Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris

Abstract

Background: Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris.

Results: Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation.

Conclusions: Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.

Keywords: Experimental evolution; Methanol; Pichia pastoris; Recombinant protein.

Fig. 1

Single clone growth rates in deep-well cultures. Three single clones from YPM (red circles) and BMM-evolved (blue squares) population 1–3 were randomly selected and growth rates were compared to the ancestral P. pastoris strain on YPM (a) and BMM (b). % growth rate relative to the ancestral strain is shown. Number of replicates per single clone, n = 2

Fig. 2

AOX1 mutations and activity in several ancestral and evolved P. pastoris strains. a Aox1 protein domains according to the recently published crystal structure (PDB ID 5HSA) [49]. Amino acid positions mutated in the evolved strains are highlighted. b Alcohol oxidase activity in P. pastoris strains. Activity was determined as described in the “Methods” section. The enzymatic activity of wildtype P. pastoris X-33 on YPM and BMM growth medium was set to 100%. The activity of the wildtype strain on glucose and methanol is shown, as well as the activity of four evolved strains with mutations of the AOX1 gene on methanol as carbon source. YPM (black bars), BMM (grey bars); Values represent averages of two biological and two technical replicates ±standard deviation

Fig. 3

Schematic presentation of cellular pathways and genetic/protein changes. The corresponding proteins of the genes where mutations were observed (as discussed in the main text) are highlighted in red. Red dots in the upper left corner of the genes/proteins indicate mutations found in clones evolved on YP-medium and blue dots indicate mutations found in clones adapted to BM-medium. Mut-pathway: AOX alcohol oxidase, Cat catalase, Dak dihydroxyacetone kinase, Das dihydroxyacetone synthase, Fba fructose-1,6-bisphosphate aldolase, Fbp fructose-1,6-bisphosphatase, Fld formaldehyde dehydrogenase, Fgh S-formylglutathione hydrolase, Fdh formate dehydrogenase, Tpi triosephosphate isomerase