Advertisement

Production of sesterterpene ophiobolin by a bifunctional terpene synthase in Escherichia coli

  • Wei Yuan
  • Shuang Lv
  • Linyue Chen
  • Yue Zhao
  • Zixin Deng
  • Kui HongEmail author
Biotechnological products and process engineering
  • 18 Downloads

Abstract

Ophiobolins (ophs) are characteristic 5-8-5 tricyclic sesterterpenes with potential pharmaceutical activities. Ophiobolin synthase is a bifunctional terpene synthase (BTS) that catalyzes both chain elongation and cyclization. In Aspergillus ustus 094102, ophiobolin accumulation was involved with not only ophiobolin synthase C25 (Au8003) but also other four gene clusters containing C15 (Au6298), C20 (Au13192 and Au11565), and C30 (Au3446) terpene synthases. In this report, overexpression of codon-optimized gene Au8003 resulted in a detectable production of oph F in E. coli. In subsequent modulation of culture conditions, pentose arabinose allowed a more than 10-fold improvement of production than that of glycerol. To achieve a higher titer, the whole mevalonate pathway and an additional copy of isopentenyl diphosphate isomerase gene were assembled, leading to approximately 24-fold and 60-fold yield increases, respectively. The above four terpene synthase genes related to ophiobolin production in strain 094102 were individually or combinatorially overexpressed with Au8003 to mimic the original fungal biosynthesis. The biosynthesis of oph scaffold was increased by short-chain terpene synthases (C15 and C20), among which the C15 synthase gene contributed the highest yield of 82.76 mg/L at 96 h; the multi-gene combinatorial results suggested that cyclization might be a rate-limiting step. Further protein engineering including fusion tags and phylogenetically based mutations on the rate-limiting cyclization part of Au8003 enabled a further yield improvement (> 150 mg/L at 96 h) in shake flasks. These multiple approaches for sesterterpene skeleton production using engineered E. coli may be applicable for cost-effective, high-yield productions of ophiobolins and other compounds synthesized by BTSs.

Keywords

Ophiobolin Sesterterpene Bifunctional terpene synthase Synthetic biology Protein engineering 

Notes

Acknowledgments

The authors are grateful to professor Tiangang Liu in Wuhan University for pMH1 and pFZ81 plasmids. We acknowledge Mr. Guofu Qiu in Wuhan University for NMR data collection. We are grateful to the assistance of Miss Yanyu Tao, Shiyu Zhu, and Mr. Jiangfeng Lu for fermentation experiments.

Funding information

This work was financially supported by grants from the National Key Research and Development Program of China (No. 2018YFC0311001) and National Natural Science Foundation of China (No. 81673331).

Compliance with ethical standards

Conflict of interest

Wuhan University has filed a patent application based on this work.

Ethical statement

This article does not contain any studies involving human participants or experimental animals.

Supplementary material

253_2019_10103_MOESM1_ESM.pdf (1.7 mb)
ESM 1 (PDF 1751 kb)

References

  1. Abdallah II, Ronald VM, Klumpenaar E, Quax WJ (2018) Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering. Sci Rep 8(9961):9961.  https://doi.org/10.1038/s41598-018-28177-4 CrossRefGoogle Scholar
  2. Arens J, Engels B, Klopries S, Jennewein S, Ottmann C, Schulz F (2013) Exploration of biosynthetic access to the shared precursor of the fusicoccane diterpenoid family. Chem Commun 49:4337–4339.  https://doi.org/10.1039/c2cc37154e CrossRefGoogle Scholar
  3. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201.  https://doi.org/10.1093/bioinformatics/bti770 CrossRefGoogle Scholar
  4. Bian G, Deng Z, Liu T (2017) Strategies for terpenoid overproduction and new terpenoid discovery. Curr Opin Biotechnol 48:234–241.  https://doi.org/10.1016/j.copbio.2017.07.002 CrossRefGoogle Scholar
  5. Brill ZG, Grover HK, Maimone TJ (2016) Enantioselective synthesis of an ophiobolin sesterterpene via a programmed radical cascade. Science 352:1078–1082.  https://doi.org/10.1126/science.aaf6742 CrossRefGoogle Scholar
  6. Bury M, Girault A, Megalizzi V, Spiegl-Kreinecker S, Mathieu V, Berger W, Evidente A, Kornienko A, Gailly P, Vandier C, Kiss R (2013) Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity. Cell Death Dis 4:e561.  https://doi.org/10.1038/cddis.2013.85 CrossRefGoogle Scholar
  7. Chai H, Yin R, Liu Y, Meng H, Zhou X, Zhou G, Bi X, Yang X, Zhu T, Zhu W, Deng Z, Hong K (2016) Sesterterpene ophiobolin biosynthesis involving multiple gene clusters in Aspergillus ustus. Sci Rep 6:27181.  https://doi.org/10.1038/srep27181 CrossRefGoogle Scholar
  8. Chen W, Ye L, Guo F, Lv Y, Yu H (2015a) Enhanced activity of an alkaline phytase from Bacillus subtilis 168 in acidic and neutral environments by directed evolution. Biotech Eng J 98:137–143.  https://doi.org/10.1016/j.bej.2015.02.021 Google Scholar
  9. Chen X, Shi J, Chen R, Wen Y, Shi Y, Zhu Z, Guo S, Li L (2015b) Molecular chaperones (TrxA, SUMO, Intein, and GST) mediating expression, purification, and antimicrobial activity assays of plectasin in Escherichia coli. Biotechnol Appl Biochem 62:606–614.  https://doi.org/10.1002/bab.1303 CrossRefGoogle Scholar
  10. Chen M, Chou WK, Toyomasu T, Cane DE, Christianson DW (2016) Structure and function of fusicoccadiene synthase, a hexameric bifunctional diterpene synthase. ACS Chem Biol 11:889–899.  https://doi.org/10.1021/acschembio.5b00960 CrossRefGoogle Scholar
  11. Chiba R, Minami A, Gomi K, Oikawa H (2013) Identification of ophiobolin F synthase by a genome mining approach: a sesterterpene synthase from Aspergillus clavatus. Org Lett 15:594–597.  https://doi.org/10.1021/ol303408a CrossRefGoogle Scholar
  12. Christianson DW (2017) Structural and chemical biology of terpenoid cyclases. Chem Rev 117:11570–11648.  https://doi.org/10.1021/acs.chemrev.7b00287 CrossRefGoogle Scholar
  13. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: A sequence logo generator. Genome Res 14:1188–1190.  https://doi.org/10.1101/gr.849004 CrossRefGoogle Scholar
  14. George KW, Alonso-Gutierrez J, Keasling JD, Lee TS (2015) Isoprenoid drugs, biofuels, and chemicals-artemisinin, farnesene, and beyond. In: Schrader J, Bohlmann J (eds) Biotechnology of Isoprenoids. Adv. Biochem. Eng./Biotechnol, vol 148. Springer, Cham, pp 355–389.  https://doi.org/10.1007/10_2014_288 CrossRefGoogle Scholar
  15. George KW, Thompson MG, Kim J, Baidoo EE, Wang G, Benites VT, Petzold CJ, Chan LJG, Yilmaz S, Turhanen P (2018) Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli. Metab Eng 47:60–72.  https://doi.org/10.1016/j.ymben.2018.03.004 CrossRefGoogle Scholar
  16. Hong K, Yang X (2013) High-yield ophiobolin compound strain Aspergillus ustus TKYX429 and application thereof. China Patent: ZL201310186483.4. 2013-8-7Google Scholar
  17. Leonard E, Ajikumar PK, Thayer K, Xiao WH, Mo JD, Tidor B, Stephanopoulos G, Prather KLJ (2010) Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc Natl Acad Sci U S A 107:13654–13659.  https://doi.org/10.1073/pnas.1006138107 CrossRefGoogle Scholar
  18. Li ZJ, Hong PH, Da YY, Li LK, Stephanopoulos G (2018) Metabolic engineering of Escherichia coli for the production of L-malate from xylose. Metab Eng 48:25–32.  https://doi.org/10.1016/j.ymben.2018.05.010 CrossRefGoogle Scholar
  19. Liu C, Bi H, Bai Z, Fan L, Tan T (2019) Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production. Appl Microbiol Biotechnol 103:239–250.  https://doi.org/10.1007/s00253-018-9472-9 CrossRefGoogle Scholar
  20. Mandell DJ, Coutsias EA, Kortemme T (2009) Sub-angstrom accuracy in protein loop reconstruction by robotics-inspired conformational sampling. Nat Methods 6:551–552.  https://doi.org/10.1038/nmeth0809-551 CrossRefGoogle Scholar
  21. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802.  https://doi.org/10.1038/nbt833 CrossRefGoogle Scholar
  22. Masi M, Dasari R, Evidente A, Mathieu V, Kornienko A (2019) Chemistry and biology of ophiobolin A and its congeners. Bioorg Med Chem Lett 29:859–869.  https://doi.org/10.1016/j.bmcl.2019.02007 CrossRefGoogle Scholar
  23. Matsuda Y, Mitsuhashi T, Lee S, Hoshino M, Mori T, Okada M, Zhang H, Hayashi F, Fujita M, Abe I (2016) Astellifadiene: Structure determination by NMR spectroscopy and crystalline sponge method, and elucidation of its biosynthesis. Angew Chem Int Ed 55:5785–5788.  https://doi.org/10.1002/anie.201601448 CrossRefGoogle Scholar
  24. Narita K, Sato H, Minami A, Kudo K, Gao L, Liu C, Ozaki T, Kodama M, Lei X, Taniguchi T, Monde K, Yamazaki M, Uchiyama M, Oikawa H (2017) Focused genome mining of structurally related sesterterpenes: enzymatic formation of enantiomeric and diastereomeric products. Org Lett 19:6696–6699.  https://doi.org/10.1021/acs.orglett.7b03418 CrossRefGoogle Scholar
  25. Nogué VS, Karhumaa K (2015) Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals. Biotechnol Lett 37:761–772.  https://doi.org/10.1007/s10529-014-1756-2 CrossRefGoogle Scholar
  26. Okada M, Matsuda Y, Mitsuhashi T, Hoshino S, Mori T, Nakagawa K, Quan Z, Qin B, Zhang H, Hayashi F (2016) Genome-based discovery of an unprecedented cyclization mode in fungal sesterterpenoid biosynthesis. J Am Chem Soc 138:10011–10018.  https://doi.org/10.1021/jacs.6b05799 CrossRefGoogle Scholar
  27. Paddon CJ, Keasling JD (2014) Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nat Rev Microbiol 12:355–367.  https://doi.org/10.1038/nrmicro3240 CrossRefGoogle Scholar
  28. Paramasivan K, Mutturi S (2017) Regeneration of NADPH coupled with HMG-CoA reductase activity increases squalene synthesis in Saccharomyces cerevisiae. J Agric Food Chem 65:8162–8170.  https://doi.org/10.1021/acs.jafc.7b02945 CrossRefGoogle Scholar
  29. Pereira B, Li ZJ, De Mey M, Lim CG, Zhang H, Hoeltgen C, Stephanopoulos G (2016) Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate. Metab Eng 34:80–87.  https://doi.org/10.1016/j.ymben.2015.12.004 CrossRefGoogle Scholar
  30. Qin B, Matsuda Y, Mori T, Okada M, Quan Z, Mitsuhashi T, Wakimoto T, Abe I (2016) An unusual chimeric diterpene synthase from Emericella variecolor and its functional conversion into a sesterterpene synthase by domain swapping. Angew Chem Int Ed 55:1658–1661.  https://doi.org/10.1002/anie.201509263 CrossRefGoogle Scholar
  31. Rowley M, Tsukamoto M, Kishi Y (1989) Total synthesis of (+)-ophiobolin C. J Am Chem Soc 111:2735–2737.  https://doi.org/10.1021/ja00189a069 CrossRefGoogle Scholar
  32. Sato H, Narita K, Minami A, Yamazaki M, Wang C, Suemune H, Nagano S, Tomita T, Oikawa H, Uchiyama M (2018) Theoretical study of sesterfisherol biosynthesis: computational prediction of key amino acid residue in terpene synthase. Sci Rep 8:2473.  https://doi.org/10.1038/s41598-018-20916-x CrossRefGoogle Scholar
  33. Schmidt MW, Baldridge KK, Boatz JA, Elbert ST, Gordon MS, Jensen JH, Koseki S, Matsunaga N, Nguyen KA, Su SJ, Windus TL, Dupuis M, Montgomery JA (1993) General atomic and molecular electronic-structure system. J Comput Chem 14:1347–1363.  https://doi.org/10.1002/jcc.540141112 CrossRefGoogle Scholar
  34. Stein A, Kortemme T (2013) Improvements to robotics-inspired conformational sampling in rosetta. PLoS One 8:e63090.  https://doi.org/10.1371/journal.pone.0063090 CrossRefGoogle Scholar
  35. Steinmetz EJ, Auldridge ME (2017) Screening fusion tags for improved recombinant protein expression in E. coli with the Expresso® solubility and expression screening system. Curr Protoc Protein Sci 90:5–27.  https://doi.org/10.1002/cpps.39 Google Scholar
  36. Sun W, Lv C, Zhu T, Yang X, Wei S, Sun J, Hong K, Zhu W, Huang C (2013) Ophiobolin-O reverses adriamycin resistance via cell cycle arrest and apoptosis sensitization in adriamycin-resistant human breast carcinoma (MCF-7/ADR) cells. Mar Drugs 11:4570–4584.  https://doi.org/10.3390/md11114570 CrossRefGoogle Scholar
  37. Tian W, Deng Z, Hong K (2017) The biological activities of sesterterpenoid-type ophiobolins. Mar Drugs 15:229.  https://doi.org/10.3390/md15070229 CrossRefGoogle Scholar
  38. Toyomasu T, Tsukahara M, Kaneko A, Niida R, Mitsuhashi W, Dairi T, Kato N, Sassa T (2007) Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi. Proc Natl Acad Sci U S A 104:3084–3088.  https://doi.org/10.1073/pnas.0608426104 CrossRefGoogle Scholar
  39. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461.  https://doi.org/10.1002/jcc.21334 Google Scholar
  40. Tsuna K, Noguchi N, Nakada M (2011) Convergent total synthesis of (+)-ophiobolin A. Angew Chem Int Ed 50:9624–9627.  https://doi.org/10.1002/anie.201104447 CrossRefGoogle Scholar
  41. Wada K, Toya Y, Banno S, Yoshikawa K, Matsuda F, Shimizu H (2017) 13C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli. J Biosci Bioeng 123:177–182.  https://doi.org/10.1016/j.jbiosc.2016.08.001 CrossRefGoogle Scholar
  42. Wang M, Chen B, Fang Y, Tan T (2017) Cofactor engineering for more efficient production of chemicals and biofuels. Biotechnol Adv 35:1032–1039.  https://doi.org/10.1016/j.biotechadv.2017.09.008 CrossRefGoogle Scholar
  43. Yang H, Liu L, Xu F (2016) The promises and challenges of fusion constructs in protein biochemistry and enzymology. Appl Microbiol Biotechnol 100:8273–8281.  https://doi.org/10.1007/s00253-016-7795-y CrossRefGoogle Scholar
  44. Zhu F, Zhong X, Hu M, Lu L, Deng Z, Liu T (2014) In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli. Biotechnol Bioeng 111:1396–1405.  https://doi.org/10.1002/bit.25198 CrossRefGoogle Scholar
  45. Zhu T, Lu Z, Fan J, Wang L, Zhu G, Wang Y, Li X, Hong K, Piyachaturawat P, Chairoungdua A, Zhu W (2018) Ophiobolins from the mangrove fungus Aspergillus ustus. J Nat Prod 81:2–9.  https://doi.org/10.1021/acs.jnatprod.7b00335 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Wei Yuan
    • 1
  • Shuang Lv
    • 1
  • Linyue Chen
    • 1
  • Yue Zhao
    • 1
  • Zixin Deng
    • 1
  • Kui Hong
    • 1
    Email author
  1. 1.Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of EducationWuhan University School of Pharmaceutical Sciences, Wuhan UniversityWuhanPeople’s Republic of China

Personalised recommendations