Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae.

William R A Brown,Zhengyao Xu

doi:10.1186/s12896-016-0241-5

William R A Brown, Zhengyao Xu

Open Access

https://doi.org/10.1186/s12896-016-0241-5

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Abstract

BackgroundPhage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology.ResultsThe activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination.ConclusionsThe results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wβ and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0241-5) contains supplementary material, which is available to authorized users.

Highlights

Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae this organism is a widely used organism in biotechnology
Assaying unidirectional phage integrases in S. cerevisiae The approach that we took to determining the relative activities of the different integrases in S. cerevisiae is analogous to the recombinase mediated cassette exchange approach that we used to assay the activities of these same integrases in vertebrate cells [7]
In contrast to the situation in vertebrate cells [7, 27, 29], we detected no site damage associated with integrase mediated site-specific recombination despite screening a total of 300 different attL and attR sites

Summary

Introduction

Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae this organism is a widely used organism in biotechnology. The most commonly used sitespecific recombinases, Cre [1] and Flp [2], belong to the tyrosine recombinase class of site-specific recombinases and promote reversible recombination between two identical 34 bp sequences in the absence of accessory proteins. This class of protein includes recombinases with a wide variety of different properties [3] Some such as the γδ, Hin and Gin recombinases promote reactions between identical sites but do so with topological specificity as a result of the participation of accessory proteins. The functional autonomy of the serine integrases together with their unidirectional nature has meant that they are an ideal complement to Cre and Flp, and several such integrases including those of the BxB1, φC31 and R4 phages are widely used for genome integration in metazoan systems such as humans, mice and Drosophila melanogaster [6]

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