Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures

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 confirmed 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).

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Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

Journal of Biotechnology ◽

10.1016/s0168-1656(00)00402-8 ◽

2001 ◽

Vol 86 (1) ◽

pp. 59-70 ◽

Cited By ~ 124

Author(s):

Stefan Minning ◽

Alicia Serrano ◽

Pau Ferrer ◽

Carles Solá ◽

Rolf D. Schmid ◽

...

Keyword(s):

Pichia Pastoris ◽

Rhizopus Oryzae ◽

Rhizopus Oryzae Lipase ◽

High Level ◽

Level Production

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Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

Microbial Cell Factories ◽

10.1186/1475-2859-6-21 ◽

2007 ◽

Vol 6 (1) ◽

pp. 21 ◽

Cited By ~ 42

Author(s):

David Resina ◽

Mónika Bollók ◽

Narendar K Khatri ◽

Francisco Valero ◽

Peter Neubauer ◽

...

Keyword(s):

Rhizopus Oryzae ◽

Transcriptional Response ◽

Lipase Production ◽

Fed Batch ◽

Rhizopus Oryzae Lipase

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Development and in silico analysis of a new nitrogen-limited feeding strategy for fed-batch cultures of Pichia pastoris based on a simple pH-control system

Biochemical Engineering Journal ◽

10.1016/j.bej.2015.02.016 ◽

2015 ◽

Vol 98 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Zahra Azimzadeh Irani ◽

Amir Maghsoudi ◽

Seyed Abbas Shojaosadati ◽

Ehsan Motamedian

Keyword(s):

Control System ◽

Pichia Pastoris ◽

In Silico ◽

Feeding Strategy ◽

In Silico Analysis ◽

Ph Control ◽

Fed Batch ◽

Batch Cultures ◽

Silico Analysis

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Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

Catalysts ◽

10.3390/catal9110961 ◽

2019 ◽

Vol 9 (11) ◽

pp. 961 ◽

Cited By ~ 2

Author(s):

Josu López-Fernández ◽

Juan J. Barrero ◽

M. Dolors Benaiges ◽

Francisco Valero

Keyword(s):

Amino Acids ◽

Ionic Strength ◽

Rhizopus Oryzae ◽

Lipase Production ◽

Biochemical Properties ◽

Mature Sequence ◽

Rhizopus Oryzae Lipase ◽

Batch Cultures ◽

Production Improvement ◽

Key Factor

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst.

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