Abstract
Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.
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27 December 2017
The original publication contains error error in the Materials and Methods section and in the acknowledgement section.
References
Anne J, Vrancken K, Van Mellaert L, Van Impe J, Bernaerts K (2014) Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans. Biochim Biophys Acta 1843(8):1750–1761. https://doi.org/10.1016/j.bbamcr.2013.12.023
Ausubel FM, Brent R, Kingston RE, Moore DO, Seidman JS, Smith JA, Struhl K (1995) Current protocols in molecular biology. Wiley, New York
Baig I, Perez M, Braña AF, Gomathinayagam R, Damodaran C, Salas JA, Méndez C, Rohr J (2008) Mithramycin analogues generated by combinatorial biosynthesis show improved bioactivity. J Nat Prod 71(2):199–207. https://doi.org/10.1021/np0705763
Bakhaeva GP, Berlin YA, Boldyreva EF, Chuprunova OA, Kolosov MN, Soifer VS, Vasiljeva TE, Yartseva IV (1968) The structure of aureolic acid (mithramycin). Tetrahedron Lett 32:3595–3598
Barkei JJ, Kevany BM, Felnagle EA, Thomas MG (2009) Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans. Chembiochem 10(2):366–376. https://doi.org/10.1002/cbic.200800646
Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen C, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O’Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417(6885):141–147. https://doi.org/10.1038/417141a
Bilyk O, Sekurova O, Zotchev SB, Luzhetskyy A (2016) Cloning and heterologous expression of the grecocycline biosynthetic gene cluster. PLoS One 11(7):e0158682. https://doi.org/10.1371/journal.pone.0158682
Blanco G, Fernández E, Fernández MJ, Braña AF, Weissbach U, Künzel E, Rohr J, Méndez C, Salas JA (2000) Characterization of two gycosyltransferases involved in early glycosylation steps during biosynthesis of the antitumor polyketide mithramycin by Streptomyces argillaceus. Mol Gen Genet 262(6):991–1000. https://doi.org/10.1007/PL00008667
Choi ES, Nam JS, Cho NP, Cho SD (2014) Modulation of specificity protein 1 by mithramycin a as a novel therapeutic strategy for cervical cancer. Sci Rep 4(1):7162. https://doi.org/10.1038/srep07162
Claus H, Decker H (2006) Bacterial tyrosinases. System Appl Microbiol 29(1):3–14. https://doi.org/10.1016/j.syapm.2005.07.012
Fernández E, Weissbach U, Reillo CS, Braña AF, Méndez C, Rohr J, Salas JA (1998) Identification of two genes from Streptomyces argillaceus encoding glycosyltransferases involved in transfer of a disaccharide during biosynthesis of the antitumor drug mithramycin. J Bacteriol 180(18):4929–4937
Fernández Lozano MJ, Remsing LL, Quirós LM, Braña AF, Fernández E, Sánchez C, Méndez C, Rohr J, Salas JA (2000) Characterization of two methyltransferases involved in the biosynthesis of the antitumor drug mithramycin by Streptomyces argillaceus. J Biol Chem 275(5):3065–3074. https://doi.org/10.1074/jbc.275.5.3065
Fujii T, Gramajo HC, Takano E, Bibb MJ (1996) redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. J Bacteriol 178(11):3402–3405. https://doi.org/10.1128/jb.178.11.3402-3405.1996
Garcia B, González-Sabín J, Menéndez N, Braña AF, Núñez LE, Morís F, Salas JA, Méndez C (2011) The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumor mithramycin. Microb Biotechnol 4(2):226–238. https://doi.org/10.1111/j.1751-7915.2010.00229.x
Gomez-Escribano JP, Song L, Fox DJ, Yeo V, Bibb MJ, Chalis GL (2012) Structure and biosynthesis of the unusual polyketide alkaloid coelimycin P1, a metabolic product of the cpk gene cluster of Streptomyces coelicolor M145. Chem Sci 3(9):2716–2720. https://doi.org/10.1039/c2sc20410j
González-Sabín J, Núñez LE, Braña AF, Méndez C, Salas JA, Gotor V, Morís F (2012) Regioselective enzymatic acylation of aureolic acids to obtain novel analogues with improved antitumor activity. Adv Synth Catal 354(8):1500–1508. https://doi.org/10.1002/adsc.201100944
Grohar PJ, Glod J, Peer CJ, Sissung TM, Arnaldez FI, Long L, Figg WD, Whitcomb P, Helman LJ, Widemann BC (2017) A phase I/II trial and pharmacokinetic study of mithramycin in children and adults with refractory Ewing sarcoma and EWS-FLI1 fusion transcript. Cancer Chemother Pharmacol 80(3):645–652. https://doi.org/10.1007/s00280–017–3382–x
Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 18:1541–1548
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics; The John Innes Foundation. Norwich, UK. 0-7084-0623-8
Kim E-S, Hong H-J, Choi C-Y, Cohen SN (2001) Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription. J Bacteriol 183:2197–2203
Knirschova R, Novakova R, Mingyar E, Bekeova C, Homerova D, Kormanec J (2015) Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces. J Microbiol Methods 113:1–3. https://doi.org/10.1016/j.mimet.2015.03.017
Lee YH, Chen BF, SY W, Leu WM, Lin JJ, Chen CW, Lo SC (1988) A trans-acting gene is required for the phenotypic expression of a tyrosinase gene in Streptomyces. Gene 65(1):71–81. https://doi.org/10.1016/0378-1119(88)90418-0
Lewis RA, Laing E, Allenby N, Bucca G, Brenner V, Harrison M, Kierzek AM, Smith CP (2010) Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization. BMC Genomics 1:682
Lombó F, Blanco G, Fernández E, Méndez C, Salas JA (1996) Characterization of Streptomyces argillaceus genes encoding a polyketide synthase involved in the biosynthesis of the antitumor mithramycin. Gene 172(1):87–91. https://doi.org/10.1016/0378-1119(96)00029-7
Lombó F, Menéndez N, Salas JA, Méndez C (2006) The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis and novel derivatives. Appl Microbiol Biotechnol 73(1):1–14. https://doi.org/10.1007/s00253-006-0511-6
Malek A, Núñez LE, Magistri M, Brambilla L, Jovic S, Carbone GM, Morís F, Catapano CV (2012) Modulation of the activity of Sp transcription factors by mithramycin analogues as a new strategy for treatment of metastatic prostate cancer. PLoS One 7(4):e35130. https://doi.org/10.1371/journal.pone.0035130
Méndez C, González-Sabín J, Morís F, Salas JA (2015) Expanding the chemical diversity of the antitumoral compound mithramycin by combinatorial biosynthesis and biocatalysis: the quest for mithralogs with improved therapeutic window. Planta Med 81(15):1326–1338. https://doi.org/10.1055/s-0035-1557876
Myronovskyi M, Rosenkranzer B, Luzhetskyy A (2014) Iterative marker excision system. Appl Microbiol Biotechnol 98(10):4557–4570. https://doi.org/10.1007/s00253-014-5523-z
Núñez LE, Nybo SE, González-Sabín J, Pérez M, Menéndez N, Braña AF, Shaaban KA, He M, Morís F, Salas JA, Méndez C (2012) A novel mitramycin analogue with high antitumor activity and less toxicity generated by combinatorial biosynthesis. J Med Chem 55(12):5813–5825. https://doi.org/10.1021/jm300234t
Nur-e-Alam M, Méndez C, Salas JA, Rohr J (2005) Elucidation of the glycosylation sequence of mithramycin biosynthesis: isolation of 3A-deolivosylpremithramycin B and its conversion to premithramycin B by glycosyltransferase MtmGII. Chembiochem 6(4):632–636. https://doi.org/10.1002/cbic.200400309
Osgood CC, Maloney N, Kidd CG, Kitchen-Goosen S, Segars L, Gebregiorgis M, Woldmichael GM, He M, Sankar S, Lessnick SL, Kang M, Smith M, Turner L, Madaj ZB, Winn ME, Núñez LE, González-Sabín J, Helman LJ, Morís F, Grohar PJ (2016) Identification of mithramycin analogues with improved targeting of EWS-FLI1 transcription factor. Clin Cancer Res 22(16):4105–4118. https://doi.org/10.1158/1078-0432.CCR-15-2624
Pandiella A, Morís F, Ocaña A, Núñez LE, Montero JC (2015) Antitumoral activity of the mithralog EC-8042 in triple negative breast cancer linked to cell cycle arrest in G2. Oncotarget 6(32):32856–32867. 10.18632/oncotarget.5942
Penn J, Li X, Whiting A, Latif M, Gibson T, Silva CJ, Brian P, Davis J, Miao V, Wrigley SK, Baltz RH (2006) Heterologous production of daptomycin in Streptomyces lividans. J Ind Microbiol Biotechnol 33(2):121–128. https://doi.org/10.1007/s10295-005-0033-8
Prado L, Fernández E, Weissbach U, Blanco G, Quirós LM, Braña AF, Méndez C, Rohr J, Salas JA (1999) Oxidative cleavage of premithramycin B is one of the last steps in the biosynthesis of the antitumor drug mithramycin. Chem Biol 6(1):19–30. https://doi.org/10.1016/S1074-5521(99)80017-9
Previdi S, Malek A, Albertini V, Riva C, Capella C, Broggini M, Carbone GM, Rohr J, Catapano CV (2010) Inhibition of Sp1-dependent transcription and antitumor activity of the new aureolic acid analogues mithramycin SDK and SK in human ovarian cancer xenografts. Gynecol Oncol 118(2):182–188. https://doi.org/10.1016/j.ygyno.2010.03.020
Remsing LL, González AM, Nur-e-Alam M, Fernández-Lozano MJ, Braña AF, Rix U, Oliveira MA, Méndez C, Salas JA, Rohr J (2003) Mithramycin SK, a novel antitumor drug with improved therapeutic index, mighramycin SA, and demycarosyl-mithramycin SK: three new products generated in the mithramycin producer Streptomyces argillaceus through combinatorial biosynthesis. J Am Chem Soc 125(19):5745–5753. https://doi.org/10.1021/ja034162h
Rezuchova B, Kormanec J (2001) A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response σ E in Escherichia coli. J Microbiol Meth 45(2):103–111. https://doi.org/10.1016/S0167-7012(01)00237-8
Rückert C, Albersmeier A, Busche T, Jaenicke S, Winkler A, Friethjonsson OH, Hreggviethsson GO, Lambert C, Badcock D, Bernaerts K, Anne J, Economou A, Kalinowski J (2015) Complete genome sequence of Streptomyces lividans TK24. J Biotechnol 199:21–22. https://doi.org/10.1016/j.jbiotec.2015.02.004
Shao L, Li J, Liu A, Chang Q, Lin H, Chen D (2013) Efficient bioconversion of echinocandin B to its nucleus by overexpression of deacylase genes in different host strains. Appl Environ Microbiol 79(4):1126–1133. https://doi.org/10.1128/AEM.02792-12
Wang L, Gao CH, Tang N, SN H, QF W (2015) Identification of genetic variations associated with epsilon-poly-lysine biosynthesis in Streptomyces albulus ZPM by genome sequencing. Sci Rep 5(1):9201. https://doi.org/10.1038/srep09201
Yamanaka K, Reynolds KA, Kersten RD, Ryan KS, Gonzalez DJ, Nizet V, Dorrestein PC, Moore BS (2014) Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc Natl Acad Sci U S A 111(5):1957–1962. https://doi.org/10.1073/pnas.1319584111
Zabala D, Braña AF, Flórez AB, Salas JA, Méndez C (2013) Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus. Metab Eng 20:187–197. https://doi.org/10.1016/j.ymben.2013.10.002
Zettler J, Xia H, Burkard N, Kulik A, Grond S, Heide L, Apel AK (2014) New aminocoumarins from the rare actinomycete Catenulispora acidiphila DSM 44928: identification, structure elucidation, and heterologous production. Chembiochem 15(4):612–621. https://doi.org/10.1002/cbic.201300712
Acknowledgements
This work was supported by the Slovak Research and Development Agency under contract APVV-15-0410 and by the VEGA grant 2/0002/16 from Slovak Academy of Sciences. The research leading to these results has received funding from the European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement STREPSYNTH (project No. 613877). This work was co–funded by the Slovak Research and Development Agency under the contract No. DO7RP-0037-12. We are grateful to A. Luzhetskyy for providing cosmid 1443::AmR and S. Zotchev for providing plasmid pCLY10 and S. cerevisiae BY4742.
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A correction to this article is available online at https://doi.org/10.1007/s00253-017-8710-x.
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Novakova, R., Núñez, L.E., Homerova, D. et al. Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains. Appl Microbiol Biotechnol 102, 857–869 (2018). https://doi.org/10.1007/s00253-017-8642-5
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DOI: https://doi.org/10.1007/s00253-017-8642-5