2.1 Construction of CRISPR-cpf1 vector for genome editing
The pJYS3_ ΔcrtYF plasmid for genome editing with CRISPR-cpf1 was
purchased from Addgene () (Jiang et al.,
2017).[15] The plasmid was modified to obtain the
pJYS3_Amp_MCS vector using the primers noted in additional file 2:
Table S1 to substitute kanamycin in over the ampicillin selection marker
genes for the sub-cloning selection in E. coli (Fig. 1). A double
target DNA system was constructed with the target DNA 1 primer set
(crRNA + 24 bp target DNA + rmB T1 terminator) and target DNA 2 primer
set (J23119 promoter + crRNA + 24 bp target DNA + sacB1 terminator). The
pJYS3_Amp_DT vector was obtained by sequentially inserting the target
DNA 1 and 2 primer sets at the HindIII-BamH1 and Xba1-Apa1 restriction
sites in the pJYS3_Amp_MCS1 vector (Fig. 1B). The DNA fragments of the
lactate dehydrogenase 1 promoter (LdhAp) and terminator (LdhAt) were
obtained from the genomic DNA of C. glutamicum with the
sense/anti-sense primer pairs of LdhAp and LdhAt,
respectively.[31] The genomic DNA isolation and
PCR procedures are described in section 2.3. The fragments were
sequentially inserted into the pCold 1 vector (Cat. No. 3362, TaKaRa,
Japan) at the Sac1-Kpn and HindIII-Xba1 sites for LdhAp and LdhAt as
homologues arms, respectively. The kanamycin gene from pJYS3_ ΔcrtYF
was amplified and inserted into the Kpn1-Xho1 restriction site between
LdhAp and LdhAt in the vector. The
[LdhAp]-[Knr]-[LdhAt] construct was
amplified with the LdhAp-sense/LdhAt-anti-sense primers pairs, and
inserted into the pJYS3_Amp_DT vector at the Xma1-Apa1 site. Finally,
the pJYS3_Amp_DT_[LdhAp-Knr-LdhAt] vector was
constructed.
Over-expression of succinic acid transporter gene onto ΔldhAmutant was conducted to improve succinic acid production. The genomic
DNA of C. glutamicum was extracted following the procedure
outlined in section 2.3. The gene encoding the succinic acid transporter
(sucE) and the ribosomal S12 protein gene (rpsL ) were
subsequently isolated and subcloned into the region between the
homologous arms on the pCold vector (Fig. 1C). To obtain streptomycin
resistance, the rpsL gene was mutated by substituting AAG
(Lys43) to AGG (Glu).[32] The
variant was denoted to rpsLm in this study. The construction
[LdhAp]-[Psod:sucE]-[Pro4:rpsLm ]-[LdhAt] on pCold
vector was amplified and inserted into the pJYS3_Amp_DT vector (Fig.
1D), and transformed into ΔldhA knock-out mutant of C. glutamiumfollowing the procedure outlined in section 2.2−2.3.