Abstract
ΒackgroundThe 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.ResultsA 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.ConclusionsCo-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.
Highlights
The methylotrophic yeast Pichia pastoris has become an important host organism for the high level production of recombinant proteins (e.g. [1,2,3])
The majority of research has been performed with P. pastoris Mut+ strains, since they have been reported to grow faster on methanol and to produce more recombinant protein (e.g. [14])
The transcription of the genes dihydroxyacetone synthase 1 (DAS1) and DAS2, which encode the key enzymes of the assimilative branch of the methonal utilization pathway (MUT) pathway, was high induced upon MeOH addition
Summary
The methylotrophic yeast Pichia pastoris has become an important host organism for the high level production of recombinant proteins (e.g. [1,2,3]). Three phenotypes of P. pastoris with regard to methanol utilization are currently available: I, Mut+ (methanol utilization plus), where both AOX genes are intact and active; II, MutS (methanol utilization slow), where AOX1 is knocked out; and III, Mut- (methanol utilization minus), which is unable to grow on methanol as the sole carbon source due to a knock-out of both AOX genes [2]. In Mut+ and MutS strains the transcription of MUT pathway genes is repressed when grown in the presence of sufficiently high concentrations of glucose or glycerol. Employing either of the two natural AOX promoters, protein expression at high levels can be induced by methanol in media lacking such fermentable carbon sources [11]. De-repression at low concentrations of glucose or glycerol can be used for strong induction, if AOX1 promoter variants are used [10]
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