Constitutive expression in komagataella phaffii of mature rhizopus oryzae lipase jointly with its truncated prosequence improves production and the biocatalyst operational stability
López Fernández, Josu (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Benaiges, M. Dolors (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Valero Barranco, Francisco (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Date:	2021
Abstract:	Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis con-firmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0. 25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).
Grants:	Agencia Estatal de Investigación PID2019-104666GB-100
Note:	Altres ajuts: Eusko Jaurlaritza/PRE_2017_1_0110
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Rhizopus oryzae lipase ; Komagataella phaffii ; Prosequence ; Flavor ; Biodiesel ; Biocatalysis
Published in:	Catalysts, Vol. 11, Issue 10 (October 2021) , art. 1192, ISSN 2073-4344

DOI: 10.3390/catal11101192

15 p, 900.6 KB

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