Metabolic engineering of Saccharomyces cerevisiae to improve succinic acid production based on metabolic profiling

Figures & data

Table 1. Microbial strains used in this study.

Strain	Genotypes	Source or reference
S. cerevisiae
BY4739	MATα leu2Δ0 lysΔ0 ura3Δ0	Open Biosystems, Inc.
S149	MATα leu2Δ0 lys2Δ0 ura3Δ0 adh1Δ::loxP adh2Δ::loxP adh3Δ::loxP adh4Δ::loxP adh5Δ::loxP	Ida et al.^50^)
BY4739sdh12	MATα leu2Δ0 lys2Δ0 ura3Δ0 SDH1::loxP-KlURA3-loxP SDH2::loxP-KlLEU2-loxP	This study
S149sdh12	MATα leu2Δ0 lys2Δ0 ura3Δ0 adh1Δ::loxP adh2Δ::loxP adh3Δ::loxP adh4Δ::loxP adh5Δ::loxP SDH1::loxP-KlURA3-loxP SDH2::loxP-KlLEU2-loxP	This study
E. coli
DH5α	F^– Φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK^–, mK^+) phoA supE44 λ^–thi-1 gyrA96 relA1	Takara Bio, Inc.

Fig. 1. Succinic acid production in the SDH1- and SDH2-disrupted S. cerevisiae strains.

Note: (A) Cell growth, (B) glucose concentration, (C) succinic acid concentration, (D) ethanol concentration, (E) glycerol concentration, and (F) fumaric acid concentration in the BY4739 (hollow circles) and BY4739sdh12 (solid circles) strains are shown. Average ± standard deviation for three independent culture experiments is shown.

Table 2. Production yields in succinic acid-producing S. cerevisiae recombinant strains.

Product	C-mol-based yield (%)
	BY4739	BY4739sdh12	S149	S149sdh12
Succinic acid	0	1.45 ± 0.11	0	2.17 ± 0.32
Ethanol	51.0 ± 1.0	51.4 ± 1.1	15.0 ± 0.5	13.2 ± 2.6
Glycerol	5.44 ± 0.32	4.05 ± 0.22	40.4 ± 10.6	22.9 ± 7.0
Fumaric acid	0.72 ± 0.08	0.066 ± 0.003	0.35 ± 0.01	0.075 ± 0.007

Note: Average ± standard deviation for three independent culture experiments is shown. For the BY4739 and BY4739sdh12 strains, the yield at 48 h is shown. For the S149 and S149sdh12 strains, the yield at 72 h is shown.

Fig. 2. Succinic acid production in ADH1–5-disrupted strain S149 and its SDH1- and SDH2-disrupted strain S149sdh12.

Note: (A) Cell growth, (B) glucose concentration, (C) succinic acid concentration, (D) ethanol concentration, (E) glycerol concentration, and (F) fumaric acid concentration in the S149 (hollow triangles) and S149sdh12 (solid triangles) strains are shown. Average ± standard deviation for three independent culture experiments is shown.

Fig. 3. Metabolic profiles of the S149 and S149sdh12 strains as analyzed by CE-TOFMS.

Note: The intracellular metabolite level (Y-axis, μmol g of dry cell⁻¹) in the S149 (left, white bar) and S149sdh12 (right, gray bar) strains is shown. Average ± standard deviation for three independent culture experiments is shown. Asterisks indicate that the intracellular content was significantly different between the S149 and S149sdh12 strains. Glc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; AcAld, acetoaldehyde, EtOH, ethanol; PGL, phospho-glucono-1,5-lactone; 6PG, 6-phosphoglycerate; Ru5P, ribulose-5-phosphate; Xu5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Cit, citrate; cis-Aco, cis-aconitate; IsoCit, isocitrate; 2OG, 2-oxoglutarate; SucCoA, succinyl-CoA; Suc, succinate; Fum, fumarate; Mal, malate; OAA, oxaloacetate.

Fig. 4. Improvement of succinic acid production in the S149sdh12 strain by expression of the S. pombe mae1 gene.

Note: (A) Cell growth, (B) glucose concentration, (C) succinic acid concentration in the S149sdh12/pNV11 (hollow squares) and S149sdh12/pNV11-mae1 (solid squares) strains are shown. Intracellular succinic acid levels in the S149sdh12/pNV11 (white bar) and S149sdh12/pNV11-mae1 (gray bar) strains are shown (D). Average ± standard deviation for three independent culture experiments is shown.

Table 3. Production yields in succinic acid-producing S. cerevisiae strains expressing the S. pombe mae1 gene.

	C-mol-based yield (%)
	S149sdh12/pNV11	S149sdh12/pNV11-mae1
Succinic acid	1.52 ± 0.06	2.36 ± 0.06
Ethanol	15.9 ± 0.5	15.9 ± 1.0
Glycerol	25.5 ± 0.3	20.0 ± 1.1
Fumaric acid	0.066 ± 0.004	0.16 ± 0.00

Note: Average ± standard deviation at 72 h for three independent culture experiments.

Ida Y, Furusawa C, Hirasawa T, Shimizu H. J. Biosci. Bioeng. 2012;113:192–195.

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