Construction of Integration Vectors for Secreted Expression of Bacterial Phytase in Yarrowia Lipolytica

Author Name(s): Daria S. Troshagina*, Anna E. Smirnova, Catherine Madzak, Aliya D. Suleimanova
Author Email: aliya.kzn@gmail.com

Abstract

Yeast Yarrowia lipolytica is a handy tool for efficient production of heterologous proteins. In this work we report the construction of integration vectors for secretion of bacterial phytase by these yeasts. Pantoea sp. 3.5.1 histidine acid phytase encoding gene sequence was codon-optimized and chemically synthetized. Optimized and native phytase gene sequences were cloned into integrative vector pINA1296, containing signal sequence of XPR2 gene. Vectors were multiplied in E.coli DH5α, isolated and linearized for successful integration into Y.lipolytica genome by homologeous recombination in pBR-region. Y.lipolytica strains with integrated bacterial native and optimized phytase genes were obtained.

Introduction

Heterologous proteins are of considerable interest for basic research, as well as for various biomedical and industrial applications. At present, various expression systems are used for the production of recombinant proteins, which can be divided into two groups – prokaryotic (bacterial) and eukaryotic, based on the usage of yeast cells, insects and mammals. Each of these systems has its advantages and disadvantages, which are based on the differences in the regulation of organism metabolism, transcription and translation mechanisms, as well as in the post-translational modifications and protein folding [1]. Therefore, the choice of an expression system depends on the properties of the target protein, as well as on the ability of the host cell to produce the protein of the desired quality with the observation of the minimum set of requirements.

The system based on E. coli is one of the prokaryotic expression systems, which has been used widely for several decades [2]. However, even with a high level of recombinant protein expression, these microorganisms can not be used as the producers for food, medical or agricultural industry because they are opportunistic pathogens and may produce various toxins along with proteins [3]. Besides, the usage of such expression systems is limited by the absence of post-translational modifications typical for eukaryotic proteins and by incorrect folding [4]. However, despite of all shortcomings, bacterial expression systems make an integral part of the initial stages of work with recombinant proteins, which is primarily related to the ease of working with bacteria, the development of genetic engineering and biotechnology methods for them, as well as the relative cost-effectiveness of the performed works.

The expression systems based on insect and mammalian cells carry out post-translational modifications of proteins, but unlike bacterial ones, are complex and high-cost systems, since they require the presence of expensive nutrient media and equipment [5].

The most optimal and compromise solution for the synthesis of recombinant proteins are yeast expression systems. First of all, yeasts are single-celled eukaryotic organisms that are well studied and variety of effective methods of genetic engineering has been developed for them. Secondly, they have the ability to support most of the posttranslational modifications of eukaryotic cells and to efficiently secrete recombinant proteins into the environment. Thirdly, yeast expression systems make it possible to obtain stable recombinant strains with a target gene integrated into the producer’s genome. High expression level is achieved in the presence of inexpensive inductors and on cheap nutrient media [4, 5].

The dimorphic yeast Yarrowia lipolytica has been used for several years as effective expression system [6]. They are characterized by a high level of heterologous protein production, a lesser degree of glycosylation of products and are unpretentious in cultivation. The purpose of this work was the construction of yeast integral vectors and creation of recombinant strains of Yarrowia lipolytica for the secretion of bacterial phytase from Pantoea sp. 3.5.1.

Summary

Thus, recombinant strains of Yarrowia lipolytica containing the gene of the bacterial histidine acid phytase from Pantoea sp. 3.5.1 in their genome were obtained. Further evaluation of bacterial phytase expression by yeast, the isolation and the purification of the enzyme, the study of its properties may be an important stage for the creation of a competitive biotechnological production of phytases effective for the preparation of individual isomers of myo-inositol phosphates for pharmacology.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (project no. 16-34-60191). The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

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