants harboring these genes were prepared. S. peucetius bldA25 harboring bldA produced 1.75 times and 1.77 times more peucemycin A (11.8 mg/L) and peucemycin B (21.2 mg/L), respectively, than the wild type. On the other hand, S. peucetius R25 harboring peuR produced 1.86 and 1.79 times more peucemycin A (12.5 mg/L) and peucemycin B (21.5 mg/L), respectively, than the wild type. Finally, strain S. peucetius bldAR25 carrying bldA and peuR produced roughly 3.52 and 2.63 times more peucemycin A (23.8 mg/L) and peucemycin B (31.5 mg/L), respectively, than the wild type. KEY POINTS: • This study identifies a new peucemycin derivative, 19-hydroxy peucemycin (peucemycin B). • The SARP regulator (PeuR) acts as a positive regulator of the peucemycin biosynthetic gene cluster. • The overexpression of peuR and heterologous expression of bldA increase the production of peucemycin derivatives.

Biosynthetic pathway of peucemycin and identification of its derivative from Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is a well-known producer of important anticancer compounds, daunorubicin and doxorubicin. In this study, we successfully identified a new macrolide, 25-hydroxy peucemycin, that exhibited an antibacterial effect on some pathogens. Based on the structure of a newly identified compound and through the inactivation of a polyketide synthase gene, we successfully identified its biosynthetic gene cluster which was considered to be the cryptic biosynthetic gene cluster. The biosynthetic gene cluster spans 51 kb with 16 open reading frames. Five type I polyketide synthase (PKS) genes encode eight modules that synthesize the polyketide chain of peucemycin before undergoing post-PKS tailoring steps. In addition to the regular starter and extender units, some modules have specificity towards ethylmalonyl-CoA and unusual butylmalonyl-CoA. A credible explanation for the specificity of the unusual extender unit has been searched for. Moreover, the enzyme responsible for the final tailoring pathway was also identified. Based on all findings, a plausible biosynthetic pathway is here proposed. KEY POINTS: • Identification of a new macrolide, 25-hydroxy peucemycin. • An FMN-dependent monooxygenase is responsible for the hydroxylation of peucemycin. • The module encoded by peuC is unique to accept the butylmalonyl-CoA as an unusual extender unit.

Increasing production of spinosad in Saccharopolyspora spinosa by metabolic engineering.

Spinosad, a combination of spinosyn A and D produced by Saccharopolyspora spinosa, is a highly efficient pesticide. There has been a considerable interest in the improvement of spinosad production because of a low yield achieved by wild-type S. spinosa. In this study, we designed and constructed a pIBR-SPN vector. pIBR-SPN is an integrative vector that can be used to introduce foreign genes into the chromosome of S. spinosa. Different combinations of genes encoding forasamine and rhamnose were synthesized and used for the construction of different recombinant plasmids. The following recombinant strains were developed: S. spinosa pIBR-SPN (only the vector), S. spinosa pIBR-SPN F (forosamine genes), S. spinosa pIBR-SPN R (rhamnose genes), S. spinosa pIBR-SPN FR (forosamine and rhamnose genes), S. spinosa pIBR-SPN FRS (forosamine, rhamnose, and SAM [S-adenosyl-L-methionine synthetase] genes), and S. spinosa MUV pIBR-SPN FR. Among these recombinant strains, S. spinosa pIBR-SPN FR produced 1394 ± 163 mg/L spinosad, which was 13-fold higher than the wild-type. S. spinosa MUV pIBR-SPN FR produced 1897 (±129) mg/L spinosad, which was seven-fold higher than S. spinosa MUV and 17-fold higher than the wild-type strain.

Genome mining Streptomyces sp. KCTC 0041BP as a producer of dihydrochalcomycin.

Streptomyces sp. KCTC 0041BP, which was isolated from a soil sample in Cheolwon, Republic of Korea, is a dihydrochalcomycin producer. In this study, we obtained the genome of S. sp. KCTC 0041BP with 7.54 Mb genome size. antiSMASH and the dbCAN2 meta server predicted that the genome would contain 26 secondary metabolite biosynthetic gene clusters (BGCs) and 285 carbohydrate-active enzymes. Besides dihydrochalcomycin, 21 compounds were successfully identified from S. sp. KCTC 0041BP, and among them, the structure of 8 compounds were proven by high-resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR). The identification of chalcomycin analogs led to a better understanding of the biosynthetic pathway of dihydrochalcomycin/chalcomycin. From the analysis of cluster 2 and solvent selection, linearmycins were determined. Linearmycins showed antibacterial activity with both Gram-positive and Gram-negative bacteria and antifungal activity. One strain many compounds (OSMAC) strategy was applied to activate the salicylic acid production in this strain. A salicylic acid biosynthetic pathway was also predicted, but not by antiSMASH. These results showed that this strain can produce many useful compounds and potentially produce novel compounds with most secondary BGCs yet to be experimentally identified.

Identification of 1,3,6,8-Tetrahydroxynaphthalene Synthase (ThnA) from Nocardia sp. CS682.

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys138, Phe188, His270, and Asn303 as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividans TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividans TK24 and S. lividans pIBR25-thnA and found that thnA was only transcribed in the mutant.