Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica.

Recombinant protein production is a multibillion-dollar market. Therefore, an important area of ​​research both in academia and industry. The use of yeast as a cell factory presents some advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is regarded as one of the most attractive host for its ability to metabolize the crude substrates, Chicken Recombinant Proteins to express the gene at high levels, and to secrete proteins in large quantities.

In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Although the construction of efficient cell factories for the synthesis of recombinant proteins is important, the development of efficient processes for the production of recombinant proteins in a bioreactor is an equally important aspect. Indeed, the sports car can not drive fast on a gravel road. 

The purpose of this study is to provide a comprehensive overview of the process tool to consider for the production of recombinant proteins in a bioreactor using Y. lipolytica as a plant cell, in order to facilitate decision-making for the future strain engineering and process.

Methyl-selective isotopic labeling using α-ketoisovalerate for yeast Pichia pastoris expression system of recombinant proteins.

Methyl-detected NMR spectroscopy is a useful tool to probe the structure and interaction of large macromolecules such as membrane proteins. The procedure for the preparation of methyl-specific isotopically-labeled protein expression system was established for Escherichia coli (E. coli), but usually not feasible to express eukaryotic protein using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and E. coli is known superior system for the expression of mammalian proteins, including secretory and membrane proteins.

 However, this system has not been optimized for methyl-specific isotopic labeling, especially for Val / Leu-methyl-specific incorporation of isotopes. To overcome this difficulty, we explored various culture conditions for yeast cells to efficiently uptake precursor Val / Leu. Among the condition searched, we found that the pH of aquaculture have an important effect on the Val / Leu precursor uptake. 

At acidic pH cultivation, absorption E.coli Recombinant Proteins precursor Val / Leu increased, and the methyl group of Val and Leu recombinant proteins synthesized 1H-13C produces an intense correlation signal. Based on these results, we present an optimized protocol for Val / Leu-methyl-13C selective incorporation by P. pastoris expression system.