Heterologous expression of Trichoderma atroviride endochitinase ech42 in Pichia pastoris at low and high dissolved oxygen tensions
Expresión heteróloga de la endoquitinasa ech42 de Trichoderma atroviride en Pichia pastoris a bajas y elevadas tensiones de oxígeno disuelto
A.S. Pérez-Martínez1,2, A.P. Barba de la Rosa1 and A. De León-Rodríguez1*
1 División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C. Camino a la Presa San José 2055, Lomas 4a secc., C.P. 78216, San Luis Potosí, S.L.P. México. *Corresponding author. E-mail: aleonr@ipicyt.edu.mx.
]]> 2 Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava # 6, Zona Universitaria, C.P. 78210, San Luis Potosí, S.L.P., México.
Received June 11, 2013.
Accepted January 23, 2014.
Abstract
The expression of recombinant Trichoderma atroviride endochitinase ech42 (rech42) in Pichia pastoris driven by the constitutive PGAP promoter was tested at low (3%) and high (40%) dissolved oxygen tension (DOT) conditions. Maximum volumetric enzyme activities of 18,600±1053 and 12,330±1000 U1−1 were attained at 3% and 40% DOT, respectively. Whereas maximum specific enzyme activities were 89.4±4.6 y 88.8±4.0 U mg−1protein. We found that conjunction of low DOT and the overexpression of rech42 affected the cell size of the P. pastoris but no the rech42 specific enzyme activity.
Keywords: chitin, cell size, recombinant endochitinase, oxygen-limited fermentation.
Resumen
]]> La expresión de la Endoquitinasa de 42 kDa de Trichoderma atroviride recombinante (rech42) en Pichia pastoris bajo la dirección del promotor constitutivo, PGAP, fue analizada a baja (3%) y alta (40%) tensión de oxígeno disuelto (TOD). Las actividades enzimáticas volumétricas máximas logradas en cada condición fueron 18,600±1053 y 12,330±1000 U 1−1, a TOD de 3% y 40%, respectivamente. Mientras que las actividades específicas máximas fueron 89.4±4.6 y 88.8±4.0 U mg−1proteína. Se encontró que la conjunción de una baja TOD y la sobreexpresión de rech42 afectó el tamaño de las células de Pichia pastoris pero no afectó la actividad enzimática específica de rech42.Palabras clave: quitina, tamaño celular, endoquitinasa recombinante, fermentación limitada por oxígeno.
DESCARGAR ARTÍCULO EN FORMATO PDF
Acknowledgements
We thank María Magdalena Martínez and Ferdinando Tristán (Nanosciences and Nanotechnology Division from IPICyT, Mexico) for helping us with AFM techniques, and Leandro G. Ordoñez for his comments. We also thank J. Simpson Williamson PhD. (Genetic Engineering Department, CINVESTAV, Mexico) for allowing us to carry out fluorescence microscopy. We thank partial financial support from CONACyT Grant No 56787 (Laboratory for Nanoscience and Nanotechnology Research-LINAN).
References
Apte-Deshpande, A., Rewanwar, S., Kotwal, P., Raiker, V.A., Padmanabhan S. (2009). Effcient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins. Biotechnology and Applied Biochemistry 54, 197-205. [ Links ]
Baumann, K., Maurer, M., Dragosits, M., Cos, O., Ferrer, P., Mattanovich, D. (2008). Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnology and Bioenginering 100, 177-183. [ Links ]
Canales, M., de la Lastra, J.M., Naranjo, V., Nijhof, A.M., Hope, M., Jongejan, F., de la Fuente, J. (2008). Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris. BMC Biotechnology 8, 14. [ Links ]
Cereghino, J.L., Cregg, J.M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews 24, 45-66. [ Links ]
De Leon-Rodriguez, A., Gonzalez-Hernandez, L., De la Rosa, A.P.B., Escalante-Minakata, P., Lopez M.G. (2006). Characterization of volatile compounds of mezcal, an ethnic alcoholic beverage obtained from Agave salmiana. Journal of Agricultural and Food Chemistry 54, 1337-1341. [ Links ]
Duran, A. and Nombela, C. (2004) Fungal cell wall biogenesis: building a dynamic interface with the environment. Microbiology 150, 3099-3103. [ Links ]
Goodrick, J.C., Xu, M., Finnegan, R., Schilling, B.M., Schiavi, S., Hoppe, H., Wan, N.C. (2001). High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system. Biotechnology and Bioengineering 74, 492-497. [ Links ]
Larsson, S., Cassland, P., Jönson, L.J. (2001). Development of Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibition in lignocellulosic hydrolysates by heterologous expression of laccase. Applied and Environmental Microbiology 67, 1163-1170. [ Links ]
Levin, D.E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 69, 262-291. [ Links ]
Li, P., Anumanthan, A., Gao, X.G., Ilangovan K., Suzara V.V., Düzgüneş N., Renugopalakrishnan V. (2007). Expression of Recombinant Proteins in Pichia pastoris. Applied Biochemistry and Biotechnology 142, 105-124. [ Links ]
Macauley-Patrick, S., Fazenda, M.L., McNeil, B., Harvey, L.M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249-270. [ Links ]
O'Brien, M., Colwell, R.R. (1987). A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-β -D-glucosamine. Applied and Environmental Microbiology 53, 1718-1720. [ Links ]
Pérez-Martinez, A.S., De Leon-Rodriguez, A., Harris, L.J., Herrera-Estrella, A., Barba de la Rosa, A.P. (2007). Overexpression, purification and characterization of the Trichoderma atroviride endochitinase, ech42, in Pichia pastoris. Protein Expression and Purification 55, 183-188. [ Links ]
Pérez-Martínez, A.S. (2007). Sobreexpresión de la endoquitinasa ech42 de Trichoderma atroviride en Pichia pastoris. Tesis de Doctorado en Ciencias en Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, México. [ Links ]
Porro, D., Sauer, M., Branduardi, P., Mattanovich, D. (2005). Recombinant protein production in yeast. Molecular Biotechnology 31, 245-259. [ Links ]
Ramirez-Coutino, L., Marin-Cervantes, M.D., Huerta, S., Revah, S., Shirai, K. (2006). Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases. Process Biochemistry 41, 1106-1110. [ Links ]
Rao, M.S., Munoz, J., Stevens, W.F. (2000). Critical factors in chitin production by fermentation of shrimp biowaste. Applied Microbiology and Biotechnology 54, 808-813. [ Links ]
Ren, H.T., Yuan, J.Q. (2005). Model-based specific growth rate control for Pichia pastoris to improve recombinant protein production. Journal of Chemical Technology and Biotechnology 80, 1268-1272. [ Links ]
Schmidt, M. (2004). Survival and cytokinesis of Saccharomyces cerevisiae in the absence of chitin. Microbiology 150, 3253-3260. [ Links ]
Schmidt, M., Varma, A., Drgon, T., Bowers, B., Cabib, E. (2003). Septins, under Cla4p regulation, and the chitin ring are required for neck integrity in budding yeast. Molecular Biology of the Cell 14, 2128-2141. [ Links ]
Sola, A., Maaheimo, H., Ylonen, K., Ferrer, P., Szyperski, T. (2004). Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris. European Journal of Biochemistry 271, 2462-2470. [ Links ]
Uchima, C.A., Arioka, M. (2012). Expression and one-step purification of recombinant proteins using an alternative episomal vector for the expression of N-tagged heterologous proteins in Pichia pastoris. Bioscience, Biotechnology and Biochemistry 76, 368-371. [ Links ]
van der Mei, H.C., Busscher, H.J., Bos, R., de Vries, J., Boonaert, C.J.P., Dufrene, Y.F. (2000) Direct probing by atomic force microscopy of the cell surface softness of a fibrillated and nonfibrillated oral streptococcal strain. Biophysical Journal 78, 2668-2674. [ Links ]
Weidner, M., Taupp, M., Hallam, S.J. (2010). Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Journal of Visualized Experiments 36, 1862. [ Links ]
Wolff, A.M., Hansen, O.C., Poulsen, U., Madrid, S., Stougaard, P. (2001). Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris. Protein Expression and Purification 22, 189-199. [ Links ]
Zhang, A.L., Zhang, T.Y., Luo, J.X., (2007). Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture. Journal of Industrial Microbiology and Biotechnology 34, 117-122. [ Links ]
]]>