Plasmid and strain construction
A pAUR101-based plasmid containing partial sequences of the CYB2gene of S. cerevisiae (for genome integration), a geneticin resistance gene (for marker) and a codon-optimized L-LDH gene from Lactobacillus casei (accession no. MF582630.1) under the control of the TDH3 promoter (for lactate dehydrogenase expression) was constructed (Figure S1a) and transformed into E. coli K-12 (NovaBlue Competent Cells, Merck, Japan), which was subsequently grown on ampicillin-supplemented LB agar. The obtained transformants were cultivated using 5 mL LB medium, and the plasmid was extracted from the cells using a LaboPassTM Plasmid Mini kit (Cosmo Genetech, South Korea). The fragment of interest was amplified by PCR using 5-CYB2.FOR (5’-ATGCTAAAATACAAACCTTTACTAAAAATCTCGAAGAAC-3’) and 3-CYB2.REV (5’-TCATGCATCCTCAAATTCTGTTAAAGTAGGT-3’) as primers and transformed into yeast cells following a LiOAc/single-stranded carrier-DNA/PEG method (Gietz et al., 2002). Transformants were obtained after incubating the cells on YPD agar containing 100 mg·L−1 geneticin at 30 °C overnight.