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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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Transcription analysis of the co-overexpressed MUT pathway genes DAS1/FLD1/TKL1 in HRP/CalB overexpressing strains. The transcript levels were normalized to the corresponding transcript levels of ARG4. The increase in AOX2 transcript levels indicated successful induction with methanol. All co-overexpression strains showed elevated transcript levels of the respective target mRNAs compared to the benchmark strains.
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Figure 4: Transcription analysis of the co-overexpressed MUT pathway genes DAS1/FLD1/TKL1 in HRP/CalB overexpressing strains. The transcript levels were normalized to the corresponding transcript levels of ARG4. The increase in AOX2 transcript levels indicated successful induction with methanol. All co-overexpression strains showed elevated transcript levels of the respective target mRNAs compared to the benchmark strains.

Mentions: We co-overexpressed three key enzymes of the MUT pathway, i.e. dihydroxyacetone synthase 1 (DAS1), formaldehyde dehydrogenase 1 (FLD1) and transketolase 1 (TKL1), to analyze possible effects on stoichiometric yields and on the strain specific parameters qs and qp. We used a MutS strain for this study, since it had been shown to be superior to the Mut+ phenotype in terms of recombinant protein production (vide supra). To be able to draw more general valid conclusions from these experiments, we used two recombinant P. pastoris MutS strains overexpressing either the reporter enzyme HRP or CalB, hereafter called benchmark strains, as a platform for co-overexpression studies with the above mentioned MUT pathway enzymes. We characterized all strains in terms of gene copy numbers of the respective reporter enzyme to exclude variations in the measured enzyme activity levels due to copy number rearrangements, such as duplications or deletions. All the generated strains had a single copy integration of the gene encoding the respective reporter enzyme. Thus, all observed variations in enzymatic activity were considered to be due to the respective co-overexpressed MUT pathway enzyme. In order to verify the successful co-overexpression of the MUT pathway enzymes, the mRNA levels of DAS1, FLD1 and TKL1 were quantified relatively to the transcript levels of the constitutively transcribed ARG4 gene via qPCR analysis (Figure 4).


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)

Transcription analysis of the co-overexpressed MUT pathway genes DAS1/FLD1/TKL1 in HRP/CalB overexpressing strains. The transcript levels were normalized to the corresponding transcript levels of ARG4. The increase in AOX2 transcript levels indicated successful induction with methanol. All co-overexpression strains showed elevated transcript levels of the respective target mRNAs compared to the benchmark strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Transcription analysis of the co-overexpressed MUT pathway genes DAS1/FLD1/TKL1 in HRP/CalB overexpressing strains. The transcript levels were normalized to the corresponding transcript levels of ARG4. The increase in AOX2 transcript levels indicated successful induction with methanol. All co-overexpression strains showed elevated transcript levels of the respective target mRNAs compared to the benchmark strains.
Mentions: We co-overexpressed three key enzymes of the MUT pathway, i.e. dihydroxyacetone synthase 1 (DAS1), formaldehyde dehydrogenase 1 (FLD1) and transketolase 1 (TKL1), to analyze possible effects on stoichiometric yields and on the strain specific parameters qs and qp. We used a MutS strain for this study, since it had been shown to be superior to the Mut+ phenotype in terms of recombinant protein production (vide supra). To be able to draw more general valid conclusions from these experiments, we used two recombinant P. pastoris MutS strains overexpressing either the reporter enzyme HRP or CalB, hereafter called benchmark strains, as a platform for co-overexpression studies with the above mentioned MUT pathway enzymes. We characterized all strains in terms of gene copy numbers of the respective reporter enzyme to exclude variations in the measured enzyme activity levels due to copy number rearrangements, such as duplications or deletions. All the generated strains had a single copy integration of the gene encoding the respective reporter enzyme. Thus, all observed variations in enzymatic activity were considered to be due to the respective co-overexpressed MUT pathway enzyme. In order to verify the successful co-overexpression of the MUT pathway enzymes, the mRNA levels of DAS1, FLD1 and TKL1 were quantified relatively to the transcript levels of the constitutively transcribed ARG4 gene via qPCR analysis (Figure 4).

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