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Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

Radoš D, Carvalho AL, Wieschalka S, Neves AR, Blombach B, Eikmanns BJ, Santos H - Microb. Cell Fact. (2015)

Bottom Line: Productivity was maximized by manipulating the aeration rate in the production phase.We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production.The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. rados@itqb.unl.pt.

ABSTRACT

Background: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions.

Results: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1).

Conclusions: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

No MeSH data available.


Related in: MedlinePlus

A summary of the stepwise systematic approach used to engineering C. glutamicum for the production of 2,3-BD. The values reflect the impact of the several steps on yield and productivity. Blue boxes refer to strain optimization steps, while the yellow box indicates the process optimization step. 2,3-BD 2,3-butanediol, Glc glucose
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Fig5: A summary of the stepwise systematic approach used to engineering C. glutamicum for the production of 2,3-BD. The values reflect the impact of the several steps on yield and productivity. Blue boxes refer to strain optimization steps, while the yellow box indicates the process optimization step. 2,3-BD 2,3-butanediol, Glc glucose

Mentions: Cell suspensions of C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) and ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) under constant air flow of 5 mL min−1 showed an increased GCR [7.2 ± 0.6 and 11.1 ± 1.0 nmol min−1 mg CDW−1 in mini-fermenter for the two respective strains (Table 4), compared to 6.1 ± 0.1 and 6.5 ± 0.5 nmol min−1 mg CDW−1 in flasks (Table 3)]; the productivity in mini-fermenter was 3.4 ± 0.3 and 5.5 ± 0.7 nmol min−1 mg CDW−1 compared to 3.1 ± 0.2 and 4.3 ± 0.4 nmol min−1 mg CDW−1 in flasks, but the yield was higher for fermentations in closed flasks (Tables 3, 4). Upon doubling of the air flow to 10 mL min−1, GCR and the productivity of C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) and ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) increased two-fold; both strains produced 2,3-BD at highest yields (0.57 ± 0.03 and 0.66 ± 0.01 mol 2,3-BD per mol glucose). Interestingly, further increase in the flow rate to 20 mL min−1 resulted in significantly lower yields and productivities (Table 4). Ethanol was produced by both strains in experiments using 5 mL min−1 air, and glycerol was absent only in the experiment using 20 mL min−1 air with the ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) strain. Other side products in these experiments were acetoin, succinate, dihydroxyacetone (DHA), pyruvate, acetate, l-alanine, α-acetolactate, α-ketoglutarate, and α-ketoisovalerate (Fig. 3, Additional file 1: Table S4). Among these, acetoin formation showed a clear dependence on oxygen availability, increasing about 3- and 5-fold when the air flow was increased from 5 to 10 mL min−1 and from 5 to 20 mL min−1, respectively. Dissolved oxygen was not controlled, but we confirmed that the oxygen concentration was below the detection limit of the oxygen electrode even at the highest aeration rate. The time course for glucose consumption and end-product formation is illustrated in Fig. 4 for the best producer strain. In summary, under an air flow of 10 mL min−1C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) produced 70 ± 8 mM 2,3-BD with a yield of 0.66 mol per mol of glucose and productivity of 11 nmol min−1 mg CDW−1, which represent a notable improvement (Fig. 5).Fig. 4


Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

Radoš D, Carvalho AL, Wieschalka S, Neves AR, Blombach B, Eikmanns BJ, Santos H - Microb. Cell Fact. (2015)

A summary of the stepwise systematic approach used to engineering C. glutamicum for the production of 2,3-BD. The values reflect the impact of the several steps on yield and productivity. Blue boxes refer to strain optimization steps, while the yellow box indicates the process optimization step. 2,3-BD 2,3-butanediol, Glc glucose
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625470&req=5

Fig5: A summary of the stepwise systematic approach used to engineering C. glutamicum for the production of 2,3-BD. The values reflect the impact of the several steps on yield and productivity. Blue boxes refer to strain optimization steps, while the yellow box indicates the process optimization step. 2,3-BD 2,3-butanediol, Glc glucose
Mentions: Cell suspensions of C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) and ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) under constant air flow of 5 mL min−1 showed an increased GCR [7.2 ± 0.6 and 11.1 ± 1.0 nmol min−1 mg CDW−1 in mini-fermenter for the two respective strains (Table 4), compared to 6.1 ± 0.1 and 6.5 ± 0.5 nmol min−1 mg CDW−1 in flasks (Table 3)]; the productivity in mini-fermenter was 3.4 ± 0.3 and 5.5 ± 0.7 nmol min−1 mg CDW−1 compared to 3.1 ± 0.2 and 4.3 ± 0.4 nmol min−1 mg CDW−1 in flasks, but the yield was higher for fermentations in closed flasks (Tables 3, 4). Upon doubling of the air flow to 10 mL min−1, GCR and the productivity of C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) and ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) increased two-fold; both strains produced 2,3-BD at highest yields (0.57 ± 0.03 and 0.66 ± 0.01 mol 2,3-BD per mol glucose). Interestingly, further increase in the flow rate to 20 mL min−1 resulted in significantly lower yields and productivities (Table 4). Ethanol was produced by both strains in experiments using 5 mL min−1 air, and glycerol was absent only in the experiment using 20 mL min−1 air with the ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,PtufbutA) strain. Other side products in these experiments were acetoin, succinate, dihydroxyacetone (DHA), pyruvate, acetate, l-alanine, α-acetolactate, α-ketoglutarate, and α-ketoisovalerate (Fig. 3, Additional file 1: Table S4). Among these, acetoin formation showed a clear dependence on oxygen availability, increasing about 3- and 5-fold when the air flow was increased from 5 to 10 mL min−1 and from 5 to 20 mL min−1, respectively. Dissolved oxygen was not controlled, but we confirmed that the oxygen concentration was below the detection limit of the oxygen electrode even at the highest aeration rate. The time course for glucose consumption and end-product formation is illustrated in Fig. 4 for the best producer strain. In summary, under an air flow of 10 mL min−1C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA) produced 70 ± 8 mM 2,3-BD with a yield of 0.66 mol per mol of glucose and productivity of 11 nmol min−1 mg CDW−1, which represent a notable improvement (Fig. 5).Fig. 4

Bottom Line: Productivity was maximized by manipulating the aeration rate in the production phase.We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production.The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. rados@itqb.unl.pt.

ABSTRACT

Background: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions.

Results: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1).

Conclusions: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

No MeSH data available.


Related in: MedlinePlus