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

Background Phage-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. Results The 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. Conclusions The 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 material The online version of this article (doi:10.1186/s12896-016-0241-5) contains supplementary material, which is available to authorized users.


Additional file figure 1; analysis of site specific integration at arrays of recombinase attachment (att) sites by PCR
A; shows PCR analysis across the recombination breakpoints of ten Leu + prototrophs generated by C31 integrase mediated site specific recombination in the assay shown in figure 1 of the main text.. B; shows PCR analysis across the recombination breakpoints of ten Leu + prototrophs generated by BxB1 integrase mediated site specific recombination in the assay shown in figure 1 of the main text..

Additional file figure 2; validation of conservative and reciprocal site specific recombination by sequencing across the recombinant products
The sequence traces are labelled with the name of the recombinant sequence and below each is the corresponding predicted sequence.

Additional file figure 3; analysis of the integrity of site specific integration at arrays of recombinase attachment (att) sites by agarose gel electrophoresis and filter hybridization ("Southern blotting")
A; shows the structure and sequence organization of the LEU2 locus containing the integrated URA3 assay construct before and after BxB1 integrase mediated site specific recombination with the donor construct containing LEU2.
B; shows analysis of the structure of the locus by restriction enzyme digestion using BamHI and BclI, blotting and filter hybridization with a single attP array. The sizes and relative intensities of the cognate fragments are consistent with the map shown in A.

Additional file figure 4; analysis of the integrity of site specific integration at a single pair of inverted sites by long range PCR
A; shows the size and sequence organization of the PCR product generated from URA3 assay construct integrated at the LEU2 locus before and after site specific recombination with the HIS3 containing donor construct. B; shows agarose gel and restriction enzyme analysis of the PCR products generated by amplification across the LEU2 locus before and after site-specific recombination with either the BxB1 or C31 integrases. Two clones isolated after recombination were analysed for each integrase.

Additional file figure 5; analysis of site specific integration at a single pair of inverted sites by PCR
A; The attB and attP sites participating in the reaction are arranged in inverted orientations (main text figure 4) and consequently the incoming His3 sequences can orient in either one of two orientations, inverted one with respect to another and to the flanking Leu2 sequences. Given this we needed to analyse each candidate recombinant with four sets of primer pairs arranged as indicated in this diagram. The lines with arrowheads indicate primers and the numbers refer to the primers used in the PCRs analysed in B. B; shows the results of the analyses for the C31 and BxB1 recombinants and demonstrates that indeed the products of the site specific integration reactions are arranged in either one of two orientations with respect to the flanking Leu2 gene Additional file  GGT GTA TTC GAT  543  TP901F  acaaatacacacactaaattaccggatcaattcgggg  atgactaaaaaggtcgctatc  544  TP901R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA GAC CTT CCG CTT CTT CTT TG  545  TP901R-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  tca TGC CAG CTG AAA CTT GAA AAT AAT G  546  TG1F  acaaatacacacactaaattaccggatcaattcgggg  atgtccgtgaaagtcgagggc  547  TG1R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA CAC CTT CCG TTT CTT CTT AG  548  TG1R-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  tca GGC AGC GGC AGT AAA GCC ATT C  549  SPBCF  acaaatacacacactaaattaccggatcaattcgggg  atggagctgaagaacatcgtc  550  SPBCR+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA GAC TTT GCG TTT CTT TTT TG  551  SPBCR-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA GTG GAA GCT GTT AGT TGC TGT C  552  RVF  acaaatacacacactaaattaccggatcaattcgggg  atgagatacaccacaccagtg  553  RVR+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA GAC CTT CCG CTT CTT CT  554  RVR-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG CG  TCA CCG CCA GTT GAC CTG CAC  R4F  acaaatacacacactaaattaccggatcaattcgggg  atgaacagaggaggaccaacag  556  R4+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA CAC TTT TCT TTT CTT TTT AG  557  R4-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  tca CTC TGC CAC ATC TCT CCA CTC  558  MR11F  acaaatacacacactaaattaccggatcaattcgggg  atgaaggtcgccatctacacc  559  MR11R+nls  ATT ACA TGA TGC G  567  A118F  acaaatacacacactaaattaccggatcaattcgggg  atgaaagccgctatctacatc  568  A118R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA CAC CTT TCT CTT TTT CTT GG  569  A118R-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA CAG CCA CTC AAT GGT CAC C  570  FC1F  acaaatacacacactaaattaccggatcaattcgggg  atgaagcgtgcagcattgtata  571  FC1R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  CTA CAC CTT GCG CTT CTT CTT G  572  FC1R-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA AAA TTT ATA TTT AAT AAT TAC CTC  573  K38F  acaaatacacacactaaattaccggatcaattcgggg  atgtggtcccacccccagttc  574  K38R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  CTA CAC CTT GCG CTT CTT C  575  K38R-nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA CGT CCT CGC CGC CCA TTT G  576  FC31F  acaaatacacacactaaattaccggatcaattcgggg  atggatacctacgccggag  577  FC31R+nls  ATT ACA TGA TGC GGC CCT CCT GCA GGG CCC TAG cg  TCA CAC TTT CCG CTT TTT CTT AG  578  FC31R-