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

BACKGROUND

Pichia pastoris is a eukaryotic expression host is widely used for the production of recombinant proteins. Adaptive evolution laboratory (ALE) has been applied in various studies in order to improve strains for the purpose of biotechnology.

 In this context, the long-term Equine Recombinant Proteins impact of the carbon source adaptation in P. pastoris has not been addressed so far. So, we did a pilot experiment to analyze the application and potential benefits of ALE to the increased growth and production of recombinant proteins in P. pastoris.

RESULTS

Adaptation to growth on methanol made in culture media replication in rich and minimal growth for 250 generations. the growth rate increased in the medium growth observed in the population and the level of a single clone. growing populations show different levels of profit growth and trade-offs in a state of non-evolutionary growth. genome resequencing reveals the potential genetic targets associated with improved growth performance on a methanol-based growth media. 

Alcohol oxidase represents a mutation hotspot since four of the seven developing P. pastoris clones harbored mutations in this gene, resulting in a decrease in AOX activity, despite an increase in the rate of growth. The selected clones show strain-dependent variation for AOX-promoter based recombinant protein expression results. One very interesting clones showed increased product titers ranging from 2.5-fold increase in shake flask batch culture 1.8-fold increase over the batch-fed cultivation.

CONCLUSION

Our data shows the correlation of complex carbon sources, the context of the growth and production of recombinant proteins. While Fruit fly Recombinant Proteins similar experiments have shown their potential in the area of ​​biotechnology others where microbes are evolutionarily engineered for improved stress resistance and growth, is now pushing the analysis dataset ALE potential for enhanced protein production in P. pastoris on a wider scale.