Construction of plasmids and strains

Standard protocols of molecular cloning, such as PCR, DNA restriction, and ligation [30] were carried out for recombinant DNA work. Techniques specific for C. glutamicum, e.g., electroporation for transformation of strains, were performed as described previously [31]. All enzymes were obtained from ThermoScientific (Schwerte, Germany). Codon-optimized synthetic genes for C. glutamicum ATCC 13032 were obtained from LifeTechnologies (Darmstadt, Germany). Genes were amplified by PCR using primers containing unique restriction sites (Table ​(Table2).2). PCR products were then used for cloning of genes into plasmid vectors using the introduced restrictions site. For the assembly of multiple genes and for elimination of undesired internal restriction sites Electra Cloning was used [32]. This cloning strategy is based on the class IIS restriction enzyme SapI, generating 5′-overhangs, which can be freely designed. In-frame gene deletions and nucleotide substitutions in the genome of C. glutamicum were performed using the pK19mobsacB system [33] by a two-step homologous recombination method described previously [34]. All constructed plasmids were finally verified by DNA sequencing at Eurofins Genomics (Ebersberg, Germany).

Oligonucleotides used in this study

Restriction sites are underlined; SapI cuts outside of its recognition site, the obtained 5′-overhangs after SapI cleavage used for Electra Cloning are shown in bold. In case of native tkt two separate PCR fragments were assembled using Electra Cloning to eliminate an internal SapI restriction site