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.