Figure Captions
Fig. 1. Construction of the vector for CRISPR/Cpf1 genome editing inC. glutamicum . (A) pJYS3_Amp_MCS vector derived from pJYS3_ ΔcrtYF was constructed. (B) A double guide crRNAs set was incorporated into the pJYS3_Amp_MCS plasmid, between the HindIII and Xba1 restriction enzyme sites. (C, D) Homologous arms with a selection marker gene (Knr, kanamycin resistance gene) and co-expression cassette (Psod:sucE-Pro4:rpsLm) were finally inserted into the JYS3_Amp_DT plasmid between the Xma1 and Apa1 restriction enzyme sites. T1 and T2, target DNA sites on the ldhA gene of C. glutamicum ; Pro1, AmpR promoter; Pro2, PlacM promoter; Pro3, J23119 promoter; Pro4, Knr promoter derived from pJYS3_ ΔcrtYF; Psod, promoter; rmB T1 term and sacB T1 term, terminator regions of the rmB and sacB genes, respectively; sT1, sacB T1 terminator; LdhAp, lactate dehydrogenase 1 (ldhA ) promoter region; ldhAt, terminator region of ldhA gene.
Fig. 2. CRISPR/Cpf1 mediated homologous recombination in C. glutamicum . (A) The double target sites were located 243 bp and 683 bp from the start codon of the ldhA gene, respectively. The first homologous recombination occurred on the LdhAp region at the first heat shock, and the second crossing-over on the LdhAt region occurred after the second heat shock. (B) The deletion of the ldhA gene and simultaneous Knr gene insertion were confirmed via PCR analysis (PCR 1 and 2). (C) Confirmation of the complete inhibition of lactic acid production in the ldhA gene deletion mutant (ΔldhA-6 ). Glucose was used as the sole carbon source.
Fig. 3. Synergistic effect of cellulase with xylanase and β-glucosidase. (A) HPAC-pretreated pine with cellulase cocktail solution alone and (B) supplement with xylanases (Xyl) and β-glucosidases (Bgls) were conducted in 1 mL citrate buffer. The incorporation of auxiliary enzymes remarkably increased the hydrolysis rate of the 2% substrate and decreased the dose of cellulase required for optimal hydrolysis (5–25 FPU). The structural recalcitrance of cellulose fiber did not substantially affect the enzymatic hydrolysis of HPAC-pretreated pine.
Fig. 4. Fermentation of the ΔldhA-6 mutant with the hydrolysates of HPAC-pretreated pine. The metabolites (succinic acid, lactic acid, and acetic acid) produced by fermentation of the 1% (A), 2% (B), 3% (C), 4% (D), and 5% hydrolysate (E) were analyzed. The cell concentrations were 10.15 (A), 15.72 (B), 16.08 (C), 21.19 (D), and 21.08 (E) g L-1 CDW (cell dried weight) for fermentation of the hydrolysates, respectively.
Fig. 5. Productivity of succinic acid from the hydrolysate of HPAC-pretreated pine. Succinic acid productivity involving in glucose consumption rate (A) and cell concentration (B) were analyzed over 9 h. SA, succinic acid (g L-1); Gc, glucose consumption rate; h, hour; Cw, cell dried weight (g L-1)
Fig. 6. Succinic acid production correlated to cell concentration. The hydrolysates were fermented with various ranges of cell densities ofΔldhA-6 mutant for 6 h, and succinic acid (A), glucose consumption rate (B), and xylose consumption rate (C) were analyzed.
Fig. 7. Over-expression of succinic acid transporter (sucE ) gene and succinic acid production in fed-batch system. (A, B) The gene was inserted onto the genomic DNA of ΔldhA-6 mutant under Psod promoter regulation through the CRISPR/cpf1 gene editing system. (C) The enhancement of succinic acid production was demonstrated in [Psod:sucE- ΔldhA ] transformant (10.00 g CDW) compared toΔldhA mutant (10.94 g CDW) when using 4% hydrolysate. (D) Comparison of the succinic acid production depending on the concentration of hydrolysates was performed with 28~30 g L-1 CDW of [Psod:sucE-ΔldhA ] transformant. (E) A fed-batch system was carried out with an initial concentration of 4% hydrolysate and 30.37 g L-1 CDW of [Psod:sucE-ΔldhA ] transformant. After 24 h of fermentation, 20 mL of 20% pine hydrolysate was added to the reaction solution, resulting in a final concentration of the 4% hydrolysate. Acetic acid was prone to be released during the first 9 h after feeding, while lactic acid was measured at the later stage of the fermentation. (F) The same volume of the 20% hydrolysate was added at 6, 9, and 24 h. P1, Psod promoter; sucE , succinic acid transporter; T1, sacB terminator; P2, Knr promoter; rpsL m, ribosomal S12 protein mutant gene for streptomycin resistant; arrows, feeding.