scholarly journals Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

Catalysts ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1192
Author(s):  
Josu López-Fernández ◽  
Maria Dolors Benaiges ◽  
Francisco Valero
Keyword(s):  
Amino Acids ◽  
Rhizopus Oryzae ◽  
Constitutive Expression ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Operational Stability ◽  
Ethyl Butyrate ◽  
Rhizopus Oryzae Lipase ◽  
Batch Cultures ◽  
Komagataella Phaffii

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).

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

Catalysts ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 961 ◽  
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.

scholarly journals Function and regulation of an aldehyde dehydrogenase essential for ethanol and methanol metabolism of the yeast, Komagataella phaffii

2020 ◽  
Author(s):  
Kamisetty Krishna Rao ◽  
Umakant Sahu ◽  
Pundi N Rangarajan
Keyword(s):  
Transcriptional Activation ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Ethanol Metabolism ◽  
Mrna Levels ◽  
Methanol Metabolism ◽  
Protein Levels ◽  
Komagataella Phaffii

AbstractThe genome of the methylotrophic yeast, Komagataella phaffii harbours multiple genes encoding putative alcohol dehydrogenases and aldehyde dehydrogenases (ALDs). Here, we demonstrate that one of the ALDs denoted as ALD-A is essential for ethanol metabolism. A zinc finger transcription factor known as Mxr1p regulates ALD-A transcription by binding to Mxr1p response elements (MXREs) in the ALD-A promoter. Mutations which abrogate Mxr1p binding to ALD-A MXREs in vitro abolish transcriptional activation from ALD-A promoter in vivo. Mxr1p regulates ALD-A expression during ethanol as well as methanol metabolism. ALD-A is essential for the utilization of methanol and Δald-a is deficient in alcohol oxidase (AOX), a key enzyme of methanol metabolism. AOX protein but not mRNA levels are down regulated in Δald-a. ALD-A and AOX localize to cytosol and peroxisomes respectively during methanol metabolism suggesting that they are unlikely interact with each other in vivo. This study has led to the identification of Mxr1p as a key regulator of ALD-A transcription during ethanol and methanol metabolism of K. phaffii. Post-transcriptional regulation of AOX protein levels by ALD-A during methanol metabolism is another unique feature of this study.

scholarly journals Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2

2021 ◽  
Vol 21 (2) ◽  
Author(s):  
Domen Zavec ◽  
Christina Troyer ◽  
Daniel Maresch ◽  
Friedrich Altmann ◽  
Stephan Hann ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Alcohol Dehydrogenase ◽  
Protein Production ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Protein Overexpression ◽  
Methylotrophic Yeasts ◽  
Methanol Metabolism ◽  
Komagataella Phaffii ◽  
Dehydrogenase Reaction

ABSTRACT Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

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