Construction of plasmids and strains

The basic DNA manipulation and strain construction was operated according to the standard molecular cloning manual.E. coli DH5α was used as the host of gene cloning. All the primers used in this study are presented in Table S1 (Supplementary Information). The suicide vector pK18mobsacB containing the sucrose lethal gene sacB was used to delete or integrate gene on chromosome.
The xylAB gene clusters from E. coli and X. campestris were amplified by primers EAB-F/EAB-R and XAB-F/XAB-R respectively, the inserted restriction sites areHind III/EcoR I and Hind III/Sac I of pXMJ19. The plasmids pXMJ19, p19Plac-EcoAB, and p19Plac-XcaAB were transformed into C. glutamicumSO26 to produce strains C. glutamicum pX、EAB、XAB. AraEfrom B. subtilis with a constitutive Peftupromoter were amplified by araE-F/araE-R and up-eftu-F/araE-eftu-R respectively. The product of fusion PCR was inserted into theEcoR V site of plasmid pk18-△iolR by Gibson assembly, and then generated the recombinant plasmid pk18- Peftu-△iolR::araE and corresponding strain XAB01.
To increase PEPC expression by lowing the acylation of PEPC, a synthesized strong promoter H36 was inserted in the upstream ofpepc gene (Yim, An, Kang, Lee, & Jeong, 2013), and then the AAG bases from 1957 bp to 1959 bp were mutated to CGC by overlap PCR, the primer up-HA-F1 / up-HA-H36-R2 amplified pepc upstream homology arm sequence, primer up-HA-H36-F3 / pepc-H36-R4 amplified H36 promoter, primer mutant-(KR)-F/down-HA-pepc-R8 realizes the base mutation of K653R, pepc-H36-F5/mutant-(KR)-R amplifies the pepc upstream homology arm sequence, primer down-HA-pepc-F9/down-HA-R10 amplified the homology arm sequence downstream of pepc . Finally, the entire fragment was combined by Gibson assembly under the primer up-HA-F1/down-HA-R10, resulted the recombinant plasmid pk18-PH36-pepc T1. Furthermore, to reduce the feedback inhibition of aspartic acid on PEPC, the same operation has been done at 895-897 bp AAC to replace GAT, amplification using pepc-H36-F5/D299N-R6 and D299N-F7/down-HA-pepc-R8 with pk18-PH36-pepc T1 as a template can realize base mutation of D299N, forming the pk18-PH36 -pepc T2, resulted in the strainC. glutamicum XAB03. Primers pXMJ19-F/pXMJ19-R and M13 fwd/M13 rev are verification primers for pXMJ19 and pK18mobsacB , respectively.
All the recombinant plasmids were constructed in E. coli DH5α and transformed into C. glutamicum by electroporation. The mutant strains were screened through two rounds of homologous recombination, and further confirmed by colony PCR and sequencing.