The insertion Green Monster (iGM) method for expression of multiple exogenous genes in yeast

Abstract

Being a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains expressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the "insertion Green Monster" (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof of principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci.

Keywords: Saccharomyces cerevisiae; flow cytometry; green fluorescent protein; multi-gene insertions; synthetic biology.

Figure 1

The insertion Green Monster (iGM) strategy. (A) Schematic map of the iGM expression vectors. (B) A universal GFP insertion cassette containing the exogenous gene under control of the GAL1-10 promoter. Each gene is cloned into the insertion cassette using the Gateway cloning reaction such that it contains a C-terminal hemagglutinin (HA) epitope tag. The insertion cassette replaces KanMX4 in a neutral locus via homologous recombination. (C) Schematic overview of the Green Monster process for sexual cycling and enrichment of strains containing multiple GFP copies using flow cytometry.

Figure 2

Testing the iGM insertion module. (A) Yeast YFR057W ORF is replaced by an insertion module containing human RPL30 gene that was cloned under control of GAL1-10 promoter and linked to an inducible GFP and the URA3 marker. Positions of the primers for genotyping are indicated by arrows. (B) Expression of the human RPL30 gene in a strain containing the RPL30 insertion module was detected by Western blotting with HA-tag–specific antibodies. Expression of the protein was induced by galactose. (C) Peptide sequences identified in a yeast strain containing the RPL30 insertion module by LC-MS/MS. RPL30 protein was immunoprecipitated with HA-tag antibody, resolved by SDS-PAGE, and subjected to in-gel trypsin digestion. The peptides detected by mass spectrometry are shown in green. (D) Expression of the mouse MsrA protein in the 3 Msr∆ strain lacking all three Msr enzymes (i.e., MsrA, MsrB, and fRMsr). The insertion module containing mouse MsrA gene under control of GAL1-10 promoter was integrated into the 3 Msr∆ strain, and expression of the protein following galactose induction was detected by Western blotting with αHA antibodies. (E) Expression of the mouse MsrA rescued the growth of 3 Msr∆ strain missing all three yeast Msrs on medium containing a source of Met-SO. Yeast strains were analyzed for growth on Met-free SC medium supplemented with 20 mg/liter Met (left) or Met-SO (right). Cells were initially grown on SC liquid medium until OD₆₀₀ reached 0.6, harvested, washed, diluted to OD₆₀₀ of 0.1 in water, and serially spotted on agar SC plates containing galactose and respective Met or Met-SO sources. The plates were incubated at 30°, and images were taken 48 hr after plating. (F) Expression of the mouse MsrA protein using iGM vectors containing alternative promoters. Insertion modules containing the mouse MsrA gene under control of ADH1, TEF, and CUP1 promoters or a version of the plasmid containing the GAL1-10 promoter that lacks the HA-tag were integrated into YER042W locus, and expression of the protein was detected by Western blotting with either HA-tag or MsrA-specific antibodies. Where indicated, logarithmically growing cultures were induced to produce the protein by addition of 100 µM CuSO₄ or 2% galactose for 4 hr.

Figure 3

Generation of the 11-insertion Green Monster strains (iGM11). (A) A diagram showing phylogenetic tree and conservation of the selenoprotein synthesis pathway in different clades. The presence of the selenoprotein synthesis machinery and number of selenoproteins encoded in the genome are indicated. (B) Individual GFP insertion strains of two different mating types were generated for the 11 human genes involved in selenoprotein synthesis. Blue rectangles represent MATa strains and red rectangles represent MATα strains. Specific positions of filled squares (▪) in each rectangle indicate inserted exogenous genes. (C) To generate yeast strains with multiple insertions of exogenous genes, individual strains carrying a single engineered locus linked to an inducible GFP marker gene were subjected to repeated cycles of the Green Monster process. The number of sexual cycling rounds and the number of obtained insertions in strains are shown on the left. (D) Representative images of a nonmutant strain and strains containing 2, 6, and 11 GFP insertion modules. Identical exposure, brightness, and contrast settings were used for fluorescence imaging. (E) Quantification of mRNA expression levels in the iGM11 mutant using RNA-seq. To analyze expression of exogenous genes, mRNA reads obtained by sequencing the transcriptome of the iGM11 mutant were aligned to the sequences of the introduced genes. The rpkm (reads per kilobase per million mapped reads) values represent the number of reads normalized to gene length and total number of mapped reads. Error bars indicate SEM. Measurements from biological replicates are shown. (F) RNA-seq read density (rpkm) aligned to the sequence of TRSP gene encoding human Sec tRNA. Error bars indicate SEM. Measurements from biological replicates are shown.

Figure 4

Strategy for identification of factors that influence Sec incorporation using MsrA selenoprotein reporter. (A) Three yeast Msr genes that catalyze the reduction of Met-SO were deleted in iGM11 strain to allow conditional selection of cells expressing functional MsrA selenoprotein. S-(Met-S-SO) and R-diastereomers (Met-R-SO) of methionine sulfoxide are reduced back to Met by MsrA and MsrB/fRMsr methionine sulfoxide reductases, respectively. (B) GPDpr-MsrA(C72U)-SECIS-HphMX4 reporter was integrated into the TRP1 locus of the iGM11 strain. The reporter contains mouse MsrA gene cloned under the control of the GPD promoter and a modified SECIS element from Toxoplasma gondii selenoprotein T (Novoselov et al. 2007). We introduced a C-terminal His-tag into the mouse MsrA sequence and replaced active site Cys72 residue with Sec. UGA codon corresponding to Sec is shown in red. (C) Mouse MsrA gene reporters possessing redox-active Cys (MsrA-Cys) or Sec (MsrA-Sec) were each separately integrated into iGM11 strain. Yeast strains were analyzed for growth on Met-free SC medium supplemented with 20 mg/liter Met (left) or Met-SO (right). Cells were initially grown on SC liquid medium until OD₆₀₀ reached 0.6, harvested, washed, diluted to OD₆₀₀ of 0.1 in water, and serially spotted on agar SC plates containing galactose and respective Met or Met-SO sources. The plates were incubated at 30°, and images were taken 48 hr after plating. (D) Expression of the reporters in the iGM11 mutant was detected by Western blotting with His-tag–specific antibodies.