Abstract
The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6′-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6′-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6′-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6′-deoxy-BLM via pilot-scale microbial fermentation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Buckel P, Buchberger A, Bock A, Wittmann HG (1977) Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin. Mol Gen Genet 158(1):47–54. https://doi.org/10.1007/BF00455118
Burger RM (1998) Cleavage of nucleic acids by bleomycin. Chem Rev 98(3):1153–1170. https://doi.org/10.1021/cr960438a
Chen C, Si S, He Q, Xu H, Lu M, Xie Y, Wang Y, Chen R (2008) Isolation and characterization of antibiotic NC0604, a new analogue of bleomycin. J Antibiot (Tokyo) 61(12):747–751. https://doi.org/10.1038/ja.2008.88
Chen Y, Smanski MJ, Shen B (2010) Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation. Appl Microbiol Biotechnol 86(1):19–25. https://doi.org/10.1007/s00253-009-2428-3
Chen Y, Yin M, Horsman GP, Shen B (2011) Improvement of the enediyne antitumor antibiotic C-1027 production by manipulating its biosynthetic pathway regulation in Streptomyces globisporus. J Nat Prod 74(3):420–424. https://doi.org/10.1021/np100825y
Chen JK, Yang D, Shen B, Murray V (2017) Bleomycin analogues preferentially cleave at the transcription start sites of actively transcribed genes in human cells. Int J Biochem Cell Biol 85:56–65. https://doi.org/10.1016/j.biocel.2017.02.001
Davies C, Bussiere DE, Golden BL, Porter SJ, Ramakrishnan V, White SW (1998) Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance. J Mol Biol 279(4):873–888. https://doi.org/10.1006/jmbi.1998.1780
Einhorn LH (2002) Curing metastatic testicular cancer. Proc Natl Acad Sci U S A 99(7):4592–4595. https://doi.org/10.1073/pnas.072067999
Galm U, Shen B (2007) Natural product drug discovery: the times have never been better. Chem Biol 14(10):1098–1104. https://doi.org/10.1016/j.chembiol.2007.10.004
Galm U, Hager MH, Van Lanen SG, Ju J, Thorson JS, Shen B (2005) Antitumor antibiotics: bleomycin, enediynes, and mitomycin. Chem Rev 105(2):739–758. https://doi.org/10.1021/cr030117g
Galm U, Wang L, Wendt-Pienkowski E, Yang R, Liu W, Tao M, Coughlin JM, Shen B (2008) In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway. J Biol Chem 283(42):28236–28245. https://doi.org/10.1074/jbc.M804971200
Hindra HT, Yang D, Rudolf JD, Xie P, Xie G, Teng Q, Lohman JR, Zhu X, Huang Y, Zhao LX, Jiang Y, Duan Y, Shen B (2014) Strain prioritization for natural product discovery by a high-throughput real-time PCR method. J Nat Prod 77(10):2296–2303. https://doi.org/10.1021/np5006168
Hindra YD, Teng Q, Dong LB, Crnovcic I, Huang T, Ge H, Shen B (2017) Genome mining of Streptomyces mobaraensis DSM40847 as a bleomycin producer providing a biotechnology platform to engineer designer bleomycin analogues. Org Lett 19(6):1386–1389. https://doi.org/10.1021/acs.orglett.7b00283
Huang SX, Feng Z, Wang L, Galm U, Wendt-Pienkowski E, Yang D, Tao M, Coughlin JM, Duan Y, Shen B (2012) A designer bleomycin with significantly improved DNA cleavage activity. J Am Chem Soc 134(32):13501–13509. https://doi.org/10.1021/ja3056535
Kawaguchi H, Tsukiura H, Tomita K, Konishi M, Saito K (1977) Tallysomycin, a new antitumor antibiotic complex related to bleomycin. I. Production, isolation and properties. J Antibiot (Tokyo) 30(10):779–788. https://doi.org/10.7164/antibiotics.30.779
Leitheiser CJ, Smith KL, Rishel MJ, Hashimoto S, Konishi K, Thomas CJ, Li C, McCormick MM, Hecht SM (2003) Solid-phase synthesis of bleomycin group antibiotics. Construction of a 108-member deglycobleomycin library. J Am Chem Soc 125(27):8218–8227. https://doi.org/10.1021/ja021388w
Ochi K (2017) Insights into microbial cryptic gene activation and strain improvement: principle, application and technical aspects. J Antibiot (Tokyo) 70(1):25–40. https://doi.org/10.1038/ja.2016.82
Ochi K, Okamoto S, Tozawa Y, Inaoka T, Hosaka T, Xu J, Kurosawa K (2004) Ribosome engineering and secondary metabolite production. Adv Appl Microbiol 56:155–184. https://doi.org/10.1016/S0065-2164(04)56005-7
Okamoto-Hosoya Y, Sato TA, Ochi K (2000) Resistance to paromomycin is conferred by rpsL mutations, accompanied by an enhanced antibiotic production in Streptomyces coelicolor A3(2). J Antibiot (Tokyo) 53(12):1424–1427. https://doi.org/10.7164/antibiotics.53.1424
Panetta A, Angelelli B, Martoni A (1999) Pilot study on induction chemotherapy with cisplatin, epirubicin, etoposide and bleomycin in cervical cancer stage Ib, IIa and IIb. Anticancer Res 19(1B):765–768
Pfister P, Hobbie S, Vicens Q, Bottger EC, Westhof E (2003) The molecular basis for A-site mutations conferring aminoglycoside resistance: relationship between ribosomal susceptibility and X-ray crystal structures. Chembiochem 4(10):1078–1088. https://doi.org/10.1002/cbic.200300657
Shen B (2015) A new golden age of natural products drug discovery. Cell 163(6):1297–1300. https://doi.org/10.1016/j.cell.2015.11.031
Shentu X, Liu N, Tang G, Tanaka Y, Ochi K, Xu J, Yu X (2016) Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering. J Antibiot (Tokyo) 69(5):406–410. https://doi.org/10.1038/ja.2015.123
Shima J, Hesketh A, Okamoto S, Kawamoto S, Ochi K (1996) Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J Bacteriol 178(24):7276–7284. https://doi.org/10.1128/jb.178.24.7276-7284.1996
Sleijfer S (2001) Bleomycin-induced pneumonitis. Chest 120(2):617–624. https://doi.org/10.1378/chest.120.2.617
Tanaka Y, Komatsu M, Okamoto S, Tokuyama S, Kaji A, Ikeda H, Ochi K (2009) Antibiotic overproduction by rpsL and rsmG mutants of various actinomycetes. Appl Environ Microbiol 75(14):4919–4922. https://doi.org/10.1128/AEM.00681-09
Tanaka Y, Kasahara K, Izawa M, Ochi K (2017) Applicability of ribosome engineering to vitamin B12 production by Propionibacterium shermanii. Biosci Biotechnol Biochem 81(8):1636–1641. https://doi.org/10.1080/09168451.2017.1329619
Tao M, Wang L, Wendt-Pienkowski E, Zhang N, Yang D, Galm U, Coughlin JM, Xu Z, Shen B (2010) Functional characterization of tlmH in Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insight into tallysomycin biosynthesis and affording a novel bleomycin analog. Mol BioSyst 6(2):349–356. https://doi.org/10.1039/b918106g
Umezawa H, Maeda K, Takeuchi T, Okami Y (1966) New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 19(5):200–209
Vila-Sanjurjo A, Lu Y, Aragonez JL, Starkweather RE, Sasikumar M, O’Connor M (2007) Modulation of 16S rRNA function by ribosomal protein S12. Biochim Biophys Acta 1769(7–8):462–471. https://doi.org/10.1016/j.bbaexp.2007.04.004
Wang L, Yun BS, George NP, Wendt-Pienkowski E, Galm U, TJ O, Coughlin JM, Zhang G, Tao M, Shen B (2007) Glycopeptide antitumor antibiotic zorbamycin from Streptomyces flavoviridis ATCC 21892: strain improvement and structure elucidation. J Nat Prod 70(3):402–406. https://doi.org/10.1021/np060592k
Wang Q, Cui K, Espin-Garcia O, Cheng D, Qiu X, Chen Z, Moore M, Bristow RG, Xu W, Der S, Liu G (2013) Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis. PLoS One 8(12):e82363. https://doi.org/10.1371/journal.pone.0082363
Wang L, Chen X, Wu G, Li S, Zeng X, Ren X, Tang L, Mao Z (2017) Enhanced epsilon-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus. Bioprocess Biosyst Eng 40(2):271–283. https://doi.org/10.1007/s00449-016-1695-5
Xu J, Tozawa Y, Lai C, Hayashi H, Ochi K (2002) A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Mol Gen Genomics 268(2):179–189. https://doi.org/10.1007/s00438-002-0730-1
Yang D, Zhu X, Wu X, Feng Z, Huang L, Shen B, Xu Z (2011) Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT-PCR. Appl Microbiol Biotechnol 89(6):1709–1719. https://doi.org/10.1007/s00253-010-3025-1
Yang D, Hindra DLB, Crnovcic I, Shen B (2017) Engineered production and evaluation of 6′-deoxy-tallysomycin H-1 revealing new insights into the structure-activity relationship of the anticancer drug bleomycin. J Antibiot (Tokyo). https://doi.org/10.1038/ja.2017.93
Yu Z, Smanski MJ, Peterson RM, Marchillo K, Andes D, Rajski SR, Shen B (2010) Engineering of Streptomyces platensis MA7339 for overproduction of platencin and congeners. Org Lett 12(8):1744–1747. https://doi.org/10.1021/ol100342m
Zhang N, Zhu X, Yang D, Cai J, Tao M, Wang L, Duan Y, Shen B, Xu Z (2010) Improved production of the tallysomycin H-1 in Streptoalloteichus hindustanus SB8005 strain by fermentation optimization. Appl Microbiol Biotechnol 86(5):1345–1353. https://doi.org/10.1007/s00253-009-2406-9
Zhu X, Zhang W, Chen X, Wu H, Duan Y, Xu Z (2010) Generation of high rapamycin producing strain via rational metabolic pathway-based mutagenesis and further titer improvement with fed-batch bioprocess optimization. Biotechnol Bioeng 107(3):506–515. https://doi.org/10.1002/bit.22819
Funding
This work was supported in part by grants from the National High Technology Research and Development Program of China 2012AA02A705, the Chinese Ministry of Education 111 Project B0803420, National Major Scientific and Technological Special Project 2011ZX09401-001, and the Natural Products Library Initiative at TSRI.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Zhu, X., Kong, J., Yang, H. et al. Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6′-deoxy-bleomycin Z production. Appl Microbiol Biotechnol 102, 1651–1661 (2018). https://doi.org/10.1007/s00253-017-8705-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8705-7