Fosfinotrisin N-asetiltransferazı Kodlayan Siyanobakteriyel Genlerin E. coli’de Klonlanması ve Heterolog İfadesi

Year 2025, Volume: 30 Issue: 2, 815 - 829, 31.08.2025

Kübra Özkul , Ebru Yılmaz , Aslıhan Kurt Kızıldoğan

https://doi.org/10.53433/yyufbed.1633412

Abstract

Fosfinotrisin N-asetiltransferaz (Pat), fosfinotrisin (PPT) içeren herbisitlere direnç sağlamak için bitkilerde aktarılan bakteriyel bir enzimdir. Pat proteininin transgenik bitkilerde ifade edilmesi, glufosinat herbisitine karşı tolerans sağlar. Siyanobakterilerdeki bu enzim dizi analizi ile tanımlanmış olmasına rağmen, aktiviteleri henüz belirlenmemiştir. Bu çalışmada, Anabaena sp. PCC7120 ve Synechocystis sp. PCC6803’de Pat enzimini kodladığı varsayılan sırasıyla alr ve sll genleri His-Taq pET28a+ ekspesyon vektörüne klonlandı. İlgili genleri içeren rekombinant vektörler proteaz içermeyen E. coli BL21 hücrelerine transfer edildi. Alr4468 ve Sll1647 polipeptitleri 1 mM IPTG ile indüklenmiş rekombinant E. coli BL21 kültürlerinde başarıyla ifade edildi. En yüksek in vitro Pat aktiviteleri IPTG ile indüklenmiş kaba hücre ekstraktları kullanılarak yapılan enzim deneylerinde elde edilmiş ve kontrollerden sırasıyla 3.47 (rAlr4468 için) ve 2.53 (rSll1647 için) kat daha yüksek bulunmuştur. Sonuç olarak, yüksek yapılı bitkilerle benzer fizyolojik özelliklere sahip olan siyanobakterilerin alr4468 ve sll1647 genleri, gelecekteki çalışmalarda herbisite dirençli yerli transgenik bitkilerin üretimi için potansiyel kaynaklar olarak kullanılabilirler.

Keywords

bar geni , Fosfinotrisin N-asetiltransferaz , Herbisit direnci , Siyanobakteriyel pat geni

Project Number

PYO.ZRT.1902.16.001

Cloning and Heterologous Expression of Cyanobacterial Genes Encoding Phosphinothricin N- acetyltransferase in E. coli

Abstract

Phosphinothricin N-acetyltransferase (Pat) is a bacterial enzyme that is introduced into plants to confer resistance to herbicides containing phosphinothricin (PPT). The expression of the Pat protein in transgenic plants allows them to tolerate the herbicide glufosinate. Although the enzyme has been identified in cyanobacteria through sequence analysis, its activities have not yet been thoroughly investigated. In this study, we cloned the putative Pat enzyme-encoding genes alr4468 and sll1647 from Anabaena sp. PCC7120 and Synechocystis sp. PCC6803, respectively, into the His-Tagged pET28a+ expression vector. The recombinant vectors containing these genes were introduced into protease-free E. coli BL21 cells. We successfully expressed the Alr4468 and Sll1647 polypeptides in the recombinant E. coli BL21 cultures that were induced with 1 mM IPTG. The highest in vitro Pat activities were measured using enzyme assays from IPTG-induced cell crude extracts, showing increases of 3.47 times for rAlr4468 and 2.53 times for rSll1647 compared to controls. As a result, the alr4468 and sll1647 genes from cyanobacteria, which exhibit similar physiological properties to those of high-structured plants, may serve as potential sources for developing herbicide-resistant transgenic plants in future research.

Keywords

bar gene , Cyanobacterial pat gene , Herbicide resistance , Phosphinothricin N-acetyltransferase

Supporting Institution

Ondokuz Mayıs University

Project Number

PYO.ZRT.1902.16.001

References

• Aragao, F. J. L., Vianna, G. R., Albino, M. M. C., & Rech, E. L. (2002). Transgenic dry bean tolerant to the herbicide glufosinate ammonium. Crop Science, 42, 1298-1302. https://doi.org/10.2135/cropsci2002.1298
• Baker, W., van den Broek, A.E., Camon, E., Hingamp, P., Sterk, P., Stoesser, G., & Tuli, M.A. (2000). The EMBL nucleotide sequence database. Nucleic Acids Research, 28(1), 19-23. https://doi.org/10.1093/nar/28.1.19
• Bartsch, K., & Tebbe, C. C. (1989). Initial steps in the degradation of phosphinothricin (glufosinate) by soil bacteria. Applied and Environmental Microbiology, 55(3) 711-716. https://doi.org/10.1128/aem.55.3.711-716.1989
• Bayer, E., Gugel K. H., Hägele, K., Hagenmeier, H., Jessipow, S., König, W. A., & Zähner, Z. (1972). Stoffwechselprodukte von Mikroorganismen. 98. Mitteilung. Phosphinothricin und Phosphinothricyl-Alanyl-Alanin. Helvetica Chimica Acta, 55(1), 224-239. https://doi.org/10.1002/hlca.19720550126
• Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
• Carbonari, C. A., Latorre, D. O., Gomes, G. L. G. C., Velini, E. D., Owens, D. K., Pan, Z., & Dayan, F.E. (2016). Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® cotton. Planta, 243, 925-933. https://doi.org/10.1007/s00425-015-2457-3
• Coudert, E., Gehant, S., de Castro, E., Pozzato, M., Baratin, D., Neto, T., Sigrist, C.J.A., Redaschi, N., & Bridge, A. (2023). Annotation of biologically relevant ligands in UniProtKB using ChEBI. Bioinformatics, 39(1). https://doi.org/10.1093/bioinformatics/btac793
• Donthi, N. R., & Dileep Kumar, A. D. (2022). Glufosinate ammonium: an overview. Pesticide Action Network.
• Falco, M. C., Tulmann-Neto, A., & Ulian, E. C. (2000). Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell Reports, 19, 1188-1194. https://doi.org/10.1007/s002990000253
• Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Nobuyuki Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M & Tabata, S. (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. sequence determination of the entire genome and assignment of potential protein coding regions. DNA Research, 3, 109-136, 185-209. https://doi.org/10.1093/dnares/3.3.109
• Kaneko, T., Nakamura, Y., Wolk, C. P., Kuritz, T., Sasamoto, S., Watanabe, A., Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takazawa, M., Yamada, M., Yasuda, M., & Tabata, S. (2001). Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Research, 8(5), 205-213. https://doi.org/10.1093/dnares/8.5.205
• Keller, G., Spatola, L., McCabe, D., Martinell, B., Swain, W., & John, M. E. (1997). Transgenic cotton resistant to herbicide bialaphos. Transgenic Research, 6, 385-392. https://doi.org/10.1023/A:1018483300902
• Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12), 1-9. https://doi.org/10.1093/molbev/msae263
• Kutyshenko, V. P., Mikoulinskaia, G. V., Chernyshov, S. V., Yegorov, A. Y., Prokhorov, D. A., & Uversky , V. N. (2019). Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5. International Journal of Biological Macromolecules, 124, 810-818. https://doi.org/10.1016/j.ijbiomac.2018.11.219
• Laemmi, U.K (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. https://doi.org/10.1038/227680a0
• Li, Z., Zhang, Z., Liu, Y., Ma, Y., Lv, X., Zhang, D., Gu, Q., Ke, H., Wu., L., Zhang, G., Ma, Z., Wang, X., & Sun, Z. (2023). Identification and expression analysis of Epsps and Bar families in cotton. Plants, 12(19), 3366. https://doi.org/10.3390/plants12193366
• Lind, L. K., Kallas, S. R., Lonneborg, A., Oquist, G., & Guastafssan, P. (1985). Cloning of the β-phycocyanin gene from Anacystis nidulans. FEBS Letter, 188(1), 27-32. https://doi.org/10.1016/0014-5793(85)80868-1
• Miki, B., & Mchugh, S. (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology, 107(3), 193-232. https://doi.org/10.1016/j.jbiotec.2003.10.011
• Mohapatra, U., McCabe, M. S., Power, J. B., Schepers, F., Van der Arend, A., & Davey, M. R. (1999). Expression of the bar gene confers herbicide resistance in transgenic lettuce. Transgenic Research, 8, 33-44. https://doi.org/10.1023/A:1008891216134
• Mulwa, R. M. S., & Mwanza, L. M. (2006). Biotechnology approaches to developing herbicide tolerans/selectivity in crops. African Journal of Biotechnology, 5(5), 396-404.
• Murakami, T., Anzai, H., Imai, S., Satho, A., Nagaoka, K., & Thompson, C., (1986). The bialaphos biyosynthetic genes of Streptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster. Molecular and General Genetics MGG, 205, 42-50. https://doi.org/10.1007/bf02428031
• Oerke, E. C. (2006). Crop losses to pests. Journal of Agricultural Science, 144(1), 31–43. https://doi.org/10.1017/S0021859605005708
• Páez-Espino, A. D., Chavarria, M., & Lorenzo, V. (2015). The two paralogue phoN (phosphinothricin acetyltransferase) genes of Pseudomonas putida encode functionally different proteins. Environmental Microbiology, 17(9), 3330-3340. https://doi.org/10.1111/1462-2920.12798
• Raven, J. A., & Allen, J. F. (2003). Genomics and chloroplast evolution: what did cyanobacteria do for plants? Genome Biology, 4, 209. https://doi.org/10.1186/gb-2003-4-3-209
• Rippka, R., Deruells, J., Waterbury, J. B., Herdman, M., & Stainer, R. Y. (1979). Generic assignment, strain histories and properties of pure cultures of cyanobacteria. The Journal of General Microbiology, 11(1), 1-61. https://doi.org/10.1099/00221287-111-1-1
• Sambrook, J., & Russell, D. W. (Eds.). (2001). Molecular cloning a laboratory manual. Third Edition. New York, Cold Spring Harbor Laboratory Press.
• Sivamani, E., Nalapalli, S., Prairie, A., Bradley, D., Richbourg, L., Tim Strebe, T., Liebler, T., Wang, D., & Que, Q. (2019). A study on optimization of pat gene expression cassette for maize transformation. Molecular Biology Reports, 46, 3009-3017. https://doi.org/10.1007/s11033-019-04737-3
• Şafak, H., Otur, Ç., & Kurt- Kızıldoğan, A. (2020). Molecular and biochemical characterization of a recombinant endoglucanase rCKT3eng, from an extreme halophilic Haloarcula sp. strain CKT3. International Journal of Biological Macromolecules, 151, 1173-1180. https://doi.org/10.1016/j.ijbiomac.2019.10.161
• Takano, H. K., Beffa, R., Preston, C., Westra, P., & Dayan, F. E. (2020). A novel insight into the mechanism of action of glufosinate: How reactive oxygen species are formed. Photosynthetic Research, 144, 361-372. https://doi.org/10.1007/s11120-020-00749-4
• Takano, H. K., & Dayan, F. E. (2020). Glufosinate-ammonium: a review of the current state of knowledge. Pest Management Science, 76(12), 3911-3925. https://doi.org/10.1002/ps.5965
• Thompson, C., Movva, R. N., Tizard, R., Crameri, R., Davies, J. E., Lauwereys, M., & Botterman, J. (1987). Characterization of the herbicide resistance gene bar from Streptomyces hygroscopicus. European Molecular Biology Organization Journal, 9, 2519-2523. https://doi.org/10.1002/j.1460-2075.1987.tb02538.x
• Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen S. G. (2012). Primer new capabilities and interfaces. Nucleic Acids Research, 40(15), 115. https://doi.org/10.1093/nar/gks596
• Vencill, V. K. (Ed.). (2002). Herbicide handbook. 8th Edition. Weed Science Society of America, 493.
• Vinnemier, J., Drogelaser, W., Pistorius, E. K., & Broer, I. (1995). Purification and partial characterisation of the Streptomyces viridochromogenes Tu494 phosphinothricin N-acetyltransferase mediating resistance to the herbicide phosphinothricin in transgenic plants. Zeitschrift fur Naturforschung C-A Journal of the Biosciences, 50, 796-805. https://doi.org/10.1515/znc-1995-11-1210
• Wild, A., & Manderschield, R. (1984). The effect of phosphinothricin on the assimilation of ammonia in plant. Zeitschrift für Naturforschung A, 39(5), 500-504. https://doi.org/10.1515/znc-1984-0539
• Wohlleben, W., Arnold, W., Broer, I., Hillemann, D., Strauch, E., & Pühler, A. (1988). Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene, 70(1), 25-37. https://doi.org/10.1016/0378-1119(88)90101-1
• Wu, G., Yuan, M., Wei, L., Zhang, Y., Lin, Y., Zhang, L., & Liu, Z. (2014). Characterization of a novel cold-adapted phosphinothricin N-acetyltransferase from the marine bacterium Rhodococcus sp. strainYM12. Journal of Molecular Catalysis B Enzymatic, 104, 23-28. https://doi.org/10.1016/j.molcatb.2014.03.001
• Yu, X., Sun, Y., Lin, C., Wang, P., Shen, Z., & Zhao, Y. (2023). Development of transgenic maize tolerant to both glyphosate and glufosinate. Agronomy, 13(1), 226. https://doi.org/10.3390/agronomy13010226
• Zhu, F., Yan, Y., Xue, X., Yu, R., & Ye, J. (2023). Identification and characterization of a phosphinothricin N-acetyltransferase from Enterobacter LSJC7. Pesticide Biochemistry and Physiology, 193, 105464. https://doi.org/10.3390/agronomy13010226