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Sinelnikov I.G., Zorov I.N., Denisenko Yu.A. Mikityuk O.D., Sinitsyn A.P., Shcherbakova L.A. A new producer of a recombinant aflatoxin-degrading enzyme obtained via heterologous expression in Pichia pastoris. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2022, Vol. 57, № 6, p. 1166-1177.
(how to cite this article)

doi: 10.15389/agrobiology.2022.6.1166eng

UDC: 636.085.19:573.6.086.83:577.21

Acknowledgements:
Supported financially by the Russian Scientific Foundation (project No. 22-16-00153)

 

A NEW PRODUCER OF A RECOMBINANT AFLATOXIN-DEGRADING ENZYME OBTAINED VIA HETEROLOGOUS EXPRESSION IN Pichia pastoris

I.G. Sinelnikov1, 2, I.N. Zorov2, Yu.A. Denisenko1, 2, O.D. Mikityuk1,
A.P. Sinitsyn1, 2, L.A. Shcherbakova1 ✉

1All-Russian Research Institute of Phytopathology, 5, ul. Institute, Bolshie Vyazemy, Odintsovsky District, Moscow Province, 143050 Russia, e-mail mod-39@list.ru, larisavniif@yahoo.com (✉ corresponding author);
2Federal Research Center Fundamentals of Biotechnology RAS, 33/2, Leninskii prospect, Moscow, 119071 Russia, e-mail sinelnikov.i@list.ru, inzorov@mail.ru, denisenkoyura@mail.ru, apsinitsyn@gmail.com

ORCID:
Sinelnikov I.G. orcid.org/0000-0001-6359-1125
Mikityuk O.D. orcid.org/0000-0003-2022-7256
Zorov I.N. orcid.org/0000-0001-6888-172X
Sinitsyn A.P. orcid.org/0000-0001-6429-8254
Denisenko Yu.A. orcid.org/0000-0003-2363-0374
Shcherbakova L.A. orcid.org/0000-0003-0254-379X

Received September 20, 2022

Contamination of food and feed with mycotoxins causes significant economic losses in the food and feed industry and poses a serious threat to the human health and animal life because of mutagenic, carcinogenic and other disruptive properties of these secondary metabolites of fungi. Enzymatic degradation of mycotoxins represents an efficient and environmentally safe alternative to the chemical decontamination of agricultural and food products. In this study, a synthetic adtz gene encoding ADTZ, an aflatoxin-degrading oxidase from Armillaria tabescens, was integrated into the genome of a Pichia pastoris GS115 strain under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter. To amplify the adtz gene, oligonucleotide sequences were constructed with specific restriction sites HindIII and NotI added to the 5' end. The adtz gene-containing pPIG-ADTZ plasmid obtained with the use of the pPIG-1 vector was linearized by digestion with restriction endonuclease ApaI, followed by transforming the cells of P. pastoris recipient strain GS115 by electroporation. The transformed yeast cell were selected on YPD medium with an antibiotic. PCR amplification, restriction analysis and Sanger sequencing confirmed insertion of the target gene. As a result, 54 transformed clones containing the target gene were obtained, and the most productive clone secreting the recombinant ADTZ-14 (2.1 mg/ml of the total extracellular protein) was selected. Recombinant ADTZ represented a monomeric protein (78±3 kDa) possessing a high affinity to aflatoxin B1 (AFB1). Saving the functional properties of the recombinant protein was shown using experiments on assessment of its ability to degrade AFB1 during short-time and prolonged incubation. The obtained protein was able to degrade AFB1 by 14 % after a 2-h incubation at 40 °С; after 72 and 120 h of incubation at 30 °С, the content of AFB1 in ADTZ-14 culture liquid (CL) reduced by 50 and 80 %, respectively, compared to content in CL of non-transformed control GS115. These data suggest a quite high biotechnological potential of a new recombinant ADTZ preparation in relation to the decontamination of agricultural products contaminated with AFB1. Thus, the earlier developed expression system intended to increase the copy number of heterologous genes in Pichia pastoris was first used to obtain a recombinant protein able to degrade AFB1. Using this approach, we transformed yeast cells with the pPIG-ADTZ plasmid and obtained 154 recombinant clones of P. pastoris, 77 % of which contained the target sequence of the adtz gene. Productivity of the best transformant (clone ADTZ-14) was 2.1 mg of protein per 1 ml of culture liquid, and about half of the pool of the extracellular proteins fell to the share of recombinant ADTZ able to degrade 80 % of AFB1 incubated in cell-free culture broth at 30 °С and pH 7.0.

Keywords: aflatoxin B1, mycotoxins, enzymatic degradation, ADTZ from Armillaria tabescens, synthetic adtz gene, recombinant proteins, heterologous expression, Pichia pastoris.

 

REFERENCES

  1. Dzhavakhiya V.G., Statsyuk N.V., Shcherbakova L.A., Popletaeva S.B. Aflatoksiny: ingibirovanie biosinteza, profilaktika zagryazneniya i dekontaminatsiya agroproduktsii [Aflatoxins: biosynthesis inhibition, conatamination prevention and decontamination of agricultural products]. Moscow, 2017 (in Russ.).
  2. Coppock R.W., Christian R.G., Jacobsen B.J. Aflatoxins. In: Veterinary toxicology. R.C. Gupta (ed.). Academic Press, 2018: 983-994 CrossRef
  3. Kononenko G.P., Zotova E.V., Burkin A.A. Advances in mycotoxicological research of forage grain crops. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2021, 56(5): 958-967 CrossRef
  4. Kononenko G.P., Burkin A.A. Toxins of micromycetes in generative organs of plants of the family Fabacea. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2021, 56(5): 968-978 CrossRef
  5. Kumar P., Mahato D.K., Kamle M., Mohanta T.K., Kang S.G.  Aflatoxins: A global concern for food safety, human health and their management. Front. Microbiol., 2017, 7: 2170 CrossRef
  6. Romani L. Immunity to fungal infections. Nat. Rev. Immunol., 2004, 4(11): 1-23 CrossRef
  7. El-Sayed R.A., Jebur A.B., Kang W., El-Demerdash F.M. An overview on the major mycotoxins in food products: characteristics, toxicity, and analysis. Journal of Future Foods, 2022, 2(2): 91-102 CrossRef
  8. Schuda P.F. Aflatoxin chemistry and syntheses. In: Syntheses of natural products. Topics in current chemistry, V. 91. Springer, Berlin, Heidelberg, 1980: 79-81 CrossRef
  9. Mahato D.K., Lee K.E., Kamle M., Devi S., Dewangan K.N., Kumar P., Kang S.G. Aflatoxins in food and feed: an overview on prevalence, detection and control strategies. Front. Microbiol., 2019, 10: 2266 CrossRef
  10. Probst C., Njapau H., Cotty P.J. Outbreak of an acute aflatoxicosis in Kenya in 2004: identification of the causal agent. Appl. Environ. Microbiol., 2007, 73(8): 2762-2764 CrossRef
  11. Medina A., Gilbert M.K., Mack B.M., O’Brian G.R., Rodriguez A., Bhatnagar D., Payne G., MaganN. Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production. Int. J. Food Microbiol., 2017, 256: 36-44 CrossRef
  12. Shcherbakova L.A., Statsyuk N.V., Mikityuk O.D., Nazarova N.A., Dzhavakhiya V.G. Aflatoxin B1 degradation by metabolites of Phoma glomerata PG41 isolated from natural substrate colonized by aflatoxigenic Aspergillus flavus. Jundishapur J. Microbiol., 2015, 8(1): e24324 CrossRef
  13. Ji C., Fan Y., Zhao L. Review on biological degradation of mycotoxins. Anim. Nutr., 2016, 2(3): 127-133 CrossRef
  14. Verheecke C., Liboz T., Mathieu F. Microbial degradation of aflatoxin B1: current status and future advances. Int. J. Food Microbiol., 2016, 237: 1-9 CrossRef
  15. Alberts J.F, Gelderblom W.C.A., Botha A., van Zyl W.H. Degradation of aflatoxin B1 by fungal laccase enzymes. Int. J. Food Microbiol., 2009, 135: 47-52 CrossRef
  16. Taylor M.C., Jackson C.J., Tattersall D.B., French N., Peat T.S., Newman J., Briggs L.J., Lapalikar G.V., Campbell P.M., Scott C., Russell R.J., Oakeshott J.G. Identification and characterization of two families of F420H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Mol. Microbiol., 2010, 78(3): 561-575 CrossRef
  17. Zhao L.H., Guan S., Gao X., Ma Q.G., Lei Y.P., Bai X.M., Ji C. Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068. J. Appl. Microbiol., 2011, 110(1): 147-155 CrossRef
  18. Wang Y., Zhao C., Zhang D., Zhao M., Zheng D., Lyu Y., Cheng W., Guo P., Cui Z. Effective degradation of aflatoxin B1 using a novel thermophilic microbial consortium TADC7. Bioresource Technology, 2017, 224: 166-173 CrossRef
  19. Guo Y.P., Qin X.J., Tang Y., Ma Q.G., Zhang J.Y., Zhao L.H. CotA laccase, a novel aflatoxin oxidase from Bacillus licheniformis, transforms aflatoxin B-1 to aflatoxin Q(1) and epi-aflatoxin Q(1). Food Chem., 2020, 325: 126877 CrossRef.
  20. Adebo  O.A., Njobeh P.B., Gbashi S., Nwinyi O.C., Mavumengwana V. Review on microbial degradation of aflatoxins. Crit. Rev.Food Sci. Nutr., 2017, 57(15): 3208-3217 CrossRef
  21. Xu H.W., Wang L.Z., Sun J.D., Wang L.P., Guo H.Y., Ye Y.L., Sun X.L. Microbial detoxification of mycotoxins in food and feed.Crit. Rev. Food Sci. Nutr.,2022, 62(18): 4951-4969 CrossRef
  22. Li C.H., Li W.Y., Hsu I.N., Liao Y.Y., Yang C.Y., Taylor M.C., Liu Y.F., Huang W.H., Chang H.H., Huang H.L., Lo S.C., Lin T.Y., Sun W.C., Chuang Y.Y., Yang Y.C., Fu R.H., Tsai R.T. Recombinant aflatoxin-degrading F420H2-dependent reductase from Mycobacterium smegmatis protects mammalian cells from aflatoxin toxicity. Toxins, 2019, 11(5): 259 CrossRef
  23. Kolosova A., Stroka J. Substances for reduction of the contamination of feed by mycotoxins: a review. World Mycotoxin Journal, 2011, 4(3): 225-256 CrossRef
  24. Loi M., Fanelli F., Zucca P., Liuzzi V.C., Quintieri L., Cimmarusti M.T., Monaci L., Haidukowski M., Logrieco A.F., Sanjust E., Mulè G. Aflatoxin B1 and M1 degradation by Lac2 from Pleurotus pulmonarius and redox mediators. Toxins, 2016, 8(9): 245 CrossRef. 
  25. Brana M.T., Sergio L., Haidukowski M., Logrieco A.F., Altomare C. Degradation of aflatoxin B-1 by a sustainable enzymatic extract from spent mushroom substrate of Pleurotus eryngii. Toxins, 2020, 12(1): 49 CrossRef
  26. Wang J., Ogata M., Hirai H., Kawagishi H. Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordid YK-624. FEMS Microbiol. Lett., 2011, 314(2): 164-169 CrossRef
  27. Yao D.S., Liang R., Liu, D.L., Gu L.Q., Ma L., Chen W.Q. Screening of the fungus whose multienzyme system has catalytic detoxification activity towards aflatoxin B1 (Part I). Ann. N.Y. Acad. Sci., 1998, 864: 579-585 CrossRef
  28. Liu D.-L., Yao D.-S., Liang Y.Q., Zhou T.-H., Song Y.-P., Zhao L., Ma L. Production, purification, and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens (E-20). Food Chem. Toxicol., 2001, 39(5): 461-466 CrossRef
  29. Cao H., Liu D., Mo X., Xie C., Yao D. A fungal enzyme with the ability of aflatoxin B1 conversion: purification and ESI-MS/MS identification. Microbiol. Res., 2011, 166(6): 475-483 CrossRef
  30. Wu Y.Z., Lu F.P., Jiang H.L., Tan C.P., Yao D.S., Xie C.F., Liu D.L. The furofuran-ring selectivity, hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase. Food Chem. Toxicol., 2015, 76: 125-131 CrossRef
  31. Xingming Y. Oral liquid medicine from ferments of Armilarilla tabescens. China PAT # CN1679642.  Priority date 11. 05.2004. Publication date 01.08.2007.
  32. Chahardooli M., Niazi A., Aram F., Sohrabi S.M. Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification. J. Sci. Food Agric., 2016, 96(2): 569-575 CrossRef
  33. Wang Y., Wang Y., Jiang J., Zhao Y., Xing F., Zhou L. High expression of zearalenone degrading enzyme in Pichia pastoris. Chinese Journal of Biotechnology, 2020, 36(2): 372-380 CrossRef
  34. Sinel’nikov I.G., Zorov I.N., Sinitsyna O.A., Sinitsyn A.P., Rozhkova A.M. Integratsionnyy vektor dlya mnogokopiynoy integratsii genov v 18Sr RNK drozhzhey Pichia pastoris. № RU 2752904C1, Mosk. FGU Issledovatel’skiy Tsentr «Osnovy Biotekhnologii» RAN (RF). Zayavl. 02.09.20. Opubl. 11.08.21. Byul. № 23 [A vector for multicopy gene integration into 18Sp RNA of the yeast Pichia pastoris. No. RU 2752904C1, Moscow. FGU Research Center «Fundamentals of Biotechnology» RAS (RF). Appl. 09.02.20. Publ. 08.11.21. Bull. № 23] (in Russ.).
  35. Lin-Cereghino J., Wong W.W., Xiong S., Giang W., Luong L.T., Vu J., Johnson S.D., Lin-Cereghino G.P. Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques, 2005, 38(1): 44-48 CrossRef
  36. Lõoke M., Kristjuhan K., Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques, 2011, 50(5): 325-328 CrossRef
  37. Waterborg J.H., Matthews H.R. The Lowry method for protein quantitation. In: Proteins. Methods in molecular biology™, vol 1. J.M. Walker (ed.). Humana Press, 1984: 1-3 CrossRef
  38. Dzhavakhiya V.G., Voinova T.M., Popletaeva S.B., Statsyuk N.V., Limantseva L.A., Shcherbakova L.A. Effect of various compounds blocking the colony pigmentation on the aflatoxin B1 production by Aspergillus flavus. Toxins, 2016, 8(11): 313 CrossRef
  39. Yang P., Xiao W., Lu S., Jiang S., Zheng Z., Zhang D., Zhang M., Jiang S., Jiang S. Recombinant expression of Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) in engineering Pichia pastoris GS115 and application in AFB1 degradation in AFB1-contaminated peanuts. Toxins, 2021, 13(5): 349 CrossRef
  40. Wang J.R., Li Y.Y., Liu D.N., Liu J.S., Li P., Chen L.Z., Xu S.D. Codon optimization significantly improves the expression level of a-amylase gene from Bacillus licheniformis in Pichia pastoris. BioMed Research International, 2015, 2015: 248680 CrossRef
  41. Liu K., Ouyang Y., Lin R., Ge C., Zhou M. Strong negative correlation between codon usage bias and protein structural disorder impedes protein expression after codon optimization. J. Biotechnol., 2022, 343: 15-24 CrossRef

 

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