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.

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.

A roadmap to improve yield of the recombinant protein production: a case study employing Komagataella phaffii as the host organism

2016 ◽  
Vol 33 ◽  
pp. S49
Author(s):  
Ayca Cankorur-Cetinkaya ◽  
Duygu Dikicioglu ◽  
Joao Dias ◽  
Jana Kludas ◽  
Juho Rousu ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Host Organism ◽  
Komagataella Phaffii

scholarly journals Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii

2020 ◽  
Vol 7 (4) ◽  
pp. 161
Author(s):  
Alexander Pekarsky ◽  
Sophia Mihalyi ◽  
Maximilian Weiss ◽  
Andreas Limbeck ◽  
Oliver Spadiut
Keyword(s):  
Recombinant Protein ◽  
Boric Acid ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Protein Quality ◽  
Negative Effects ◽  
Komagataella Phaffii ◽  
Cobalt Dichloride ◽  
The Impact ◽  
Reach Regulation

The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways.

scholarly journals A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

2000 ◽  
Vol 66 (10) ◽  
pp. 4253-4257 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Tatsuro Miyaji ◽  
Hiroya Yurimoto ◽  
Yasuyoshi Sakai ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Alcohol Oxidase ◽  
Pectate Lyase ◽  
Methylotrophic Yeast ◽  
Pectin Methylesterase ◽  
Candida Boidinii ◽  
Methanol Metabolism ◽  
Genes Encoding ◽  
Dihydroxyacetone Synthase ◽  
In Cells

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

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 The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris

2018 ◽  
Vol 293 (43) ◽  
pp. 16647-16660 ◽  
Author(s):  
Trishna Dey ◽  
Kamisetty Krishna Rao ◽  
Jesminara Khatun ◽  
Pundi N. Rangarajan
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Signaling Pathway ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Leucine Zipper ◽  
Alcohol Oxidase ◽  
Transcriptional Regulator ◽  
Methylotrophic Yeast ◽  
Methanol Metabolism ◽  
Methylotrophic Yeast Pichia Pastoris

Rtg1p and Rtg3p are two basic helix–loop–helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae. Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris. We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris. We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

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