Nebuilder hifi dna assembly method

All plasmids and oligonucleotide primers used in this study are listed in Supplementary Tables 1 and 2, respectively. Plasmids were assembled using either the USER cloning method (Bitinaite et al., 2007 (link)), NEBuilder HiFi DNA assembly method (New England Biolabs) or restriction enzyme-based cloning techniques (Sambrook et al., 1989 ). Plasmid DNA preparation was carried out using the QIAprep® Spin Miniprep Kit (Qiagen). Gel purified linearized DNA was extracted using the QIAquick® Gel Extraction Kit (Qiagen). Genomic DNA was isolated with the GenElute™ Bacterial Kit (Sigma-Aldrich). All restriction endonucleases, T4 DNA ligase and NEBuilder® HiFi DNA Assembly Master Mix were acquired from New England Biolabs. DNA sequences were verified by Sanger sequencing (Eurofins Genomics). A detailed assembly description for each plasmid is provided in the Supplementary information.

A codon-optimized L-LDH from Lactobacillus casei (GenBank accession number MF582630.1) was selected as the LDH gene source. All DNA fragments were assembled by following the NEBuilder HiFi DNA Assembly method (NEB, USA) and transformed into E. coli JM109. Plasmid-harboring transformants were cultivated overnight (35 °C, 200 rpm) using LB medium supplemented with 0.1 g L⁻¹ of ampicillin as a selection marker. Plasmids were extracted using a LaboPass™ Plasmid Mini kit (Cosmo Genetech, Korea). Digested plasmids were transformed into a wild-type strain by following a LiAc/single-stranded carrier DNA/PEG method^{46 (link)}. Each transformant was selected on a YPD agar containing selection marker(s).