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Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.

Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A - Microb. Cell Fact. (2012)

Bottom Line: A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A.Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed.A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria.

ABSTRACT

Unlabelled: ΒACKGROUND: The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.

Results: A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.

Conclusions: Co-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

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Methanol utilization (MUT) pathway in P. pastoris. A, MUT pathway overview (adapted from [5] and [16]); B, catalytic reaction of DAS1, DAS2 and hypothetically TKL1; C, catalytic reaction of FLD1; D, catalytic reaction of TKL1 in the pentose phosphate pathway. ADH: methylformate synthase. AOX: alcohol oxidase. CAT: catalase. DAK: dihydroxyacetone kinase. DHA: dihydroxyacetone. DHAP: dihydroxyacetone phosphate. F1,6BP: fructose-1,6-bisphosphate. F6P: fructose-6-phosphate. FBA: fructose-1,6-bisphosphate aldolase. FBP: fructose-1,6-bisphosphatase. FLD: formaldehyde dehydrogenase. FDH: formate dehydrogenase. FGH: S-formylglutathione hydrolase. GAP: glyceraldehyde-3-phosphate. GSH: glutathione. Pyr: pyruvate. PPP: pentose phosphate pathway. Ri5P: ribose-5-phosphate. SeHe7P: sedoheptulose-7-phosphate. TCA: tricarboxylic acid cycle. Xu5P: xylulose-5-phosphate.
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Figure 1: Methanol utilization (MUT) pathway in P. pastoris. A, MUT pathway overview (adapted from [5] and [16]); B, catalytic reaction of DAS1, DAS2 and hypothetically TKL1; C, catalytic reaction of FLD1; D, catalytic reaction of TKL1 in the pentose phosphate pathway. ADH: methylformate synthase. AOX: alcohol oxidase. CAT: catalase. DAK: dihydroxyacetone kinase. DHA: dihydroxyacetone. DHAP: dihydroxyacetone phosphate. F1,6BP: fructose-1,6-bisphosphate. F6P: fructose-6-phosphate. FBA: fructose-1,6-bisphosphate aldolase. FBP: fructose-1,6-bisphosphatase. FLD: formaldehyde dehydrogenase. FDH: formate dehydrogenase. FGH: S-formylglutathione hydrolase. GAP: glyceraldehyde-3-phosphate. GSH: glutathione. Pyr: pyruvate. PPP: pentose phosphate pathway. Ri5P: ribose-5-phosphate. SeHe7P: sedoheptulose-7-phosphate. TCA: tricarboxylic acid cycle. Xu5P: xylulose-5-phosphate.

Mentions: Regardless of which phenotype is used, AOX catalyzes the first reaction in the MUT pathway, oxidizing methanol to formaldehyde while reducing O2 to H2O2 (Figure 1A). Formaldehyde is then either oxidized to CO2 in the dissimilative branch of the MUT pathway giving 2 NADH molecules per molecule formaldehyde, or is condensed with xylulose-5-phosphate and subsequently converted to dihydroxyacetone and glyceraldehyde-3-phosphate in the assimilative branch of the MUT pathway (Figure 1A; see also [5]).


Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.

Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A - Microb. Cell Fact. (2012)

Methanol utilization (MUT) pathway in P. pastoris. A, MUT pathway overview (adapted from [5] and [16]); B, catalytic reaction of DAS1, DAS2 and hypothetically TKL1; C, catalytic reaction of FLD1; D, catalytic reaction of TKL1 in the pentose phosphate pathway. ADH: methylformate synthase. AOX: alcohol oxidase. CAT: catalase. DAK: dihydroxyacetone kinase. DHA: dihydroxyacetone. DHAP: dihydroxyacetone phosphate. F1,6BP: fructose-1,6-bisphosphate. F6P: fructose-6-phosphate. FBA: fructose-1,6-bisphosphate aldolase. FBP: fructose-1,6-bisphosphatase. FLD: formaldehyde dehydrogenase. FDH: formate dehydrogenase. FGH: S-formylglutathione hydrolase. GAP: glyceraldehyde-3-phosphate. GSH: glutathione. Pyr: pyruvate. PPP: pentose phosphate pathway. Ri5P: ribose-5-phosphate. SeHe7P: sedoheptulose-7-phosphate. TCA: tricarboxylic acid cycle. Xu5P: xylulose-5-phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3295664&req=5

Figure 1: Methanol utilization (MUT) pathway in P. pastoris. A, MUT pathway overview (adapted from [5] and [16]); B, catalytic reaction of DAS1, DAS2 and hypothetically TKL1; C, catalytic reaction of FLD1; D, catalytic reaction of TKL1 in the pentose phosphate pathway. ADH: methylformate synthase. AOX: alcohol oxidase. CAT: catalase. DAK: dihydroxyacetone kinase. DHA: dihydroxyacetone. DHAP: dihydroxyacetone phosphate. F1,6BP: fructose-1,6-bisphosphate. F6P: fructose-6-phosphate. FBA: fructose-1,6-bisphosphate aldolase. FBP: fructose-1,6-bisphosphatase. FLD: formaldehyde dehydrogenase. FDH: formate dehydrogenase. FGH: S-formylglutathione hydrolase. GAP: glyceraldehyde-3-phosphate. GSH: glutathione. Pyr: pyruvate. PPP: pentose phosphate pathway. Ri5P: ribose-5-phosphate. SeHe7P: sedoheptulose-7-phosphate. TCA: tricarboxylic acid cycle. Xu5P: xylulose-5-phosphate.
Mentions: Regardless of which phenotype is used, AOX catalyzes the first reaction in the MUT pathway, oxidizing methanol to formaldehyde while reducing O2 to H2O2 (Figure 1A). Formaldehyde is then either oxidized to CO2 in the dissimilative branch of the MUT pathway giving 2 NADH molecules per molecule formaldehyde, or is condensed with xylulose-5-phosphate and subsequently converted to dihydroxyacetone and glyceraldehyde-3-phosphate in the assimilative branch of the MUT pathway (Figure 1A; see also [5]).

Bottom Line: A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A.Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed.A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria.

ABSTRACT

Unlabelled: ΒACKGROUND: The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.

Results: A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.

Conclusions: Co-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

Show MeSH
Related in: MedlinePlus