Construction of recombinant yeast strains

A 1350 bp DNA fragment covering the coding region of arginase gene CAR1 (Accession number {"type":"entrez-nucleotide","attrs":{"text":"M10110","term_id":"171160","term_text":"M10110"}}M10110) and its upstream and downstream regions was amplified from the genomic DNA of S. cerevisiae YS58 by PCR with primer pair CAR1-F1/CAR1-R1. The amplified fragment was digested with KpnI/SalI, and then inserted into the corresponding sites of plasmid pUC18 to generate pUC18C1. Phosphoribosylanthranilate isomerase gene TRP1 (Accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_001136","term_id":"330443520","term_text":"NC_001136"}}NC_001136) was amplified from the genomic DNA of S. cerevisiae CE25 with primer pair TRP1-F/TRP1-R. After digested with AflII/EcoNI, TRP1 was inserted into the corresponding sites of plasmid pUC18C1 to generate recombinant plasmid pUc1T1, in which 692 bp coding sequence of CAR1 was replaced by 1067 bp of TRP1. Disruption cassette car1::TRP1 was amplified from plasmid pUc1T1 with primer pair CAR1-F1/CAR1-R1, and transformed into S. cerevisiae YS58 to disrupt the chromosomal CAR1 by double-crossover homologous recombination. Transformants were screened using TRP1 as the selectable marker. The disruption of CAR1 was confirmed by PCR and sequence analysis. The resulting mutant was designated as yeast strain YS58-car1.

The geneticin (G418) resistance cassette was amplified from plasmid pFA6a-kanMX4⁴⁰ with primer pair KanMX-F/KanMX-R, and inserted into plasmid YCp50⁴¹ after digested by SalI/ApaI to generate plasmid pYCPG. The ADH1 promoter was amplified from S. cerevisiae YS58 genomic DNA with primer pair ADH1-F/ADH1-R, and then inserted into the EcoRI and BamHI sites of pYCPG to generate plasmid pYCPGA1. For overexpression of CAR1 or ARG4, the DNA fragment covering the coding region and its terminator region of CAR1 or ARG4 (Accession number {"type":"entrez-nucleotide","attrs":{"text":"K01813","term_id":"171078","term_text":"K01813"}}K01813) was amplified from S. cerevisiae YS58 genomic DNA with primer pair CAR1-F2/CAR1-R2 or ARG4-F1/ARG4-R1. The amplified DNA fragments were digested by BamHI/SalI, and then inserted into the corresponding sites of pYCPGA1 to generate plasmid pYGAC1 or pYGAA4 respectively, in which the expression of CAR1 or ARG4 was driven by ADH1 promoter. Plasmid pYGAC1 or pYGAA4 was introduced respectively into S. cerevisiae YS58 to generate recombinant strain YS58-ARG4 or YS58-CAR1. As a control, S. cerevisiae YS58 was transformed with vector pYCPGA1 to generate control strain YS58-V. All PCR products were verified by DNA sequencing. All yeast strains were confirmed by G418 resistance and PCR analysis. The genetic stability of yeast strains was analyzed as described previously⁴².