Abstract
BackgroundPichia 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.ResultsAdaptation 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.ConclusionsOur 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.
Highlights
Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production
Glucose- and glycerol based expression systems are widely applied for P. pastoris [11], methanol-induced expression of recombinant proteins mediated by strongly inducible promoters such as the alcohol oxidase 1 (Aox1) gene promoter [12] can be considered as one of the core features of P. pastoris
Recent studies led to a better understanding of the methanol utilization process in general; e.g. methanol utilization is predominantly regulated on the transcriptional rather than on the translational level in P. pastoris [18], whereas other data show that methanol metabolic processes are confined to peroxisomes [19]
Summary
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. Moser et al Microb Cell Fact (2017) 16:49 and the correlations of recombinant protein production and process-relevant environmental factors [5,6,7] Classical approaches, such as co-chaperone overexpression in order to increase production efficiency in bacteria and yeasts [8,9,10], can be complemented by systems-wide analysis in order to identify novel targets for strain and process engineering. Transcriptional regulators involved in the expression of Mut proteins have been identified, clarifying how they work in concert to promote important steps such as Mut protein expression and peroxisome proliferation [20,21,22]
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