scholarly journals Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

2021 ◽  
Vol 83 (1) ◽  
pp. 115-120
Author(s):  
D. S. Bytyak ◽  
O. S. Korneeva ◽  
E. A. Motina
Keyword(s):  
Phospholipase A2 ◽  
Methylotrophic Yeast ◽  
Specific Rate ◽  
Methylotrophic Yeasts ◽  
Aox1 Promoter ◽  
Basal Salt Medium ◽  
Methanol Induction ◽  
Enzyme Preparations ◽  
Komagataella Phaffii

Currently, there is a significant increase in interest in the industrial production of enzyme preparations (and other recombinant proteins) using various microorganisms, including methylotrophic yeasts such as Komagataella phaffii. At the same time, the most significant productivity of the target proteins is achieved by methanol induction of heterologous genes cloned under the control of the AOX1 promoter. Thus, the efficiency of biosynthesis is largely determined by the metabolism of methanol. In this connection, the aim of the work is to develop an optimal strategy for methanol induction of the AOX1 promoter of Komagataella phaffii. The object of the study is the culture of the recombinant phospholipase A2 producing strain Komagataella phaffii. The studies were carried out in a laboratory fermenter Infors Minifors (Switzerland) on a liquid nutrient medium BSM (Basal Salt Medium) We used the generally accepted methods of studying the characteristics of metabolic activity, including the calculation of specific characteristics and productivity of the strain. The result of the study is the determination of the specific rate of consumption of methanol used as a carbon source, which was 19.2±1.8 mg/g*h. Also, the specific growth rate of Komagataella phaffii was determined and amounted to 0.24 h-1.Based on the data obtained during the research, a strategy for the induction of the AOX1 promoter in the cultivation of the methylotrophic yeast Komagataella phaffii was developed by maintaining the methanol concentration in the range of 0.6 to 2% based on the concentration of dissolved oxygen in the medium. The developed strategy of induction of the AOX1 promoter made it possible to obtain at least 1.87 g / l of recombinant protein (phospholipase A2) during cultivation of Komagataella phaffii for 96 h, which is 3.7 times higher than the known results.

scholarly journals Comparative expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide

2021 ◽  
Vol 83 (1) ◽  
pp. 263-269
Author(s):  
D. S. Bytyak ◽  
Y. A. Gladchenko ◽  
A. V. Ryapolova ◽  
O. S. Korneeva ◽  
E. A. Motina
Keyword(s):  
Phospholipase A2 ◽  
Specific Activity ◽  
Methylotrophic Yeast ◽  
Enzyme Preparations ◽  
Secretion Signal ◽  
Komagataella Phaffii ◽  
Alpha Factor ◽  
Streptomyces Violaceoruber ◽  
The Cost ◽  
Genetic Constructs

Currently, the Russian market of phospholipase A2 enzyme preparations is represented by commercial preparations of foreign manufacturers: Nagase (Japan) and Maxapal (the Netherlands). However, the growing demand and the need to reduce the cost of production of phospholipase A2 require the development of new super-producers of phospholipase A2. In this connection, the aim of the work is to compare the expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide. The object of the study is the recipient yeast strain Komagataella phaffii X-33. The studies were conducted in accordance with generally accepted norms and approaches. Phospholipase A2 genes from Streptomyces violaceoruber were used for this worK. The target sequences were synthesized in the company "Eurogen" (Russia) and cloned as part of the TE vector pUC57. In the course of the work, the genetic constructs pPICZaA-Pla2 and PPICZmf4iA-Pla2 containing the Streptomyces violaceoruber phospholipase A2 gene were assembled under the native signal a-MF and its modification mf4i. The transformation of the yeast Komagataella phaffii X-33 with the obtained genetic constructs was also carried out. As a result of the conducted studies, it was shown that on average, there were no significant differences in the level of expression and specific activity of recombinant phospholipase A2 in methylotrophic yeast K. Phaffii X-33 when using the native a-MF secretion signal and its modified version mf4i. However, the use of the secretion factor mf4i allows for higher production of phospholipase A2 in individual clones. The obtained data indicate the prospects of using the secretion factor mf4i to create super-producers of enzymes based on yeast K. Phaffii X-33.

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.

Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants

Biochemistry ◽  
1991 ◽  
Vol 30 (29) ◽  
pp. 7283-7297 ◽  
Author(s):  
Otto G. Berg ◽  
Bao Zhu Yu ◽  
Joe Rogers ◽  
Mahendra Kumar Jain
Keyword(s):  
Phospholipase A2 ◽  
Rate Constants ◽  
Kinetic Rate ◽  
Kinetic Rate Constants ◽  
Interfacial Catalysis

P3-173: Determination of a specific lipoprotein-associated phospholipase A2 activity in human cerebrospinal fluid

2010 ◽  
Vol 6 ◽  
pp. S500-S500
Author(s):  
Liang Ye ◽  
Tina Li ◽  
Daniel Lee ◽  
Curtis Huang ◽  
Chenbing Guan ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Phospholipase A2 ◽  
Human Cerebrospinal Fluid ◽  
Phospholipase A2 Activity

scholarly journals New Sensitive Kinetic Spectrophotometric Methods for Determination of Omeprazole in Dosage Forms

2009 ◽  
Vol 2009 ◽  
pp. 1-11 ◽  
Author(s):  
Ashraf M. Mahmoud
Keyword(s):  
Reference Method ◽  
Fixed Time ◽  
Dosage Forms ◽  
Charge Transfer Complexes ◽  
Specific Rate ◽  
Spectrophotometric Methods ◽  
H Nmr ◽  
Nmr Techniques ◽  
Kinetic Spectrophotometric

New rapid, sensitive, and accurate kinetic spectrophotometric methods were developed, for the first time, to determine omeprazole (OMZ) in its dosage forms. The methods were based on the formation of charge-transfer complexes with both iodine and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The variables that affected the reactions were carefully studied and optimized. The formed complexes and the site of interaction were examined by UV/VIS, IR, and1H-NMR techniques, and computational molecular modeling. Under optimum conditions, the stoichiometry of the reactions between OMZ and the acceptors was found to be 1 : 1. The order of the reactions and the specific rate constants were determined. The thermodynamics of the complexes were computed and the mechanism of the reactions was postulated. The initial rate and fixed time methods were utilized for the determination of OMZ concentrations. The linear ranges for the proposed methods were 0.10–3.00 and 0.50–25.00   with the lowest LOD of 0.03 and 0.14   for iodine and DDQ, respectively. Analytical performance of the methods was statistically validated; RSD was <1.25% for the precision and <1.95% for the accuracy. The proposed methods were successfully applied to the analysis of OMZ in its dosage forms; the recovery was 98.91–100.32%  0.94–1.84, and was found to be comparable with that of reference method.

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