Construction of Knockout Mutants

In E. coli, the galR, galS, zwf, and pfkA genes were knocked-out by the modified one-step inactivation method (Song and Lee, 2013). First, the linear DNA fragments for gene knockout were prepared by two-step polymerase chain reaction (PCR; MiniAmp^TM Thermal Cycler, Thermo Fisher, Singapore), using template plasmid pMtrc9 (Kim et al., 2008; Nogrado et al., 2019) containing the lox66-cat-lox71 cassette. For example, to construct the linear DNA fragments for knockout of the galR gene, primers galR-KO-F1, galR-KO-R1, galR-KO-F2, and gal-KO-R2 were used (Supplementary Table 1). In the first PCR with primers galR-KO-F1 and galR-KO-R1, using pMtrc9 as a template, the 50-bp homologous arm sequences of the target gene were franked into the PCR products. In the next PCR with primers galR-KO-F2 and galR-KO-R2, additional 50-bp homologous extension were generated. Thus, the resulting PCR fragments contained 100-bp homologous arm sequences matched to the upstream and downstream regions of the galR gene. The other PCR fragments for knockout of the galS, zwf, and pfkA genes were also prepared by the same methods using the corresponding primers (Supplementary Table 1).

Next, E. coli harboring plasmid pCW611 (Song and Lee, 2013) was prepared by transformation of the plasmid into the target strain. Then, E. coli competent cells harboring plasmid pCW611 were prepared. For the preparation of competent cells, a colony was inoculated into LB medium supplemented with Ap, and cultured for 12 h at 30°C. One milliliter of cell cultures was transferred into 100 mL LB medium supplemented with Ap and 10 mM of arabinose (Sigma, USA) for induction of λ-red recombinase, and incubated at 30°C. After that, the electro-competent cells were prepared by the previous methods (Sambrook et al., 1989).

In final, the linear PCR fragments containing 100-bp homologous arm for the galR gene knockout were transformed into the competent E. coli cells by electroporation (Sambrook et al., 1989). Cells were then incubated at 30°C for 1 h, and spread on LB agar plate containing Cm. Recombinants were screened by colony PCR using primers galR-KO-F2, and galR-KO-R2 (Supplementary Table 1). Other primers used in this study were listed in Supplementary Table 1. The confirmed colonies were cultured on LB agar plate containing Cm and Ap. For the pop-out of the cat gene from chromosome, then, the colony was suspended in 1 mL LB broth, and spreaded on LB agar plate containing Ap together with IPTG for induction of Cre recombinase. The recombinants generated by pop-out recombination were screened by colony PCR using primers galR-KO-F1, and galR-KO-R1 (Supplementary Table 1). Then, plasmid pCW611 was cured by incubation at 42°C in the final E. coli strains.