Expression of a synthetic protein with a high proportion of essential amino acids by Pichia pastoris

  • Y. Bartolo-Aguilar
  • C. Chávez-Cabrera CECyTE Michoacán
  • J.C. Cancino-Díaz
  • R. Marsch
Keywords: essential amino acids, synthetic peptide, human nutrition, Pichia pastoris

Abstract

It is widely known that an adequate intake of proteins with essential amino acids stimulates the production of essential nonprotein substances for the body, such as serotonin, thyroid hormones, and glutathione. Biotechnology applications can focus on food and its benefits for human health, either by improving the nutritional value of existing foods or developing new alternatives that can help resolve the effects of poor nutrition. The yeast Pichia pastoris has been designated as a GRAS (generally recognized as safe) microorganism and is currently used to produce a large number of recombinant proteins. In this study, a 100% synthetic protein was expressed intracellularly as a protein with high nutritional quality that contains a 75% ratio of essential amino acids using the expression system of the yeast P. pastoris. This approach might offer future possibilities for using the modified yeast as a single-cell protein source in the livestock industry and the food supplement market to reduce total protein intake.

References

Angov, E., Hillier, C. J., Kincaid, R. L. and Lyon, J. A. (2008). Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PloS ONE, 3(5), e2189. https://doi.org/10.1371/journal.pone.0002189.

Baeshen, N. A., Baeshen, M. N., Sheikh, A., Bora, R. S., Ahmed, M. M., Ramadan, H. A., Saini, K. S. and Redwan, E. M. (2014). Cell factories for insulin production. Microbial Cell Factories 13, 141. https://doi.org/10.1186/s12934-014-0141-0.

Bartolo-Aguilar, Y., Dendooven, L., Chávez-Cabrera, C., Flores-Cotera, L. B., Hidalgo-Lara, M. E., Villa-Tanaca, L. and Marsch, R. (2017). Autolysis of Pichia pastoris induced by cold. AMB Express 7, 95. https://doi.org/10.1186/s13568-017-0397-y.

Bussey, H. and Umbarger, H. E. (1969). Biosynthesis of branched-chain amino acids in yeast: regulation of synthesis of the enzymes of isoleucine and valine biosynthesis. Journal of Bacteriology 98(2), 623–628. https://doi.org/10.1128/jb.98.2.623-628.1969.

Çelik, E. and Çalık, P. (2012). Production of recombinant proteins by yeast cells. Biotechnology Advances 30(5), 1108–1118. https://doi.org/10.1016/j.biotechadv.2011.09.011.

CFR - Code of Federal Regulations Title 21. (1993). U.S. Food and Drug Administration. Retrieved from https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=573.750

Chakraborty, S., Chakraborty, N. and Datta, A. (2000). Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proceedings of the National Academy of Sciences 97(7), 3724–3729. https://doi:10.1073/pnas.97.7.3724.

Chávez-Cabrera, C., Marsch, R., Bartolo-Aguilar, Y., Flores-Bustamante, Z. R., Hidalgo-Lara, M. E., Martínez-Cárdenas, A., Cancino-Díaz, J.C., Sánchez, S. and Flores-Cotera, L. B. (2015). Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma. FEMS Yeast Research 15(6), fov054. https://doi.org/10.1093/femsyr/fov054.

Ciofalo, V., Barton, N., Kreps, J., Coats, I. and Shanahan, D. (2006). Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil. Regulatory Toxicology and Pharmacology 45(1), 1-8. https://doi.org/10.1016/j.yrtph.2006.02.001.

Cregg, J. M. (2007). Pichia protocols. Totowa, New Jersey: Humana Press.

Cummings, N. E., Williams, E. M., Kasza, I., Konon, E. N., Schaid, M. D., Schmidt, B. A., Poudel, C., Sherman, D. S. and Yu, D. (2017). Restoration of metabolic health by decreased consumption of branched-chain amino acids. The Journal of Physiology 596 (4), 623–645. https://doi:10.1113/JP275075.

Falco, S.C., Keeler, S.J. and Rice, J.A. (1994). Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants (U.S. Patent 5,559,223). Justia Patents. https://patents.justia.com/patent/5559223.

FAO. (2013). Dietary protein quality evaluation in human nutrition. Report of an FAO Expert Consultation. FAO Food and Nutrition Paper, No. 92. Rome, FAO. Retrieved from http://www.fao.org/ag/humannutrition/35978-02317b979a686a57aa4593304ffc17f06.pdf.

FAO, IFAD, UNICEF, WFP and WHO. (2018). The State of Food Security and Nutrition in the World 2018. Building climate resilience for food security and nutrition. Rome, FAO. Retrieved from http://www.fao.org/3/i9553en/i9553en.pdf.

Galili, G. and Amir, R. (2012). Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality. Plant Biotechnology Journal 11(2), 211–222. https://doi:10.1111/pbi.12025.

Gomes, A. R., Byregowda, S. M., Veeregowda, B. M. and Balamurugan, V. (2016). An overview of heterologous expression host systems for the production of recombinant proteins. Advances in Animal and Veterinary Sciences 4(7), 346–356. https://doi.org/10.14737/journal.aavs/2016/4.7.346.356.

Hofacker, I. L., Priwitzer, B., Stadler, P. F. (2004). Prediction of locally stable RNA secondary structures for genome-wide surveys. Bioinformatics 20(2), 186–190. https://doi.org/10.1093/bioinformatics/btg388.

Institute of Medicine. (2005). Dietary reference intakes for energy, carbohydrates, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). Washington, DC: The National Academies Press. https://doi.org/10.17226/10490.

Ivanciuc, O., Schein. C. H. and Braun, W. (2003). SDAP: Database and Computational Tools for Allergenic Proteins. Nucleic Acids Research 31, 359-362. https://doi: 10.1093/nar/gkg010.

Johnson, E. A. (2013). Biotechnology of non-Saccharomyces yeasts-the ascomycetes. Applied Microbiology and Biotechnology 97, 503–517. https://doi.org/10.1007/s00253-012-4497-y.

Karbalaei, M., Rezaee, S. A. and Farsiani, H. (2020). Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology 235(9), 5867–5881. https://doi.org/10.1002/jcp.29583.

Keefe, D. M. (2018). GRAS Notice GRN 737 Agency Response Letter | FDA. U.S. Food and Drug Administration. https://www.fda.gov/media/116243

Kidd, M. T. and Tillman, P. B. (2016). Key principles concerning dietary amino acid responses in broilers. Animal Feed Science and Technology 221, 314–322. https://doi:10.1016/j.anifeedsci.2016.05.012.

Kim, H., Yoo, S. J. and Kang, H. A. (2015). Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Research 15, 1-16. https://doi.org/10.1111/1567-1364.12195.

Kozak, M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13-37. https://doi.org/10.1016/j.gene.2005.06.037.

Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157(1), 105–132. https://doi:10.1016/0022-2836(82)90515-0.

Martínez-Hernández, S., Marin-Muñoz, M., Ventura-Juarez, J. and Jauregui, J. (2019). Fed-batch cultivation and operational conditions for the production of a recombinant anti-amoebic vaccine in Pichia pastoris system. Revista Mexicana de Ingeniería Química 19(2), 691-705. https://doi.org/10.24275/rmiq/Bio725.

Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M. and Turner, D. H. (2004). Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proceedings of the National Academy of Sciences 101(19), 7287–7292. doi:10.1073/pnas.0401799101.

Newgard, C. B., An, J., Bain, J. R., Muehlbauer, M. J., Stevens, R. D., Lien, L. F., Haqq, A. M., Shah, S. H., Arlotto, M., Slentz, C. A., Rochon, J., Gallup, D., Ilkayeva, O., Wenner, B. R., Yancy, W. S., Jr, Eisenson, H., Musante, G., Surwit, R. S., Millington, D. S., Butler, M. D., … Svetkey, L. P. (2009). A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metabolism 9(4), 311–326. https://doi.org/10.1016/j.cmet.2009.02.002.

Porro, D., Gasser, B., Fossati, T., Maurer, M., Branduardi, P., Sauer, M. and Mattanovich, D. (2011). Production of recombinant proteins and metabolites in yeasts. Applied Microbiology and Biotechnology 89(4), 939–948. https://doi.org/10.1007/s00253-010-3019-z.

Rashad, M. M., Moharib, S. A. and Jwanny, E. W. (1990). Yeast conversion of mango waste or methanol to single cell protein and other metabolites. Biological Wastes 32, 277–284. https://doi.org/10.1016/0269-7483(90)90059-2.

Rath, A., Glibowicka, M., Nadeau, V. G., Chen, G., and Deber, C. M. (2009). Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences 106(6), 1760–1765. https://doi:10.1073/pnas.0813167106.

Rheeder, R.A.L. (2014). Anexploration of synthetic biology: A preliminary Christian ethical assessment of the advantages and disadvantages of synthetic biology. In die Skriflig 48(2), 1-10 pages. http:// dx.doi.org/10.4102/ids.v48i2.722.

Rentería-Martínez, O., Páez-Lerma, J., Rojas-Contreras, J., López-Miranda, J., Martell-Nevárez, M. and Soto-Cruz, N. (2021). Enhancing isoamyl acetate biosynthesis by Pichia fermentans. Revista Mexicana de Ingeniería Química 20(2), 621-633. https://doi.org/10.24275/rmiq/Bio2125.

Ritala, A., Häkkinen, S.T., Toivari, M. and Wiebe, M.G. (2017). Single Cell Protein-State-of-the-Art, Industrial Landscape and Patents 2001-2016. Frontiers in Microbiology 8. https://doi.org/10.3389/fmicb.2017.02009.

Sambrook, J. and Russel, D. (2001). Molecular cloning a laboratory manual. 3a ed. Cold Spring Harbor Laboratory Press. New York.

Sánchez-Crisóstomo, M. I., Rojo-López, M. I., Sharma, A., Cancino-Diaz, J. C., Jaimes-Díaz, H., Ariza-Ortega, J. A., Madrigal-Santillán, E. and Betanzos-Cabrera, G., (2019). Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes. Molecular Biology Reports 46, 1593–1601. https://doi.org/10.1007/s11033-019-04604-1.

Schulte-Herbrüggen, B., Cowlishaw, G., Homewood, K., Rowcliffe, J.M. (2017). Rural protein insufficiency in a wildlife-depleted West African farm-forest landscape. PLoS ONE. 12(12), e0188109. https://doi.org/10.1371/journal.pone.0188109.

Thompson, C. A. (2010). FDA approves kallikrein inhibitor to treat hereditary angioedema. American Journal of Health-System Pharmacy 67(2), 93–93. https://doi.org/10.2146/news100005.

Wu, J.C., Gardner, D.P., Ozer, S., Gutell, R.R. and Ren, P. (2009). Correlation of RNA secondary structure statistics with thermodynamic stability and applications to folding. Journal of Molecular Biology 391(4), 769-783. https://doi: 10.1016/j.jmb.2009.06.036.

Wu, F., Zhang, Q. and Wang, X. (2018). Design of adjacent transcriptional regions to tune gene expression and facilitate circuit construction. Cell Systems. 6(2), 206–215.e6. https://doi:10.1016/j.cels.2018.01.010.

Wu, G. (2009). Amino acids: metabolism, functions, and nutrition. Amino Acids, 37, 1-17. https://doi.org/10.1007/s00726-009-0269-0.

Yang, M. S., Espinoza, N. O., Nagpala, P. G., Dodds, J. H., White, F. F., Schnorr, K. L. and Jaynes, J. M. (1989). Expression of a synthetic gene for improved protein quality in transformed potato plants. Plant Science 64(1), 99–111. https://doi:10.1016/0168-9452(89)90156-8.

Published
2021-07-05
How to Cite
Bartolo-Aguilar, Y., Chávez-Cabrera, C., Cancino-Díaz, J., & Marsch, R. (2021). Expression of a synthetic protein with a high proportion of essential amino acids by Pichia pastoris. Revista Mexicana De Ingeniería Química, 20(3), Bio2419. https://doi.org/10.24275/rmiq/Bio2419