Plasmid design and construction
All plasmids used in this study are compiled in Table 1 and illustrated in Figure 1 , whereas all primers are listed inSupplementary Table S1 . Codon-optimized L-LDH fromLactobacillus casei (GenBank accession number MF582630.1),LcLLDH , was selected as an LDH gene source. DNA fragments were assembled via the NEBuilder HiFi DNA Assembly method (NEB, County Road Ipswich, MA) and transformed into E. coli JM109 (recA1endA1gyrA96thi -1 ,hsdR17 (rK - mK +),e14 - (mcrA -), supE44relA1 , D(lac-proAB )/F’ [traD36proAB +lac IqlacZ D M15]). Plasmid-harboring transformants were cultivated overnight (37 °C, 190 rpm) using LB medium supplemented with 0.1 g∙L-1 of ampicillin (Sigma-Aldrich, USA) as a selection marker. Plasmids were extracted using a LaboPassTM Plasmid Mini kit (Cosmo Genetech, Seoul, Korea).
Introduction of the LDH gene into S. cerevisiae genome using a genome-integrated plasmid yields a higher accumulation of lactic acid than using an episomal type plasmid (Ishida et al., 2005). Therefore, the former plasmid type was employed to construct all engineered strains in this experiment. For the simultaneous expression of the LDH gene and disruption of PDC genes, partial coding sequences of PDC1 /PDC5 genes were cloned into plasmids containing an LDH expression cassette. The sequence ofLDH was fused with the TDH3 promoter and terminator sequences at both its upstream and downstream regions, respectively, to construct constitutively expressed LDH systems: pAUR101-TDH3pro-LcLLDH-dPDC1 and pAUR101-TDH3pro-LcLLDH-dPDC5. After the integration of the respective gene-expressing cassettes into the genome, there can be a possibility that both promoters control the expression of the LDH gene. The orientation ofPTDH3 -LcLLDH -TTDH3was flipped to avoid that effect, as shown in Figure 1(B) . Moreover, pAUR101-BTCC3PDC1-LcLLDH has a sequence of LDH gene integrated between the promoter and terminator of PDC1 gene cloned from the genome, and we intended to utilize this native glucose-dependent promoter. In contrast, pP01-BTCC3PDC5KO contains only the partial coding sequence of PDC5 and KanMx , a marker gene, to distort the expression of the corresponding gene without the introduction of the LDH gene.