Abstract
Improvement of the production of vincamine in endophytic fungus VINI-7 was performed by using the inactivated protoplast fusion method. The preparation conditions of protoplasts were optimized by systematic trials with various parameters, and inactivated protoplast fusion was subsequently performed. The mycelium in logarithmic growth phase was treated with 1500 U/mL lywallzyme, 1500 U/mL lysozyme, 2000 U/mL cellulase, and 1000 U/mL snailase solution for 3 h at 30 °C and had the best conditions, in which the concentration of the protoplast was 3.17 × 107 cells/mL. Protoplasts were inactivated by heat, ultraviolet, microwave, sodium nitrite, and diethyl sulfate, respectively. Subsequently, protoplasts inactivated by different methods were subjected to respective protoplast fusion. The results showed that the yield of vincamine in fusants inactivated by mutagens was generally higher than that of fusants inactivated by heat. The highest yield of vincamine in two fusants (U-U1 and N-N1) was 31.6 and 38.7 mg, which increased to 162.24 and 221.16%, respectively, as compared to the parent strain (12.05 mg). LC-MS/MS analysis showed that U-U1 and N-N1 fusants could produce vincamine. Furthermore, the results of genetic stability experiments indicated that U-U1 and N-N1 were genetically stable.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ando K, Hironaka N, Shuto K (2003) Effects of vinconate on scopolamine-induced memory impairment in rhesus monkeys. Nihon shinkei seishin yakurigaku zasshi 23:43–46
Cui YX, Liu JJ, Liu Y, Cheng QY, Yu Q, Chen X, Ren XD (2014) Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei. Biotechnol Lett 36(12):2495–2499
Feng W, Liang J, Wang B, Chen J (2019) Improvement of kojic acid production in Aspergillus oryzae AR-47 mutant strain by combined mutagenesis. Bioprocess Biosyst Eng 42(5):753–761
Khattab A, Bazaraa WA (2005) Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production. J Ind Microbiol Biotechnol 32:289–294
Li LM, Zheng TT, Sui Y, Li X, Sun JH, Zhang LH, Zhu CY, Chen YQ (2017) DES mutagenesis of antagonistic streptomyces of botryosphaeria dothidea and optimization of its fermentation conditions. Plant Dis Pests 8(1):15–23
Li XH, Yang HJ, Roy B, Park E, Jiang LJ, Wang D, Miao YG (2009) Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet. Microbiol Res 165:190–198
Manda V, Avula B, Dale O, Chittiboyina A, Khan IA, Walker LA, Khan SI (2015) Studies on pharmacokinetic drug interaction potential of vinpocetine. Medicines 2:93–105
Na R, Jiajia L, Dongliang Y, Yingzi P, Juan H, Xiong L, Nana Z, Jing Z, Yitian L (2016) Indentification of vincamine indole alkaloids producing endophytic fungi isolated from Nerium indicum, Apocynaceae. Microbiol Res 192:114–121
Peng R, Yang GJ, Wang QM, Du YY, Li J (2013) Isolation and mutagenesis of a novel phenol-degrading strain. Adv Mater Res 647:588–594
Peng R, Fu Y, Zou J, Qiu H, Gan L, Yi H, Yao R, Luo X (2016) Improvement of polysaccharide and triterpenoid production of Ganoderma lucidum through mutagenesis of protoplasts. Biotechnol Biotechnol Equip 30(2):381–387
Ren N, Liu J, Yang D, Liu X, Zhou J, Peng Y (2018) Preparation and regeneration of protoplasts from the ethyl vincamine producing fungus CH1 (Geomyces sp.). Nat Prod Commun 13(2):145–148
Shin WS, Park B, Lee D, Oh MK, Chun GT, Kim S (2017) Enhanced production of itaconic acid through development of transformed fungal strains of Aspergillus terreus. J Microbiol Biotechnol 27(2):306–315
Song X, Zhang Y, Zhu X, Wang Y, Chu J, Zhuang Y (2017) Mutation breeding of high avermectin B1a-producing strain by the combination of high energy carbon heavy ion irradiation and sodium nitrite mutagenesis based on high throughput screening. Biotechnol Bioprocess Eng 22(5):539–548
Stierle A, Strobel GA, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 260:214–216
Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5:535–544
Vaiyapuri P, Mathivanan N, Sagadevan E, Murugesan K, Lalithakumari D (2007) Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum. Bioresour Technol 97:2330–2334
Wu P, Zhao X, Pan S (2014) Intraspecific protoplast fusion of Brettanomyces anomalus for improved production of an extracellular β-glucosidase. Biotechnol Biotechnol Equip 28:878–881
Yin H, Gao Y (2010) Opitimization of fermentation conditions of a mutagenized berberine-producing strain. J Fungal Res 1:44–47
Yin H, Sun Y-H (2011) Vincamine-producing endophytic fungus isolated from Vinca minor. Phytomedicine 18:802–805
Yin H, Zhao Q, Sun FM, An T (2009) Gentiopicrin-producing endophytic fungus isolated from Gentiana macrophylla. Phytomedicine 16:793–797
Zhang M, Xiao Y, Zhu R, Zhang Q, Wang SL (2012) Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion. Bioprocess Biosyst Eng 35:1455–1465
Zhao K, Zhou DP, Ping WX, Ge JP (2004) Study on preparation and regeneration of protoplast from taxol-producing fungus Nodulisporum sylviforme. Nat Sci 2:52–59
Zhao J, Ma D, Luo M, Wang W, Zhao C, Zu Y, Fu Y, Wink M (2014) In vitro antioxidant activities and antioxidant enzyme activities in HepG2 cells and main active compounds of endophytic fungus from pigeon pea [Cajanus cajan (L.) Mill sp.]. Food Res Int 56:243–251
Zhou XW, Wei YM, Zhu HF, Wang ZN, Lin JA, Liu L, Tang KX (2008) Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp. Afr J Biotechnol 7(12):2017–2024
Zou ZS, Zhao YY, Zhang TZ, Xu JX, He AY, Deng Y (2018) Efficient isolation and characterization of a cellulase hyperproducing mutant strain of Trichoderma reesei. J Microbiol Biotechnol 28(9):1473–1481
Funding
This study received funding from Hunan Provincial Department of Education (18C1431), Natural Science Foundation of Hunan Province (2019JJ70061), and Innovation Center Program for Effective Utilization of Hunan Province (422190011).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(DOCX 1362 kb)
Rights and permissions
About this article
Cite this article
Xu, S., Ren, N., Liu, J. et al. Improvement of vincamine production of endophytic fungus through inactivated protoplast fusion. Int Microbiol 23, 441–451 (2020). https://doi.org/10.1007/s10123-020-00117-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10123-020-00117-1