Article Figures & Data
Figures
- FIG 1
A. succinogenes metabolism and genetic modifications. Gray arrows indicate native flux from pentose and hexose sugars. Green arrows indicate overexpression targets. Red arrows indicate genetic knockout targets. 13DPG, 3-phospho-d-glyceroyl phosphate; 2PG, d-glycerate 2-phosphate; 3PG, 3-phospho-d-glycerate; 6PGC, 6-phospho-d-gluconate; AC, acetate; ACALD, acetaldehyde; ACCOA, acetyl-CoA; ACTP, acetyl phosphate; DHAP, dihydroxyacetone phosphate; E4P, d-erythrose 4-phosphate; ETOH, ethanol; F6P, d-fructose 6-phosphate; FDP, d-fructose 1,6-bisphosphate; FOR, formate; FUM, fumarate; G3P, glyceraldehyde 3-phosphate; G6P, d-glucose 6-phosphate; GLC, d-glucose; MAL, l-malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; R5P, alpha-d-ribose 5-phosphate; RU5P, d-ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; SUCC, succinate; XU5P, d-xylulose 5-phosphate; XYL, d-xylose; XU, d-xylulose; LAC, d-lactate; pflB, pyruvate formate lyase; ackA, acetate kinase; PCK, phosphoenolpyruvate carboxykinase; MDH, malate dehydrogenase; FUM, fumarase.
- FIG 2
Example of a knockout cassette, ΔpflB (top) and plasmid vector for the overexpression of SA pathway genes (bottom). A.s. Ori frag, DNA fragment required for replication in Actinobacillus succinogenes.
- FIG 3
Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) compared to those of the knockout strains ΔackA, ΔpflB, and ΔpflB ΔackA. Scatter plots present bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production: SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H). The bar graphs show SA titers (I), yields and metabolic yields (J), and overall and maximum instantaneous productivities (K). SA “yield” is the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in all cases (since sugars were mostly consumed at that time). The maximum instantaneous productivity was observed at 12 h for the control and 48 h for the knockout strains.
- FIG 4
Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) and overexpression constructs such as the single-gene overexpression strains pFUM, pPCK, and pMDH and the triple-gene overexpression strain pPFM. The scatter plots show bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production, SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H), for the wild-type, pMDH, and pPFM strains. The scatter plots corresponding to pMDH and pPFM strains are shown in Fig. S1 in the supplemental material. The bar graphs show (I) SA titers, (J) yields and metabolic yields, and (K) overall and maximum instantaneous productivities for the five strains. SA “yield” is calculated as the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield but considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in wild type, pMDH, and pPFM and at 72 h (and marked with an asterisk) in pFUM and pPCK. The maximum instantaneous productivity was observed at 12 h for the control, 25 h for pMDH and pPFM, 21 h for pFUM, and 24 h for pPCK.
- FIG 5
Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) compared to those of the ΔackA, ΔpflB, and ΔpflB ΔackA strains carrying pPMF. The scatter plots show bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production: SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H). The bar graphs show SA titers (I), yields and metabolic yields (J), and overall and maximum instantaneous productivities (K). SA “yield” is calculated as the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield but considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in the wild-type strain and at 144 h in the rest of the strains. The maximum instantaneous productivity was observed at 12 h for the control, ∼72 h for the ΔackA and ΔpflB strains carrying pPMF, and 98 h for the ΔpflB ΔackA strain carrying pPMF.
Tables
- TABLE 1
Bacterial strains and plasmids
Strain or plasmid Description Reference or source Strains Escherichia coli DH5-α F− Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK− mK−) phoA supE44 λ−thi-1 gyrA96 relA1 Invitrogen Actinobacillus succinogenes 130Z Wild type ΔpflB::bla 130Z derivative; contains loxP-bla-lox loxP integration replacing pflB; Ampr This study ΔpflB ΔpflB::bla derivative; contains one loxP site This study ΔackA 130Z derivative; contains ackA knockout This study ΔpflBΔackA 130Z derivative; contains pflB and ackA knockouts This study Plasmids pLGZ920 E. coli-A. succinogenes shuttle vector; Ampr; contains PpckA 22 pL920Cm pLGZ920 derivative; Cmr; Cmr gene under the control of Pbla This study pPCK pLGZ920 derivative; Ampr; pckA under the control of PpckA This study pMDH pLGZ920 derivative; Ampr; mdh under the control of Pmdh This study pFUM pLGZ920 derivative; Ampr; fum under the control of PpckA This study pPMF pLGZ920 derivative; Cmr; pckA under the control of PpckA; mdh under the control of Pmdh; fum under the control of Pfum This study pLCreCm pLGZ920 derivative; Cmr; cre under the control of PpckA This study - TABLE 2
Primers used in this study
Primer Sequence (5′ to 3′) Usage YC-1 CGTTAACCGTGGGAATCAGTTTGTTAGGAATG pflB up fragment; Gibson Assembly YC-2 CGAAGTTATTGTAATACTTCCTTTTGCTAGTATTGATAATGAAATCCTGTAAG pflB up fragment; Gibson Assembly YC-3 CCATAACTTCGTATAATGTATGCTATACGAAGTTATTTGGGGTAACGTAATAAAAATG pflB down fragment; Gibson Assembly YC-4 TCTCTCCTTCGCGGAATAAAATATCCACTTC pflB down fragment; Gibson Assembly YC-5 CTAGCAAAAGGAAGTATTACAATAACTTCGTATAATGTATGCTATACGAAGTTATAATTCTTGAAGACGAAAGGGCCTCGTG bla-loxP fragment; Gibson Assembly YC-6 CGTATAGCATACATTATACGAAGTTATGGGGTCTGACGCTCAGTGGAACGAAAACTC bla-loxP fragment; Gibson Assembly YC-7 GCAGCAATAGAGGAAACACGGTTTG pckA fragment YC-8 GGATTTGGTACCGTGCCGGCGGCCTAATAACCTG pckA fragment, KpnI site underlined YC-9 CGAACCGAAGCGTTCCTGCGCGAGTAACGC mdh fragment; blunt-end ligation YC-10 GCTTCCCATTAATCAAACGGCGG mdh fragment; blunt-end ligation YC-11 CTCAAACAAACCGTGTTTCCTCTATTGCTGC pLGZ920 to linearize for mdh cloning YC-12 AATTATCAATGAGGTGAAGTATGACATTTCGTATTGAAAAAGACAC fum CDS fragment; Gibson Assembly YC-13 GAATTCGAGCTCGGTACCCGGGGATCCCTGACCGTCTTCGGTGAATACTGATATAG fum CDS fragment; Gibson Assembly YC-14 ACTTCACCTCATTGATAATTTAAAATTAAAAATCC pLGZ920 to linearize for fum cloning YC-15 GGATCCCCGGGTACCGAGCTCGAATTCACTG pLGZ920 to linearize for mdh or fum cloning YC-16 CGGTACCGAGCTCGAATTCACTGGCCGTCG pPCK to linearize for mdh and fum cloning YC-17 GTCATCTTAACAGGTTATTAGGCCGCCGGCA pPCK to linearize for mdh and fum cloning YC-18 CCTTCGGCCGGCCCTGCCGTTTCGGAAAACTCACGCTTTACCCG fum fragment; FseI site underlined YC-19 CCTTCGGCCGGCCCATAAAGAATCCAAGATAAACGAATTGGC fum fragment; FseI site underlined
Supplemental material
- Supplemental file 1 -
Evaluation of different fermentation and metabolic parameters (Fig. S1 and S2), t test data (Table S1), details of the carbon balance calculations (Table S2), and details of the degree of reduction balance (Table S3).
PDF, 204K
- Supplemental file 1 -
