Fig. 3.

DBTL cycles of threonine pathway engineering through the PS-Brick scheme. a The metabolic pathway for threonine synthesis from glucose. b Three rounds of DBTL cycle of metabolic engineering were performed step by step through four iterative rounds of PS-Brick assembly from pOthr vector. The most efficient mutant of feedback-resistant ThrA was screened through the first round of PS-Brick. The bottleneck gene for threonine synthesis was identified and overexpressed through the second round of PS-Brick. Four threonine exporters under the control of the same promoter and RBS were prioritized through the third and fourth rounds of PS-Brick. PR indicates the native promoter and RBS, and T indicates the native terminator. c The threonine production with 20 ThrA mutants. The assembled plasmids harbouring the thrABC operon containing the saturation mutated thrA gene (pACYC184-thrA⁴³³BC) were transformed into the E. coli K12 MG1655 strain, respectively. The threonine titers at the 12 h of shake flask fermentation were measured. *Denotes the wild-type control. d Effects of the ppc, aspA, aspC, asd and pntAB genes overexpression on the threonine production. The assembled plasmids pACYC184-thrA⁴³³ BC-asd/ppc/aspA/aspC/pntAB were transformed into the E. coli K12 MG1655 strain, respectively. The threonine titres at the 12 h of shake flask fermentation were measured. e Effects of overexpression of the four exporters on the threonine production. The assembled plasmids pACYC184-thrA⁴³³BC-asd-P_TBCD1- rhtA/rhtB/rhtC/yeaS were transformed into the E. coli K12 MG1655 strain, respectively. The threonine titres at the 12 h of shake flask fermentation were measured. Prioritization of isoenzymes for threonine efflux. Promoter (P_T) and RBS (BCD1) was inserted in front of start codon ATG of the four exporter gene. The relative concentration was calculated by dividing the measured concentration of threonine produced with the engineered strains by that with control strain in the same batch of flask fermentation. Data shown are mean values from three biological replicates, and the standard deviations are presented