(1) Background: Manumycins are small actinomycete polyketides with prominent cancerostatic and immunosuppressive activities via inhibition of various eukaryotic enzymes. Their overall activity towards human cells depends on the structural variability of both their polyketide chains, mainly the upper one. In our genetic screening project to find novel producers of anti-inflammatory manumycins, the strain Saccharothrix espanaensis DSM44229 was identified as containing a novel manumycin-type biosynthetic gene cluster (BGC). (2) Methods: The biosynthetic genes appeared to be silent under all assayed laboratory conditions. Several techniques were used to activate the BGC, including: (i) heterologous expression in various hosts, (ii) overexpression of putative pathway-specific regulatory genes, and (iii) overexpression of a bottleneck cyclizing aminolevulinate synthase gene in both natural and heterologous producers. (3) Results: Multiple novel manumycin-type compounds were produced at various levels by genetically-modified strains, sharing a tetraene lower chain structure with a colabomycin subgroup of manumycins, but possessing much shorter and saturated upper chains. (4) Conclusions: A cryptic manumycin-type BGC was successfully activated by genetic means to gain production of novel manumycin-type compounds for future comparative activity assays. Heterologously produced compounds were identical to those found after final activation of the BGC in the original strain, proving the intactness of the cloned BGC.

Faculty of Fisheries and Protection of Waters University of South Bohemia Zátiší 728 2 38925 Vodňany Czech Republic

Faculty of Science University of South Bohemia Branišovská 1645 31a 37005 České Budějovice Czech Republic

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Studničkova 7 12800 Prague Czech Republic

Institute of Microbiology Academy of Sciences of the Czech Republic Vídeňská 183 14220 Prague Czech Republic

Institute of Soil Biology Biology Centre Academy of Sciences of the Czech Republic Na Sádkách 702 7 37005 České Budějovice Czech Republic

Zobrazit více v PubMed

Katz L., Baltz R.H. Natural product discovery: Past, present, and future. J. Ind. Microbiol. Biotechnol. 2016;43:155–176. doi: 10.1007/s10295-015-1723-5. PubMed DOI

Nguyen C.T., Dhakal D., Pham V.T.T., Nguyen H.T., Sohng J.K. Recent Advances in Strategies for Activation and Discovery/Characterization of Cryptic Biosynthetic Gene Clusters in Streptomyces. Microorganisms. 2020;8:616. doi: 10.3390/microorganisms8040616. PubMed DOI PMC

Kang H.S., Kim E.S. Recent advances in heterologous expression of natural product biosynthetic gene clusters in Streptomyces hosts. Curr. Opin. Biotechnol. 2021;69:118–127. doi: 10.1016/j.copbio.2020.12.016. PubMed DOI

Zeeck A., Schroder K., Frobel K., Grote R., Thiericke R. The Structure of Manumycin. 1. Characterization, Structure Elucidation and Biological-Activity. J. Antibiot. 1987;40:1530–1540. doi: 10.7164/antibiotics.40.1530. PubMed DOI

Zeeck A., Frobel K., Heusel C., Schroder K., Thiericke R. The Structure of Manumycin. 2. Derivatives. J. Antibiot. 1987;40:1541–1548. doi: 10.7164/antibiotics.40.1541. PubMed DOI

Ali B.R.S., Pal A., Croft S.L., Taylor R.J.K., Field M.C. The farnesyltransferase inhibitor manumycin A is a novel trypanocide with a complex mode of action including major effects on mitochondria. Mol. Biochem. Parasitol. 1999;104:67–80. doi: 10.1016/S0166-6851(99)00131-0. PubMed DOI

Kainuma O., Asano T., Hasegawa M., Kenmochi T., Nakagohri T., Tokoro Y., Isono K. Inhibition of growth and invasive activity of human pancreatic cancer cells by a farnesyltransferase inhibitor, manumycin. Pancreas. 1997;15:379–383. doi: 10.1097/00006676-199711000-00008. PubMed DOI

Yeung S.C.J., Xu G.P., Pan J.X., Christgen M., Bamiagis A. Manumycin enhances the cytotoxic effect of paclitaxel on anaplastic thyroid carcinoma cells. Cancer Res. 2000;60:650–656. PubMed

Hara M., Akasaka K., Akinaga S., Okabe M., Nakano H., Gomez R., Wood D., Uh M., Tamanoi F. Identification of Ras Farnesyltransferase Inhibitors by Microbial Screening. Proc. Natl. Acad. Sci. USA. 1993;90:2281–2285. doi: 10.1073/pnas.90.6.2281. PubMed DOI PMC

She M.R., Yang H.L., Sun L.L., Yeung S.C.J. Redox control of manumycin a-induced apoptosis in anaplastic thyroid cancer cells - Involvement of the xenobiotic apoptotic pathway. Cancer Biol. Ther. 2006;5:275–280. doi: 10.4161/cbt.5.3.2383. PubMed DOI

Tanaka T., Tsukuda E., Uosaki Y., Matsuda Y. EI-1511-3, -5 and EI-1625-2, novel interleukin-1 beta converting enzyme inhibitors produced by Streptomyces sp. E-1511 and E-1625. 3. Biochemical properties of EI-1511-3, -5 and EI-1625-2. J. Antibiot. 1996;49:1085–1090. doi: 10.7164/antibiotics.49.1085. PubMed DOI

Bernier M., Kwon Y.K., Pandey S.K., Zhu T.N., Zhao R.J., Maciuk A., He H.J., DeCabo R., Kole S. Binding of manumycin A inhibits I kappa B kinase beta activity. J. Biol. Chem. 2006;281:2551–2561. doi: 10.1074/jbc.M511878200. PubMed DOI

Costantini C., Weindruch R., Della V.G., Puglielli L. A TrkA-to-p75NTR molecular switch activates amyloid beta-peptide generation during aging. Biochem. J. 2005;391:59–67. doi: 10.1042/BJ20050700. PubMed DOI PMC

Striz I., Krasna E., Petrickova K., Brabcova E., Kolesar L., Slavcev A., Jaresova M., Petricek M. Manumycin and asukamycin inhibition of IL-1beta and IL-18 release from human macrophages by caspase-1 blocking. Allergy. 2008;63:142–143.

Hrdy J., Sukenikova L., Petraskova P., Novotna O., Kahoun D., Petricek M., Chronakova A., Petrickova K. Inhibition of Pro-Inflammatory Cytokines by Metabolites of Streptomycetes—A Potential Alternative to Current Anti-Inflammatory Drugs? Microorganisms. 2020;8:621. doi: 10.3390/microorganisms8050621. PubMed DOI PMC

Honnappa C.G., Kesavan U.M. A concise review on advances in development of small molecule anti-inflammatory therapeutics emphasising AMPK: An emerging target. Int. J. Immunopathol. Pharmacol. 2016;29:562–571. doi: 10.1177/0394632016673369. PubMed DOI PMC

Petrickova K., Chronakova A., Zelenka T., Chrudimsky T., Pospisil S., Petricek M., Kristufek V. Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: Outcomes for genetic screening techniques. Front. Microbiol. 2015;6:814. doi: 10.3389/fmicb.2015.00814. PubMed DOI PMC

Petrickova K., Pospisil S., Kuzma M., Tylova T., Jagr M., Tomek P., Chronakova A., Brabcova E., Andera L., Kristufek V., et al. Biosynthesis of Colabomycin E, a New Manumycin-Family Metabolite, Involves an Unusual Chain-Length Factor. ChemBioChem. 2014;15:1334–1345. doi: 10.1002/cbic.201400068. PubMed DOI

Hopwood D.A., Bibb M.J., Chater K.F., Kieser T., Bruton C.J., Kieser H.M., Lydiate D.J., Ward J.M., Schrempf H. Genetic Manipulation of Streptomyces. A Laboratory Manual. The John Innes Foundation; Norwich, UK: 1985.

Sambrook J., Fritz E.F., Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY, USA: 1989.

Gust B., Chandra G., Jakimowicz D., Yuqing T., Bruton C.J., Chater K.F. Lambda red-mediated genetic manipulation of antibiotic-producing Streptomyces. Adv. Appl. Microbiol. 2004;54:107–128. doi: 10.1016/S0065-2164(04)54004-2. PubMed DOI

Kieser T., Bibb M.J., Buttner M.J., Chater K.F., Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation; Norwich, UK: 2000.

Floriano B., Bibb M. afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2) Mol. Microbiol. 1996;21:385–396. doi: 10.1046/j.1365-2958.1996.6491364.x. PubMed DOI

Gomez-Escribano J.P., Bibb M.J. Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb. Biotechnol. 2011;4:207–215. doi: 10.1111/j.1751-7915.2010.00219.x. PubMed DOI PMC

Rodriguez-Garcia A., Combes P., Perez-Redondo R., Smith M.C.A., Smith M.C.M. Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces. Nucl. Acids Res. 2005;33:e87. doi: 10.1093/nar/gni086. PubMed DOI PMC

Singh M.P., Petersen P.J., Weiss W.J., Kong F., Greenstein M. Saccharomicins, novel heptadecaglycoside antibiotics produced by Saccharothrix espanaensis: Antibacterial and mechanistic activities. Antimicrob. Agents Chemother. 2000;44:2154–2159. doi: 10.1128/AAC.44.8.2154-2159.2000. PubMed DOI PMC

Strobel T., Al-Dilaimi A., Blom J., Gessner A., Kalinowski J., Luzhetska M., Puhler A., Szczepanowski R., Bechthold A., Ruckert C. Complete genome sequence of Saccharothrix espanaensis DSM 44229(T) and comparison to the other completely sequenced Pseudonocardiaceae. BMC Genom. 2012;13:465. doi: 10.1186/1471-2164-13-465. PubMed DOI PMC

Rui Z., Petrickova K., Skanta F., Pospisil S., Yang Y.L., Chen C.Y., Tsai S.F., Floss H.G., Petricek M., Yu T.W. Biochemical and Genetic Insights into Asukamycin Biosynthesis. J. Biol. Chem. 2010;285:24915–24924. doi: 10.1074/jbc.M110.128850. PubMed DOI PMC

Grote R., Zeeck A., Beale J.M. Metabolic Products of Microorganisms. 245. Colabomycins, New Antibiotics of the Manumycin Group from Streptomyces griseoflavus. 2. Structure of Colabomycin-A. J. Antibiot. 1988;41:1186–1195. doi: 10.7164/antibiotics.41.1186. PubMed DOI

Xie P.F., Sheng Y., Ito T., Mahmud T. Transcriptional regulation and increased production of asukamycin in engineered Streptomyces nodosus subsp. asukaensis strains. Appl. Microbiol. Biotechnol. 2012;96:451–460. doi: 10.1007/s00253-012-4084-2. PubMed DOI

Cecrdlova E., Petrickova K., Kolesar L., Petricek M., Sekerkova A., Svachova V., Striz I. Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes. Immunol. Lett. 2016;169:8–14. doi: 10.1016/j.imlet.2015.11.010. PubMed DOI

Yang W.L., Del Villar K., Urano J., Mitsuzawa H., Tamanoi F. Advances in the development of farnesyltransferase inhibitors: Substrate recognition by protein farnesyltransferase. J. Cell. Biochem. 1997;67:12–19. doi: 10.1002/(SICI)1097-4644(1997)27+<12::AID-JCB5>3.0.CO;2-4. PubMed DOI

Dixit D., Sharma V., Ghosh S., Koul N., Mishra P.K., Sen E. Manumycin inhibits STAT3, telomerase activity, and growth of glioma cells by elevating intracellular reactive oxygen species generation. Free Radic. Biol. Med. 2009;47:364–374. doi: 10.1016/j.freeradbiomed.2009.04.031. PubMed DOI

Zhang J., Jiang H., Xie L., Hu J., Li L., Yang M., Cheng L., Liu B., Qian X. Antitumor effect of manumycin on colorectal cancer cells by increasing the reactive oxygen species production and blocking PI3K-AKT pathway. Onco Targets Ther. 2016;9:2885–2895. doi: 10.2147/OTT.S102408. PubMed DOI PMC

Schaffert L., Marz C., Burkhardt L., Droste J., Brandt D., Busche T., Rosen W., Schneiker-Bekel S., Persicke M., Puhler A., et al. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microb. Cell Fact. 2019;18:114. doi: 10.1186/s12934-019-1162-5. PubMed DOI PMC

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018;35:1547–1549. doi: 10.1093/molbev/msy096. PubMed DOI PMC