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.