Supplementary Methods

Fragments for thrBCdisruption

DNA fragments containing upstream and downsteam region of thrBC genes were amplified from MG1655 genome using the primer pairs thrAdn_OL /thrAF_isceI_sac and thrCdn_IsceI_psti/thrCF_OL. The fragments were joined by over-lap PCR with an EcoRV site separating them added by the primers. Then the joined fragment was cloned into pMD19-T simple. The plasmid obtained was named pMD19-thrBCdel.

A1.1 kb PCR fragment from pMDICI using primers mdF/mdR containing chloroamphenicol resistancegene flanked by two I-SceI recognition sites was blunt cloned into pMD19-thrBCdel at EcorV site, to yield pMD19-thrBC-Cm.

A 1.7 kb PCR fragment was amplified from pMD19-thrBC-Cm, using the primers pair M13F / M13R. This fragment was transformed into the recipient strain by electroporation

Donor plasmid with three fragments.

Donor fragments for cadA deletion and gdhA integration were amplifiedusing the primer pair cadAF_ol/cadAR_sacspe and gnttF_kpnXho/gntTR_OL and joined by over-lap PCR. Then the joined PCR fragment was subcloned into pKSI-1 at PstI/XhoI sites, yielding pKSIcgg.

0.7kb SacI/PstI fragment from pMD19-thrBCdel for thrBC deletion was then subcloned into pKSIcgg, yielding pKSIcggt. The final resulting plasmid pKSIcggt were used as the donor plasmid used for marker recycling and genetic modification. (Fugure 2A)

Marker recycling and modification

To regenerate the marker, donor plasmid was transformed into an intermediate strain with helper plasmid pREDTKI.*

Several resulting colonies were first incubated in LB liquid medium with 0.5% glucose. Forty microliters of overnight seed culture were inoculated in a test tubecontaining 4 mL LB medium with 50μg/mL kanamycin and10mmol/L IPTG andthe test tube was cultivated at 30 ºC and 200 rpm for ~2 h. IPTG was added to the mixture to a final concentration of 20mmol/L and the test tube was cultivated for 6~8 h or overnight. Forty microliters of culture were inoculated into another test tube containing 4 mL LB medium with 20 mM IPTG, 10mM L-arabinose and 50μg/mL kanamycin. The test tube was cultivated for 6~8 h or overnight. The culture was harvested, suspended in sterile water, diluted and spreadon LB plates supplemented with 20mmol/L IPTG, 10mmol/L L-arabinose and 50μg/mL kanamycin. Colonies appeared after overnight incubation at 30 ºC. Then the colonies were analyzed for sensitivephenotype, and the final clones were confirmed by PCR.