An overview of the plasmids constructed in this study is reported in Table
2 , the detailed maps of the plasmids are contained in Additional file
1 . The gene coding for α-santalene synthase (SanSynopt) was codon optimized for expression in S. cerevisiae and synthesized by DNA 2.0 (Menlo Park, CA, USA) (Additional file
2 ), cut with NotI/PacI and ligated into NotI/PacI restricted vector pICK01 containing tHMG1[12 (link)] resulting in plasmid pISP15 (Figure
1 ).
To simultaneously integrate multiple genes into the yeast genome a series of plasmids containing the genes, constitutive strong promoters, terminators, marker gene sequences and the required region for genomic integration were constructed. All endogenous S. cerevisiae genes were PCR amplified using genomic DNA of strain CEN.PK113-5D as template. Primers used for amplification are provided in Additional file
3 . All PCRs were performed using high fidelity Phusion™ DNA polymerase (Finnzymes, Vantaa, Finland). The ERG20 gene [GenBank: NM_001181600] was amplified using primer pair 1/2, subsequently digested with BamHI/NheI and ligated into the vector pSP-GM2
[55 (link)] restricted with the respective enzymes downstream of the TEF1 promoter resulting in plasmid pIGS01. A 711 bp upstream flanking region (AD1) selected for genomic integration was amplified using primer pair 3/4, cut with MreI/Kpn2I and ligated into vector pIGS01 restricted with the respective enzymes resulting in plasmid pIGS02. Plasmid pIGS03 was obtained by cloning gene GDH2 [GenBank: NM_001180275] amplified with primer pair 5/6 into pIGS02 downstream of the PGK1 promoter using PacI/NotI restriction sites. A downstream flanking region of 653 bp (AD2) was amplified with primers 7/8, digested with AscI/AvrII and ligated into pIGS03. The resulting plasmid was named pIGS04. To complete the plasmid for integration the Kluyveromyces lactis (Kl) URA3 gene [GenBank: Y00454] was amplified with primers 9 and 10 using plasmid pWJ1042
[56 (link)] as template, cut with FseI and ligated into pIGS04 after restriction with the respective enzyme. The resulting plasmid was designated pIGS05, digested with MreI/AscI and the resulting fragment used for integration into the yeast genome as described below. The 5´ region of the Kl URA3 gene was amplified with primers 11 and 12, cut with AvrII/AscI and cloned into pIGS03 restricted with the respective enzymes resulting in plasmid pIGS06. Amplification of the catalytic domain of the HMG-CoA reductase gene (tHMG1) [GenBank: NM_001182434] was performed using primer pair 13/14, the resulting fragment cleaved with NheI/BamHI and cloned downstream of the TEF1 promoter into NheI/BamHI restricted pSP-GM2 resulting in pIGS07. A mutant allele upc2-1 of the UPC2 gene [GenBank: NC_001180521] was created by use of primer pair 15/16. To introduce the pleiotropic mutation G888D, the corresponding codon GGT was mutated to GAT generating the amino acid substitution. Subsequently, the PCR amplified upc2-1 was cloned downstream of the PGK1 promoter into pIGS07 using NotI/PacI resulting in plasmid pIGS08. An 829 bp downstream flanking region (AD3) selected for genomic integration was amplified using primer pair 17/18 cut with MreI/Kpn2I and ligated into vector pIGS08 restricted with the respective enzymes resulting in plasmid pIGS09. The 3´ region of Kl URA3 (overlapping with the 5´region described above) was amplified with primers 19 and 20, cut with AvrII/AscI and cloned into pIGS09 restricted with the respective enzymes resulting in plasmid pIGS10. All plasmids were verified by sequencing (Sigma-Aldrich, St. Luis, MO). Subsequently, plasmids pIGS06 and pIGS10 were restricted with MreI/AscI, the cassettes isolated from the vector backbone and used for yeast transformation (see below).
To simultaneously integrate multiple genes into the yeast genome a series of plasmids containing the genes, constitutive strong promoters, terminators, marker gene sequences and the required region for genomic integration were constructed. All endogenous S. cerevisiae genes were PCR amplified using genomic DNA of strain CEN.PK113-5D as template. Primers used for amplification are provided in Additional file
[55 (link)] restricted with the respective enzymes downstream of the TEF1 promoter resulting in plasmid pIGS01. A 711 bp upstream flanking region (AD1) selected for genomic integration was amplified using primer pair 3/4, cut with MreI/Kpn2I and ligated into vector pIGS01 restricted with the respective enzymes resulting in plasmid pIGS02. Plasmid pIGS03 was obtained by cloning gene GDH2 [GenBank: NM_001180275] amplified with primer pair 5/6 into pIGS02 downstream of the PGK1 promoter using PacI/NotI restriction sites. A downstream flanking region of 653 bp (AD2) was amplified with primers 7/8, digested with AscI/AvrII and ligated into pIGS03. The resulting plasmid was named pIGS04. To complete the plasmid for integration the Kluyveromyces lactis (Kl) URA3 gene [GenBank: Y00454] was amplified with primers 9 and 10 using plasmid pWJ1042
[56 (link)] as template, cut with FseI and ligated into pIGS04 after restriction with the respective enzyme. The resulting plasmid was designated pIGS05, digested with MreI/AscI and the resulting fragment used for integration into the yeast genome as described below. The 5´ region of the Kl URA3 gene was amplified with primers 11 and 12, cut with AvrII/AscI and cloned into pIGS03 restricted with the respective enzymes resulting in plasmid pIGS06. Amplification of the catalytic domain of the HMG-CoA reductase gene (tHMG1) [GenBank: NM_001182434] was performed using primer pair 13/14, the resulting fragment cleaved with NheI/BamHI and cloned downstream of the TEF1 promoter into NheI/BamHI restricted pSP-GM2 resulting in pIGS07. A mutant allele upc2-1 of the UPC2 gene [GenBank: NC_001180521] was created by use of primer pair 15/16. To introduce the pleiotropic mutation G888D, the corresponding codon GGT was mutated to GAT generating the amino acid substitution. Subsequently, the PCR amplified upc2-1 was cloned downstream of the PGK1 promoter into pIGS07 using NotI/PacI resulting in plasmid pIGS08. An 829 bp downstream flanking region (AD3) selected for genomic integration was amplified using primer pair 17/18 cut with MreI/Kpn2I and ligated into vector pIGS08 restricted with the respective enzymes resulting in plasmid pIGS09. The 3´ region of Kl URA3 (overlapping with the 5´region described above) was amplified with primers 19 and 20, cut with AvrII/AscI and cloned into pIGS09 restricted with the respective enzymes resulting in plasmid pIGS10. All plasmids were verified by sequencing (Sigma-Aldrich, St. Luis, MO). Subsequently, plasmids pIGS06 and pIGS10 were restricted with MreI/AscI, the cassettes isolated from the vector backbone and used for yeast transformation (see below).
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