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.