Transcriptional regulatory proteins in central carbon metabolism of Pichia pastoris and Saccharomyces cerevisiae

Kalender, Özge; Çalı́k, Pınar

doi:10.1007/s00253-020-10680-2

Cited by 23 publications

(18 citation statements)

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“…This could indicate that, when combining both the heterologous gene copy number increase and hypoxic conditions, the transcriptional machinery is working closer to its upper rate limit, apart from the potential protein processing limitation mentioned previously. A plausible explanation for such an effect could be a finite availability of TDH3-related transcription factors, such as Gal4-like (CRA1), found to be crucial in the regulation of respiro-fermentative metabolism through the interaction with P GAP and possibly other glycolytic promoters (Ata et al, 2017(Ata et al, , 2018Kalender and Çalık, 2020). Dramatically increasing the number of transcription factor binding sites, which occurs in MCC, with a limited pool of these transcription factors, could cause an attenuation of the transcription of all P GAP regulated genes, as previously described for P AOX1 (Cámara et al, 2017;Garrigós-Martínez et al, 2019).…”

Section: Effect Of Gene Dosage On Gene Expression and Crl1 Productionmentioning

confidence: 99%

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Gasset

Garcia-Ortega

Garrigós-Martínez

et al. 2022

Front. Bioeng. Biotechnol.

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The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris. Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive PGAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient (RQ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate (qP) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher qP values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy.

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Section: Effect Of Gene Dosage On Gene Expression and Crl1 Productionmentioning

confidence: 99%

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Gasset

Garcia-Ortega

Garrigós-Martínez

et al. 2022

Front. Bioeng. Biotechnol.

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“…phaffii (Kalender & Calik, 2020). Results from this study revealed that the gss1 gene might also be involved in the inhibition of glycerol metabolism by glucose in K. phaffii.…”

Section: Screening Of Mps and Tfsmentioning

confidence: 62%

“…A synergistic carbon substrate coutilization strategy that could properly modulate the balance between cell growth and product biosynthesis is regarded as a promising method for high-level biochemical production from glucose. However, glucose was mainly used for cell growth in K. phaffii through glycolysis and PPP (Figure 1) (Baumann et al, 2010;Kalender & Calik, 2020). To block the carbon flux from glucose towards glycolysis and PPP, the pfk1 and pfk2…”

Section: Control Of Carbon Flux From Glucose To Glycolysis and Pppmentioning

confidence: 99%

“…In view of CCR, efforts have been made to alleviate its effects to allow synergetic utilization of multiple carbon sources. Methods include disruption of mediator proteins (MPs) (Kalender & Calik, 2020) and transcriptional factors (TFs), and overexpression of metabolism-related genes (Chen et al, 2013). K. phaffii regulates its gene-specific response to different carbon sources mainly at the transcriptional level (Prielhofer et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…K. phaffii regulates its gene-specific response to different carbon sources mainly at the transcriptional level (Prielhofer et al, 2015). Several MPs and TFs, such as GT1, HXT1, GSS1, and MIG1, that are involved in the inhibition of methanol metabolism in K. phaffii growing on either glucose or glycerol have been characterized (Kalender & Calik, 2020).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Wang

Zhao

Luo

et al. 2022

Yeast

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Komagataella phaffii GS115 is a proven heterologous expression system and has recently been exploited for the production of value‐added biochemicals from glucose through metabolic engineering. A major challenge for high‐level biochemical production is the appropriate distribution of carbon flux between cell growth and product biosynthesis. In this study, we report the development of a synergetic glucose and glycerol coutilization strategy for K. phaffii, potentially enabling this strain to consume glycerol for growth while conserving more glucose for product formation. First, several potential genes encoding mediator proteins and transcriptional factors that were considered to be associated with carbon catabolite repression in K. phaffii were screened, and deletion of gss1, a glucose sensor, appeared to be able to eliminate the glucose‐induced repression of glycerol utilization in a mixed glucose–glycerol medium. Transcriptome comparisons between the parent strain and the Δgss1 mutant were then performed, and the glycerol‐metabolism genes that were subjected to glucose regulation were identified. Second, coutilization of glucose and glycerol in K. phaffii was achieved by overexpressing genes relevant to glycerol metabolism, namely, gt1, gut1, and gut2. Furthermore, knockout or knockdown of pfk and zwf genes resulted in a reduction of carbon flux from glucose towards glycolysis and the pentose phosphate pathway. With these efforts, the cell metabolism of the final strain was divided into growth and production modules. This study describes a promising strategy to address the challenge of carbon flux distribution in K. phaffii, and would be valuable in engineering this strain as a versatile fermentation platform for biochemical production.

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Effect of Ca²⁺ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation

Xie,

Zheng,

Sun

et al. 2024

J Sci Food Agric

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BACKGROUNDSaccharomyces cerevisiae is a fungus widely used in the food industry and biofuel industry, whereas it is usually exposed to high sugar stress during the fermentation process. Ca2+ is a key second messenger of the cell, it can regulate cell metabolism. The present study investigated the effect of the Ca2+ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation.RESULTSThe expression of genes encoding hexokinase was up‐regulated in the high sugar environment, the activity of hexokinase was increased, glucose transmembrane transport capacity was enhanced, the ability of glucose to enter into glycolytic metabolism was increased, and the expression of genes related to pentose phosphate metabolism, glycerol metabolism and trehalose metabolism was up‐regulated in the high glucose with Ca2+ group.CONCLUSIONCa2+ signal regulates the cellular metabolism of glycerol and trehalose and optimizes the allocation of carbon flow by regulating the key enzymes and related gene expression to enhance the resistance of yeast to high sugar stress. © 2024 Society of Chemical Industry.

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Transcriptional regulatory proteins in central carbon metabolism of Pichia pastoris and Saccharomyces cerevisiae

Cited by 23 publications

References 243 publications

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Effect of Ca²⁺ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation

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Transcriptional regulatory proteins in central carbon metabolism of Pichia pastoris and Saccharomyces cerevisiae

Cited by 23 publications

References 243 publications

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Effect of Ca2+ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation

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Product

Resources

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Effect of Ca²⁺ signal on the activity of key enzymes of carbon metabolism and related gene expression in yeast under high sugar fermentation