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Construction of Cordycepin High-Production Strain and Optimization of Culture Conditions

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Abstract

This study aimed to increase cordycepin production by over-expressing bio-synthetic enzyme genes, including the adenylosuccinate synthase, adenylosuccinate lyase, and 5′-nucleotidase genes. Research data showed that the extracellular and intracellular cordycepin concent of 24 recombinant strains were higher than those of C. militaris WT, indicating that over-expression of key enzyme genes increased cordycepin production. Among them, the CM-adss-5 strain had highest cordycepin production, and the extracellular and intracellular cordycepin concent were 1119.75 ± 1.61 and 65.56 ± 0.97 mg/L, which were 1.26 and 2.61 times that of C. militaris WT. This study also optimized the culture conditions of CM-adss-5 strain through single factor experiments to obtain the best culture conditions. The best culture condition was 25 °C constant temperature, 180-rpm shaking culture, fermentation period 12 days, inoculate amount 5%, initial pH 6, seed age 108 h, and liquid volume 110/250 mL. Then, the extracellular and intracellular cordycepin content of CM-adss-5 strain reached 2581.96 ± 21.07 and 164.08 ± 1.44 mg/L, which were higher by 130.6% and 150.3%, respectively. Therefore, our research provides a way to efficiently produce cordycepin for the development of cordycepin and its downstream products.

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References

  1. Kunhorm P, Chaicharoenaudomrung N, Noisa P (2019) Enrichment of cordycepin for cosmeceutical applications: culture systems and strategies. Appl Microbiol Biotechnol 103(4):1681–1691. https://doi.org/10.1007/s00253-019-09623-3

    Article  CAS  Google Scholar 

  2. Cui JD (2015) Biotechnological production and applications of Cordyceps militaris, a valued traditional Chinese medicine. Crit Rev Biotechnol 35(4):475–484. https://doi.org/10.3109/07388551.2014.900604

    Article  CAS  Google Scholar 

  3. Das SK (2010) Medicinal uses of the mushroom Cordyceps militaris: current state and prospects. Fitoterapia 81(8):961–968. https://doi.org/10.1016/j.fitote.2010.07.010

    Article  Google Scholar 

  4. Ng TB, Wang HX (2005) Pharmacological actions of Cordyceps, a prized folk medicine. J Pharm Pharmacol 57(12):1509–1519. https://doi.org/10.1211/jpp.57.12.0001

    Article  CAS  Google Scholar 

  5. Xia YL, Luo FF, Shang YF, Chen PL, Lu YZ, Wang CS (2017) Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin. Cell Chem Biol 24(12):1479–1489. https://doi.org/10.1016/j.chembiol.2017.09.001

    Article  CAS  Google Scholar 

  6. Ashraf SA, Elkhalifa AO, Siddiqui AJ, Patel M, Awadelkareem AM, Snoussi M, Ashraf MS, Adnan M, Hadi S (2020) Cordycepin for health and wellbeing: a potent bioactive metabolite of an entomopathogenic medicinal fungus Cordyceps with its nutraceutical and therapeutic potential. Molecules 25(12):2735–2756. https://doi.org/10.3390/molecules25122735

    Article  CAS  Google Scholar 

  7. Chamyuang S, Owatworakit A, Honda Y (2019) New insights into cordycepin production in Cordyceps militaris and applications. Ann Transl Med 7(3):78–80

    Article  Google Scholar 

  8. Qin P, Li XK, Yang H et al (2019) Therapeutic potential and biological applications of cordycepin and metabolic mechanisms in cordycepin-producing fungi. Molecules 24(12):2231–2256. https://doi.org/10.3390/molecules24122231

    Article  CAS  Google Scholar 

  9. Govindula A, Pai A, Baghel S, Mudgal J (2021) Molecular mechanisms of cordycepin emphasizing its potential against neuroinflammation: an update. Eur J Pharmacol 908(4):174364. https://doi.org/10.1016/j.ejphar.2021.174364

    Article  CAS  Google Scholar 

  10. Luo QY, Cao HF, Liu SK, Wu M, Li SS, Zhang ZQ, Chen AJ, Shen GH, Wu HJ, Li ML, Liu XY, Jiang Y, Bi JF, He ZY (2020) Novel liquid fermentation medium of Cordyceps militaris and optimization of hydrothermal reflux extraction of cordycepin. J Asian Nat Prod Res 22(2):167–178. https://doi.org/10.1080/10286020.2018.1539080

    Article  CAS  Google Scholar 

  11. Tang JP, Qian ZQ, Wu H (2018) Enhancing cordycepin production in liquid static cultivation of Cordyceps militaris by adding vegetable oils as the secondary carbon source. Biores Technol 268:60–67. https://doi.org/10.1016/j.biortech.2018.07.128

    Article  CAS  Google Scholar 

  12. Mao XB, Eksriwong T, Chauvatcharin S et al (2005) Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Process Biochem 40(5):1667–1672. https://doi.org/10.1016/j.procbio.2004.06.046

    Article  CAS  Google Scholar 

  13. Das SK, Masuda M, Hatashita M et al (2010) Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation. Process Biochem 45(1):129–132. https://doi.org/10.1016/j.procbio.2009.08.008

    Article  CAS  Google Scholar 

  14. Shih IL, Tsai KL, Hsieh C (2007) Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochem Eng J 33(3):193–201. https://doi.org/10.1016/j.bej.2006.10.019

    Article  CAS  Google Scholar 

  15. Sari N, Suparmin A, Kato T et al (2016) Improved cordycepin production in a liquid surface culture of Cordyceps militaris isolated from wild strain. Biotechnol Bioprocess Eng 21(5):595–600. https://doi.org/10.1007/s12257-016-0405-0

    Article  CAS  Google Scholar 

  16. Xu JW, Ji SL, Li HJ, Zhou JS, Duan YQ, Dang LZ, Mo MH (2015) Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous alpha-phosphoglucomutase gene. Bioprocess Biosyst Eng 38(2):399–405. https://doi.org/10.1007/s00449-014-1279-1

    Article  CAS  Google Scholar 

  17. Zhang H, Wang YX, Tong XX, Wallace Y, Cao J, Wang F, Peng C, Guo JL (2020) Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris. Chin J Nat Med 18(5):393–400. https://doi.org/10.1016/S1875-5364(20)30046-7

    Article  CAS  Google Scholar 

  18. Liu TF, Liu ZY, Yao XY, Huang Y, Qu QS, Shi XS, Zhang HM, Shi XY (2018) Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae. R Soc Open Sci 5(12):181247–181261. https://doi.org/10.1098/rsos.181247

    Article  CAS  Google Scholar 

  19. Zheng P, Xia YL, Xiao GH, Xiong CH, Hu X, Zhang SW, Zheng HJ, Huang Y, Zhou Y, Wang SY, Zhao GP, Liu XZ, St Leger RJ, Wang CS (2011) Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional chinese medicine. Genome Biol 12(11):R116-135. https://doi.org/10.1186/gb-2011-12-11-r116

    Article  CAS  Google Scholar 

  20. Vongsangnak W, Raethong N, Mujchariyakul W, Nguyen NN, Leong HW, Laoteng K (2017) Genome-scale metabolic network of Cordyceps militaris useful for comparative analysis of entomopathogenic fungi. Gene 626:132–139. https://doi.org/10.1016/j.gene.2017.05.027

    Article  CAS  Google Scholar 

  21. Chen YJ, Wu YQ, Liu L, Feng JH, Zhang TC, Qin S, Zhao XY, Wang CX, Li DM, Han W, Shao MH, Zhao P, Xue JF, Liu XM, Li HJ, Zhao EW, Zhao W, Guo XJ, Jin YF, Cao YM, Cui LW, Zhou ZQ, Xia QY, Rao ZH, Zhang YZ (2019) Study of the whole genome, methylome and transcriptome of Cordyceps militaris. Sci Rep 9:898–912. https://doi.org/10.1038/s41598-018-38021-4

    Article  CAS  Google Scholar 

  22. Masuda M, Urabe E, Honda H, Sakurai A, Sakakibara M (2007) Enhanced production of cordycepin by surface culture using the medicinal mushroom Cordyceps militaris. Enzyme Microb Technol 40(5):1199–1205. https://doi.org/10.1016/j.enzmictec.2006.09.008

    Article  CAS  Google Scholar 

  23. Lennon MB, Suhadolnik RJ (1976) Biosynthesis of 3′deoxyadenosine by Cordyceps militaris: mechanism of reduction. Biochim Biophys Acta 425(4):532–536. https://doi.org/10.1016/0005-2787(76)90017-4

    Article  CAS  Google Scholar 

  24. Lin S, Liu ZQ, Xue YP, Baker PJ, Wu H, Xu F, Teng Y, Brathwaite ME, Zheng YG (2016) Biosynthetic pathway analysis for improving the cordycepin and cordycepic acid production in Hirsutella sinensis. Appl Biochem Biotechnol 179(4):633–649. https://doi.org/10.1007/s12010-016-2020-0

    Article  CAS  Google Scholar 

  25. Kato T, Ahmad S, Park EY (2017) Functional analysis of ribonucleotide reductase from Cordyceps militaris expressed in Escherichia coli. Appl Biochem Biotechnol 182(4):1307–1317. https://doi.org/10.1007/s12010-017-2400-0

    Article  CAS  Google Scholar 

  26. Yin YL, Yu GJ, Chen YJ, Jiang S, Wang M, Jin YX, Lan XQ, Liang Y, Sun H (2012) Genome-wide transcriptome and proteome analysis on different developmental stages of Cordyceps militaris. PLoS ONE 7(12):e51853-51867. https://doi.org/10.1371/journal.pone.0051853

    Article  CAS  Google Scholar 

  27. Tang JP, Liu YT, Zhu L (2014) Optimization of fermentation conditions and purification of cordycepin from Cordyceps militaris. Prep Biochem Biotechnol 44(1):90–106. https://doi.org/10.1080/10826068.2013.833111

    Article  CAS  Google Scholar 

  28. Xie CY, Liu GX, Gu ZX, Fan GJ, Zhang L, Gu YJ (2009) Effects of culture conditions on mycelium biomass and intracellular cordycepin production of Cordyceps militaris in natural medium. Ann Microbiol 59(2):293–299. https://doi.org/10.1007/BF03178331

    Article  CAS  Google Scholar 

  29. Kang C, Wen TC, Kang JC, Meng ZB, Li GR, Hyde KD (2014) Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid static culture. Sci World J. https://doi.org/10.1155/2014/510627

    Article  Google Scholar 

  30. Li HJ, Zhang DH, Yue TH, Jiang LX, Yu XY, Zhao P, Li T, Xu JW (2016) Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of vitreoscilla hemoglobin gene. J Biotechnol 217:132–137. https://doi.org/10.1016/j.jbiotec.2015.11.011

    Article  CAS  Google Scholar 

  31. Zhou JS, Bai Y, Dai RJ, Guo XL, Liu ZH, Yuan S (2018) Improved polysaccharide production by homologous co-overexpression of phosphoglucomutase and udp glucose pyrophosphorylase genes in the mushroom Coprinopsis cinerea. J Agric Food Chem 66(18):4702–4709. https://doi.org/10.1021/acs.jafc.8b01343

    Article  CAS  Google Scholar 

  32. Frandsen RJN (2011) A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation. J Microbiol Methods 87(3):247–262. https://doi.org/10.1016/j.mimet.2011.09.004

    Article  CAS  Google Scholar 

  33. Han GM, Shao Q, Li CP, Zhao K, Jiang L, Fan J, Jiang HY, Tao F (2018) An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus. J Microbiol 56(5):356–364. https://doi.org/10.1007/s12275-018-7349-3

    Article  CAS  Google Scholar 

  34. Figueiredo JG, Goulin EH, Tanaka F, Stringari D, Kava-Cordeiro V, Galli-Terasawa LV, Staats CC, Schrank A, Glienke C (2010) Agrobacterium tumefaciens-mediated transformation of Guignardia citricarpa. J Microbiol Methods 80(2):143–147. https://doi.org/10.1016/j.mimet.2009.11.014

    Article  CAS  Google Scholar 

  35. Cai LN, Xu SN, Lu T, Lin DQ, Yao SJ (2020) Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity. Chin J Chem Eng 28(4):1120–1128. https://doi.org/10.1016/j.cjche.2019.11.012

    Article  CAS  Google Scholar 

  36. Nakamura M, Kuwahara H, Onoyama K, Iwai H (2012) Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletotrichum sansevieriae. Curr Microbiol 65(2):176–182. https://doi.org/10.1007/s00284-012-0140-5

    Article  CAS  Google Scholar 

  37. Cai LN, Xu SN, Lu T et al (2020) Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity. Chin J Chem Eng 28(4):1120–1128. https://doi.org/10.1016/j.cjche.2019.11.012

    Article  CAS  Google Scholar 

  38. Zhang P, Liu TT, Zhou PP, Li ST, Yu LJ (2011) Agrobacterium tumefaciens-mediated transformation of a taxol-producing endophytic fungus, Cladosporium cladosporioides MD2. Curr Microbiol 62(4):1315–1320. https://doi.org/10.1007/s00284-010-9864-2

    Article  CAS  Google Scholar 

  39. Liu H, Zhang ZY, Liang SJ, Guo L, Sun SY, Pan KH, Yang GP (2020) Transcriptome responses of Hygromycin B resistance gene-transformed, Hygromycin B-adaptive and Wild Nannochloropsis oceanica Strains to Hygromycin B. J Ocean Univ China 19(2):453–458. https://doi.org/10.1007/s11802-020-4252-4

    Article  CAS  Google Scholar 

  40. Martinez-Cruz J, Romero D, de Vicente A, Perez-Garcia A (2017) Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens. New Phytol 213(4):1961–1973. https://doi.org/10.1111/nph.14297

    Article  CAS  Google Scholar 

  41. Deng L, Zhou TY, Pi L, Zhao XH, Han T, Li YK, Han F (2013) Optimization of microwave-assisted extraction of cordycepic acid and cordycepin from cultured Cordyceps militaris by response surface methodology. Asian J Chem 25(14):8065–8071. https://doi.org/10.14233/ajchem.2013.15056

    Article  CAS  Google Scholar 

  42. Chang CY, Lue MY, Pan TM (2005) Determination of adenosine, cordycepin and ergosterol contents in cultivated Antrodia camphorata by HPLC method. J Food Drug Anal 13(4):338–342. https://doi.org/10.1263/jbb.100.685

    Article  CAS  Google Scholar 

  43. Chen YS, Liu BL, Chang YN (2011) Effects of light and heavy metals on Cordyceps militaris fruit body growth in rice grain-based cultivation. Korean J Chem Eng 28(3):875–879. https://doi.org/10.1007/s11814-010-0438-6

    Article  CAS  Google Scholar 

  44. Shashidhar MG, Manohar B (2016) Acidified hot water extraction of adenosine, cordycepin, and polysaccharides from the chinese caterpillar mushroom, Ophiocordyceps sinensis CS1197 (Ascomycetes): application of an artificial neural network and evaluation of antioxidant and antibacterial activities. Int J Med Mushrooms 18(10):915–926. https://doi.org/10.1615/intjmedmushrooms.v18.i10.70

    Article  CAS  Google Scholar 

  45. Soltani M, Abd Malek R, Ware I, Ramli S, Elsayed EA, Aziz R, El-Enshasy HA (2017) Optimization of cordycepin extraction from Cordyceps militaris fermentation broth. J Sci Ind Res 76(6):355–361

    CAS  Google Scholar 

  46. Qin P, Wang ZY, Lu DX, Kang HM, Li G, Guo R, Zhao YH, Han RB, Ji B, Zeng Y (2019) Neutral lipid content in lipid droplets: potential biomarker of cordycepin accumulation in cordycepin-producing fungi. Molecules 24(18):3363–3376. https://doi.org/10.3390/molecules24183363

    Article  CAS  Google Scholar 

  47. Lee SK, Lee JH, Kim HR, Chun Y, Yoo HY, Park C, Kim SW (2019) Improved cordycepin production by Cordyceps militaris KYL05 using casein hydrolysate in submerged conditions. Biomolecules 9(9):461–471. https://doi.org/10.3390/biom9090461

    Article  CAS  Google Scholar 

  48. Li Q, Li YM, Han S, Liu YZ, Song DX, Hao D, Wang JJ, Qu YH, Zheng YX (2013) Optimization of fermentation conditions and properties of an exopolysaccharide from Klebsiella sp H-207 and application in adsorption of hexavalent chromium. PLoS ONE 8(1):53542–53552. https://doi.org/10.1371/journal.pone.0053542

    Article  CAS  Google Scholar 

  49. Tang JP, Qian ZQ, Zhu L (2015) Two-step shake-static fermentation to enhance cordycepin production by Cordyceps militaris. Iaeds15: International Conference in Applied Engineering and Management 46: 19–24. https://doi.org/10.3303/CET1546004.

  50. Mao XB, Zhong JJ (2004) Hyperproduction of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors. Biotechnol Prog 20(5):1408–1413. https://doi.org/10.1021/bp049765r

    Article  CAS  Google Scholar 

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Acknowledgements

Great appreciation is given to all members of our laboratory for their enthusiastic participation in the research.

Funding

This work was supported by a key project funded by Science and Technology Department of Zhejiang Province [Nos. 2018C02017, 2020C02055, 2020C02040, and 2020C02041].

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Author notes

  1. Hui Zhang, Ping Chen, Lin Xu and De Xu have contributed equally to this work.

Authors and Affiliations

  1. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, People’s Republic of China

    Hui Zhang

  2. The College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Ping Chen, Lin Xu, De Xu & Shengli Yang

  3. Zhejiang Skyherb Biotechnology Inc., Anji, 313300, People’s Republic of China

    Wendi Hu & Yong Cheng

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  1. Hui Zhang
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Contributions

YSL and ZH conceived and designed research. CP conducted experiments. XL, XD, HWD, and CY analyzed data, CP wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Hui Zhang or Shengli Yang.

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Zhang, H., Chen, P., Xu, L. et al. Construction of Cordycepin High-Production Strain and Optimization of Culture Conditions. Curr Microbiol 80, 12 (2023). https://doi.org/10.1007/s00284-022-03110-1

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