Advertisement

Improved milbemycin production by engineering two Cytochromes P450 in Streptomyces bingchenggensis

  • Haiyan Wang
  • Xu Cheng
  • Yuqing Liu
  • Shanshan Li
  • Yanyan Zhang
  • Xiangjing WangEmail author
  • Wensheng XiangEmail author
Biotechnological products and process engineering

Abstract

Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis.

Keywords

Milbemycins Post-PKS step CYPs Cytochromes P450 Streptomyces bingchenggenis 

Notes

Acknowledgments

We would like to thank Professor Mervyn Bibb (John Innes Centre, Norwich, UK) for providing S. coelicolor M1146, Professor Mark Buttner (John Innes Centre, Norwich, UK) for providing plasmid pIJ10500, and Doctor Weishan Wang (Chinese Academy of Sciences, Beijing, China) for providing promoter pKasO*.

Author contributions

Haiyan Wang, Xu Cheng, Xiangjing Wang, and Wensheng Xiang designed the research. Material preparation, data collection, and analysis were performed by Xu Cheng, Haiyan Wang, and Yuqing Liu. The first draft of the manuscript was written by Haiyan Wang and Xu Cheng. Shanshan Li and Yanyan Zhang modified the manuscript. All authors read and approved the final manuscript.

Funding information

This work received financial support from the National Natural Science Foundation of China (Grant Nos. 31601701 and 31572070) and General Financial Grant from China Postdoctoral Science Foundation (No. 2016 M600152).

Compliance with ethical standards

Conflict of interest

The authors have filed a provisional patent for this work to the China National Intellectual Property Administration (CNIPA). W. X., X.C., H.W., Y.L., and X.W. are inventors on the provisional patent application (CN202010032599.2, filed 13 January 2020).

Ethical approval

This article does not contain any studies with human or animals performed by any of the authors.

Supplementary material

253_2020_10410_MOESM1_ESM.pdf (518 kb)
ESM 1 (PDF 518 kb)

References

  1. Chamberland S, LEE M, Lomovskaya O (1997) Fungal efflux pump inhibitors. International Patent: PCT/US98/20916Google Scholar
  2. Child SA, Naumann EF, Bruning JB, Bell SG (2018) Structural and functional characterisation of the cytochrome P450 enzyme CYP268A2 from Mycobacterium marinum. Biochem J 475(4):705–722.  https://doi.org/10.1042/BCJ20170946 CrossRefPubMedGoogle Scholar
  3. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791.  https://doi.org/10.1111/j.1558-5646.1985.tb00420.x CrossRefPubMedPubMedCentralGoogle Scholar
  4. Goegelman RT (1985) Antihelmintic macrocyclic lactones and their production by fermentation. Eur Pat 86303294:2Google Scholar
  5. Hayes B, Schnitzler B, Wiseman S, Snyder DE (2015) Field evaluation of the efficacy and safety of a combination of spinosad and milbemycin oxime in the treatment and prevention of naturally acquired flea infestations and treatment of intestinal nematode infections in dogs in Europe. Vet Parasitol 207(1):99–106.  https://doi.org/10.1016/j.vetpar.2014.11.011 CrossRefPubMedGoogle Scholar
  6. He H, Ye L, Li C, Wang H, Guo X, Wang X, Zhang Y, Xiang W (2018) SbbR/SbbA, an important ArpA/AfsA-like system, regulates milbemycin production in Streptomyces bingchenggensis. Front Microbiol 9:1064.  https://doi.org/10.3389/fmicb.2018.01064 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Horbal L, Marques F, Nadmid S, Mendes MV, Luzhetskyy A (2018) Secondary metabolites overproduction through transcriptional gene cluster refactoring. Metab Eng 49:299–315.  https://doi.org/10.1016/j.ymben.2018.09.010 CrossRefPubMedGoogle Scholar
  8. Huang J, Zha W, An T, Dong H, Huang Y, Wang D, Yu R, Duan L, Zhang X, Peters RJ, Dai Z, Zi J (2019) Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid. Appl Microbiol Biotechnol 103(17):7029–7039.  https://doi.org/10.1007/s00253-019-10004-z CrossRefPubMedGoogle Scholar
  9. Ikeda H, Omura S (1997) Avermectin biosynthesis. Chem Rev 97(7):2591–2610.  https://doi.org/10.1021/cr960023p CrossRefPubMedGoogle Scholar
  10. Kasey CM, Zerrad M, Li Y, Cropp TA, Williams GJ (2018) Development of transcription factor-based designer macrolide biosensors for metabolic engineering and synthetic biology. ACS Synth Biol 7(1):227–239.  https://doi.org/10.1021/acssynbio.7b00287 CrossRefPubMedGoogle Scholar
  11. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics, vol 412 John Innes Foundation NorwichGoogle Scholar
  12. Kim MS, Cho WJ, Song MC, Park SW, Kim K, Kim E, Lee N, Nam SJ, Oh KH, Yoon YJ (2017) Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis. Microb Cell Factories 16(1):9–16.  https://doi.org/10.1186/s12934-017-0626-8 CrossRefGoogle Scholar
  13. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874.  https://doi.org/10.1093/molbev/msw054 CrossRefGoogle Scholar
  14. Li L, Wei K, Liu X, Wu Y, Zheng G, Chen S, Jiang W, Lu Y (2019) aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes. Metab Eng 52:153–167.  https://doi.org/10.1016/j.ymben.2018.12.001 CrossRefGoogle Scholar
  15. Liu W, Zhang Q, Guo J, Chen Z, Li J, Wen Y, Elliot MA (2015) Increasing avermectin production in Streptomyces avermitilis by manipulating the expression of a novel TetR-family regulator and its target gene product. Appl Environ Microbiol 81(15):5157–5173.  https://doi.org/10.1128/aem.00868-15 CrossRefPubMedPubMedCentralGoogle Scholar
  16. McKellar QA, Benchaoui HA (1996) Avermectins and milbemycins. J Vet Pharmacol Ther 19(5):331–351.  https://doi.org/10.1111/j.1365-2885.1996.tb00062.x CrossRefPubMedGoogle Scholar
  17. Merola VM, Eubig PA (2018) Toxicology of avermectins and milbemycins (macrocyclic lactones) and the role of P-glycoprotein in dogs and cats. Vet Clin North Am Small Anim Pract 48(6):991–1012.  https://doi.org/10.1016/j.cvsm.2018.07.002 CrossRefPubMedGoogle Scholar
  18. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New YorkGoogle Scholar
  19. Nelson DR (2009) The cytochrome p450 homepage. Hum Genomics 4(1):59–65CrossRefGoogle Scholar
  20. Niu G, Chater KF, Tian Y, Zhang J, Tan H (2016) Specialised metabolites regulating antibiotic biosynthesis in Streptomyces spp. FEMS Microbiol Rev 40(4):554–573.  https://doi.org/10.1093/femsre/fuw012 CrossRefPubMedGoogle Scholar
  21. Nonaka K, Kumasaka C, Okamoto Y, Maruyama F, Yoshikawa H (1999) Bioconversion of milbemycin-related compounds: biosynthetic pathway of milbemycins. J Antibiot 52(2):109–116.  https://doi.org/10.7164/antibiotics.52.109 CrossRefPubMedGoogle Scholar
  22. Pogorevc D, Panter F, Schillinger C, Jansen R, Wenzel SC, Muller R (2019) Production optimization and biosynthesis revision of corallopyronin A, a potent anti-filarial antibiotic. Metab Eng 55:201–211.  https://doi.org/10.1016/j.ymben.2019.07.010 CrossRefPubMedGoogle Scholar
  23. Rudolf JD, Chang C, Ma M, Shen B (2017) Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function. Nat Prod Rep 34(9):1141–1172.  https://doi.org/10.1039/c7np00034k CrossRefPubMedPubMedCentralGoogle Scholar
  24. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425.  https://doi.org/10.1093/oxfordjournals.molbev.a040454 CrossRefPubMedGoogle Scholar
  25. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set)Google Scholar
  26. Sherwood EJ, Bibb MJ (2013) The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba. Proc Natl Acad Sci U S A 110(27):E2500–E2509.  https://doi.org/10.1073/pnas.1305392110 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Sun P, Zhao Q, Yu F, Zhang H, Wu Z, Wang Y, Wang Y, Zhang Q, Liu W (2013) Spiroketal formation and modification in avermectin biosynthesis involves a dual activity of AveC. J Am Chem Soc 135(4):1540–1548.  https://doi.org/10.1021/ja311339u CrossRefPubMedGoogle Scholar
  28. Takiguchi Y, Mishima H, Okuda M, Terao M, Aoki A, Fukuda R (1980) Milbemycins, a new family of macrolide antibiotics: fermentation, isolation and physico-chemical properties. J Antibiot 33(10):1120–1127.  https://doi.org/10.7164/antibiotics.33.1120 CrossRefPubMedGoogle Scholar
  29. Tatsuta K (2016) Celebrating the 2015 Nobel Prize in Physiology or Medicine of Dr Satoshi Omura. J Antibiot 69(1):1.  https://doi.org/10.1038/ja.2015.113 CrossRefPubMedGoogle Scholar
  30. Wang X, Wang X, Xiang W (2009) Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically induced mutants. World J Microbiol Biotechnol 25(6):1051–1056.  https://doi.org/10.1007/s11274-009-9986-5 CrossRefGoogle Scholar
  31. Wang X, Wang C, Sun X, Xiang W (2010a) 5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization. Biotechnol Lett 32(10):1497–1502.  https://doi.org/10.1007/s10529-010-0320-y CrossRefPubMedGoogle Scholar
  32. Wang X, Yan Y, Zhang B, An J, Wang J, Tian J, Jiang L, Chen Y, Huang S, Yin M, Zhang J, Gao A, Liu C, Zhu Z, Xiang W (2010b) Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis. J Bacteriol 192(17):4526–4527.  https://doi.org/10.1128/JB.00596-10 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Wang X, Zhang B, Yan Y, An J, Zhang J, Liu C, Xiang W (2013) Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray. Genome 56(11):677–689.  https://doi.org/10.1139/gen-2013-0098 CrossRefPubMedGoogle Scholar
  34. Wang H, Zhang J, Zhang Y, Zhang B, Liu C, He H, Wang X, Xiang W (2014) Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis. Appl Microbiol Biotechnol 98(23):9703–9712.  https://doi.org/10.1007/s00253-014-5970-6 CrossRefPubMedGoogle Scholar
  35. Wang W, Li S, Li Z, Zhang J, Fan K, Tan G, Ai G, Lam SM, Shui G, Yang Z, Lu H, Jin P, Li Y, Chen X, Xia X, Liu X, Dannelly HK, Yang C, Yang Y, Zhang S, Alterovitz G, Xiang W, Zhang L (2020) Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces. Nat Biotechnol 38(1):76–83.  https://doi.org/10.1038/s41587-019-0335-4 CrossRefGoogle Scholar
  36. Wei K, Wu Y, Li L, Jiang W, Hu J, Lu Y, Chen S (2018) MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus. Appl Microbiol Biotechnol 102(20):8841–8853.  https://doi.org/10.1007/s00253-018-9280-2 CrossRefPubMedGoogle Scholar
  37. Zhang J, An J, Wang J, Yan Y, He H, Wang X, Xiang W (2013) Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin. Appl Microbiol Biotechnol 97:1–11.  https://doi.org/10.1007/s00253-013-5255-5 CrossRefGoogle Scholar
  38. Zhang Y, He H, Liu H, Wang H, Wang X, Xiang W (2016) Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microb Cell Factories 15(1):152.  https://doi.org/10.1186/s12934-016-0552-1 CrossRefGoogle Scholar
  39. Zhu T, Cheng X, Liu Y, Deng Z, You D (2013) Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens. Metab Eng.  https://doi.org/10.1016/j.ymben.2013.06.003 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina
  2. 2.School of Life ScienceNortheast Agricultural UniversityHarbinChina

Personalised recommendations