Ravi Kumar Singarapu Bala Reddy Bheema Reddy Mathur Rajesh


Phytates present in plant-derived feed can chelate nutrients and reduce their bioavailability for monogastric animals such as poultry and swine. The addition of hydrolase, phytase can alleviate this problem but is hindered by its cost. The goal of the current study is to clone, express and purify the phytase gene from Bacillus sp. (DS11) into Schizochytrium sp. ATCC 20888 is also a good producer of Docosahexaenoic acid (DHA). This is expected to enhance animal nutrition and reduce phosphate pollution. The DNA sequence analysis using multiple sequence alignments showed significant similarity to the phytase gene from Bacillus sp. (DS11). Subsequently, specific primers were designed based on the consensus sequence of the Bacillus phytase gene obtained from sequencing. The coding DNA sequence was determined to have a length of 1152 base pairs. Phytase gene was successfully cloned into the pRI201-AN DNA vector and transformed into Schyzochytrium sp. Screening on G418 plates showed 53 resistant colonies and from this 11 prominent colonies were chosen for further testing. Out of this, 8 colonies tested positive, with colony PCR having 1.5 kb with a phytase activity of 1.77 U/ml of crude lysate. Further purification with Ni-NTA affinity chromatography provided a specific activity of 15.59 U/mg. This appears to be the first ever reported recombinant phytase produced in Schizochytrium sp. The phytase recommendations are 250U/Kg of feed preparation for broiler & swine diets  . It was also determined that 72.64 U/5.2 gm of wet biomass and 1.80% of w/w microalgae would fulfil these requirements per kg of feed preparation.




Assay, Cloning, Docosahexaenoic acid, Omega-3-fatty acid, Phytase, Phytic acid, Schizochytrium sp

Adachi, T., Sahara, T., Okuyama, H. & Morita, N. (2017).Glass Bead-based Genetic Transformation:An Efficient Method for Transformation of Thraustochytrid Microorganisms. Journal of Oleo Science. 66(7), 791-795. doi: 10.5650/jos.ess17084.
Afinah, S., Yazid, A. M., Anis Shobirin M. H., & Shuhaimi, M. (2010). Phytase: application in food industry, International Food Research Journal. 17, 13-21
Alagawany, M., Elnesr, S.S., Farag, M.R., Abd El-Hack, M.E., Khafaga, A.F., Taha, A.E., Tiwari, R., Yatoo, M.I., Bhatt, P., Khurana, S.K. & Dhama, K.(2019). Omega-3 and omega-6 fatty acids in poultry nutrition: effect on production performance and health. Animals, 9(8), p.573. doi: 10.3390/ani9080573.
Patel, A., Karageorgou, D., Katapodis, P., Sharma, A., Rova, U., Christakopoulos, P. & Matsakas, L.(2021). Bioprospecting of thraustochytrids for omega-3 fatty acids: A sustainable approach to reduce dependency on animal sources. Trends in Food Science & Technology, 115, pp.433-444.. https://doi.org/10.1016/j.tifs.2021.06.044.
Antonyuk, S.V., Olczak, M., Olczak, T., Ciuraszkiewicz, J,&Strange, R.W., (2014).The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold. IUCrJ. 2014. 1(Pt 2):101-9. doi: 10.1107/S205225251400400X.
Bergkessel, M., Guthrie, C., & Colony PCR. Methods in Enzymology. 2013. 529:299-309. doi: 10.1016/B978-0-12-418687-3.00025-2.
Bozzo, G.G., Raghothama, K.G., Plaxton, W.C., (2002).Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell culturesEuropean Journal of Biochemistry 269(24): p. 6278-6286. doi: 10.1046/j.1432-1033.2002.03347.x
Cangussu, A.S.R., Aires Almeida, D., Aguiar, R.W.D.S., Bordignon-Junior, S.E., Viana, K.F., Barbosa, L.C.B., Cangussu, E.W.D.S., Brandi, I.V., Portella, A.C.F., Santos, G.R.D. & Sobrinho, E.M., (2018). Characterization of the catalytic structure of plant phytase, protein tyrosine phosphatase-like phytase, and histidine acid phytases and their biotechnological applications. Enzyme research,  . 11, 2018:8240698. doi: 10.1155/2018/8240698.
Chen, Y.& Hu, H., (2019).High efficiency transformation by electroporation of the freshwater alga Nannochloropsislimnetica. World Journal of Microbiology and Biotechnologyl. 35:1–10. doi: 10.1007/s11274-019-2695-9.
Cheng, R., Ma, R., Li, K., Rong, H., Lin, X., Wang, Z., Yang, S. & Ma, Y.(2012). Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiological research, 167(3), pp.179-186. doi: 10.1016/j.micres.2011.05.003.
Cheng, R.B., Lin, X.Z., Wang, Z.K., Yang, S.J., Rong, H.& Ma, Y., (2011).Establishment of a transgene expression system for the marine microalga Schizochytriumsps by 18S rDNA-targeted homologous recombination. World Journal of Microbiology and Biotechnology. 7, 737–41. DOI: 10.3923/pjn.2010.471.474
Corrêa, T.L.R., & de, Araújo, E.F,. (2020).Fungal phytases: from genes to applications. Brazilian Journal of Microbiology. 3:1009-1020. doi: 10.1007/s42770-020-00289-y.
Dechow, F.J, (1989).Separation and Purification Techniques in Biotechnology. Noyes Publications, New Jersey, 1989. 416- 478.
Dionisio, G., Madsen, C.K., Holm, P.B., Welinder, K.G,, Jørgensen, M & Stoger, E., (2011), Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice. Plant Physiology.156(3),1087–1100. doi: 10.1104/pp.110.164756.
Dokuzparmak, E., Sirin, Y., Cakmak, U. and Saglam Ertunga, N., (2017). Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16. International Journal of Food Properties, 20(5), 1104-1116.
Fernanda,Erpel, F., Restovic, F.&Arce-Johnson, P.,(2016), Development of phytase-expressing chlamydomonasreinhardtii for monogastric animal nutrition. BMC Biotechnology. 2016. 16:29. doi: 10.1186/s12896-016-0258
Hakim, A., (2013).The potential of heterotrophic microalgae(Schizochytrium sp.) as a source of dha.Squalen Bull. Mar. Fish. Postharvest Biotechnol..7: p. 29.
Heinonen, J.K.& Lahti, R.J., (1981). A new convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Analytical Biochemistry. 113, 313-317. doi:10.1016/0003-2697(81)90082-8
Holmqvist, M., Stensjö, K., Oliveira, P., Lindberg, P.& Lindblad, P., (2009).Characterization of the hupSL promoter activity in NostocpunctiformeATCC 29133. BMC Microbiology. 9: 54. doi: 10.1186/1471-2180-9-54.
Hu, X., Tang, X., Bi, Z., Zhao, Q & Ren. L, (2021).Adaptive evolution of microalgae Schizochytrium sp. under high temperature for efficient production of docosahexaeonic acid. Algal Research54. 102212. 10.1016/j.algal.2021.102212..
Huang, H., Luo, H., Yang, P., Meng, K., Wang,Y.& Yuan, T., (2006).A novel phytase with preferable characteristics from Yersinia intermediaBiochemical and Biophysical Research Communications. 350(4):884–889. doi: 10.1016/j.bbrc.2006.09.118.
Huang, P.W., Xu, Y.S., Sun, X.M., Shi, T.Q., Gu, Y., Ye, C.& Huang, H, (2021).Development of an efficient Gene Editing Tool in Schizochytrium sp. and improving its lipid and terpenoid biosynthesis, Frontiers in Nutrition. 8:795651. doi: 10.3389/fnut.2021.795651.
Hui, He., Rongqun, Li., Yi, Chen., Ping, Pan., Wenjuan, Tong., Xueyan Dong., Yueming, Chen.&Daojun, Yu.,(2017) Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Scientific Reports. 7: 45199 https://doi.org/10.1038/srep45199
Jaeger, D., Hübner, W., Huser, T., Mussgnug, JH. &Kruse, O,. (2017).Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidiumneglectum. Journal of Biotechnology. 249:10–15. doi: 10.1016/j.jbiotec.2017.03.011.
Kim,Y.O., Lee, J.K, Kim, H.K., Yu, J.H.& Oh, T.K., (1998).Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiology Letters.162(1):185-91. doi: 10.1111/j.1574-6968.1998.tb12997.x.
Kindle, K.L., (1990).High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences USA. 87:1228–1232. doi: 10.1073/pnas.87.3.1228.
Lott, J.N.A., Ockenden, I., Raboy, V.&Batten, G.D., (2000) Phytic acid and phosphorus in crop seeds and fruits: a global estimate. Seed Science Research 10:11–33
Maga, J.A.,(1982).Phytate: its chemistry, occurrence, food interactions, nutritional significance, and methods of analysis. Journal of Agricultural and Food Chemistry.30: p. 1-9. DOI: https://doi.org/10.1017/S0960258500000039
Moza, M.I., & Postolache, C., (2021). Optimized Protocol for Cyanobacterial 16S rRNA Analysis in Danube Delta Lakes. bioRxiv. 2021. pp.2021-04.doi: https://doi.org/10.1101/2021.04.23.441086
Mullaney, E.C.D., & Ullah, A., (2000).Advances in phytase research. Advances in Applied Microbiologyax., 47: p. 157-99.DOI: 10.1016/s0065-2164(00)47004-8
Padmavathi, T., Bhargavi, R., Priyanka, P.R., Niranjan, N.R. and Pavitra, P.V., 2018. Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification. Journal of Genetic Engineering and Biotechnology, 16(2), pp.357-362.
Ranjan, B.& Satyanarayana, T., (2016).Recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile: expression of the codon-optimized phytase gene in Pichia pastoris and applications. Molecular Biotechnology.58, 137–147. doi: 10.1007/s12033-015-9909-7
Reddy, C.S., Achary, V.M.M., Manna, M., Singh, J., Kaul, T.& Reddy, M.K., (2015).Isolation and molecular characterization of thermostable phytase from Bacillus
subtilis (BSPhyARRMK33). Applied Biochemistry and Biotechnology.175, 3058-3067.DOI 10.1007/s12010-015-1487-4
Reddy, C.S., Kim, S.C.& Kaul, T., (2017) Genetically modified phytase crops role in sustainable plant and animal nutrition and ecological development: a review. 3 Biotechology. Jul;7(3):195. doi: 10.1007/s13205-017-0797-3. PMID: 28667635;
Saadi, M.I., Doosti, A., Jalali, H., Abdolyousefi, E.N., Hooshiyar, M., Tabrizi, R.& Noshadi E.. (2021). Cloning of Bacillus subtilis phytase gene construct in Escherichia coli. Iranian Journal of Microbiology. 5:664-670. doi: 10.18502/ijm.v13i5.7433.
Sakaguchi, K., Matsuda, T., Kobayashi, T., Ohara, J., Hamaguchi, R.&Abe, E., (2012), Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for thraustochytrids. Applied and Environmental Microbiology. 78, 3193-3202. Doi:10.1128/AEM.07129-11
Singh, N., Kuhar, S., Priya, K., Jaryal, R.& Yadav, R. (2018). Phytase: The Feed Enzyme, an Overview. In: Gahlawat, S., Duhan, J., Salar, R., Siwach, P., Kumar, S., Kaur, P. (eds) Advances in Animal Biotechnology and its Applications. Springer, Singapore. https://doi.org/10.10 07/978-981-10-4702-2_17
Shimogawara, K., Fujiwara, S., Grossman, A.& Usuda, H., (1998).High-efficiency transformation of Chlamydomonasreinhardtii by electroporation. Genetics. 148:1821–1828. doi: 10.1093/genetics/148.4.1821.
Trivedi, S., Husain, I. & Sharma, A. (2022). Purification and characterization of phytase from Bacillus subtilis P6: Evaluation for probiotic potential for possible application in animal feed. Food Frontiers, 3(1), pp.194-205.
Vohra, A. & Satyanarayana, T., (2003).Phytases: microbial sources, production, purification, and potential biotechnological applications. Crit Rev Biotechnol, 23(1): p. 29-60. doi: 10.1080/713609297.
Wang, F., Bi, Y. Diao, J. et al (2019). Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31. Biotechnol Biofuels 12, 141. https://doi.org/10.1186/s13068-019-1484-x
Zhang, C.& Hu, H., (2014).High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. Mar Genom.16:63–66. doi: 10.1016/j.margen.2013.10.003
Zhang S, He Y, Sen B, Chen X, Xie Y, Keasling JD, Wang G (2018) Alleviation of reactive oxygen species enhances PUFA accumulation in Schizochytrium sp. through regulating genes involved in lipid metabolism. Metab Eng Commun, 6:39–48
Zhao, T., Yong, X., Zhao, Z., Dolce, V., Li, Y. &Curcio, R.(2021). Research status of Bacillus phytase. 3 Biotech, 11(9), p.415.. https://doi.org/10.1007/s13205-021-02964-9
Research Articles

How to Cite

Heterologous expression of phytase in Schizochytrium sp. as a fortified feed additive for the Livestock industry. (2023). Journal of Applied and Natural Science, 15(3), 1245-1253. https://doi.org/10.31018/jans.v15i3.4858