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.