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Biological synthesis of coumarins in Escherichia coli.

Yang SM, Shim GY, Kim BG, Ahn JH - Microb. Cell Fact. (2015)

Bottom Line: The solubility of F6'H was critical for the final yield.Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains.The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143-701, Korea. lcanldyl@nate.com.

ABSTRACT

Background: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.

Results: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.

Conclusions: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

No MeSH data available.


Related in: MedlinePlus

Production of umbelliferone from glucose using B-CM6. A, standard umbelliferone; B, reaction product from B-CM6 (P1, 2-hydroxy p-coumaric acid; P2, p-coumaric acid; P3, umbelliferone).
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Fig5: Production of umbelliferone from glucose using B-CM6. A, standard umbelliferone; B, reaction product from B-CM6 (P1, 2-hydroxy p-coumaric acid; P2, p-coumaric acid; P3, umbelliferone).

Mentions: Umbelliferone and esculetin are synthesized from p-coumaric acid, and caffeic acid, respectively. p-Coumaric acid is synthesized from tyrosine by the action of tyrosine ammonia lyase (TAL). Subsequently, 3′-hydroxylation of p-coumaric acid leads to the synthesis of caffeic acid. Therefore, for the synthesis of umbelliferone, an additional gene, TAL is needed, which was previously cloned in our lab [26]. E. coli BL21 (DE3) was transformed with the TAL gene along with pG-pIbF6′H2-Os4CL. The resulting strain, B-CM6, was used to synthesize umbelliferone. The analysis of the culture filtrate by HPLC showed a peak (peak 3 in Figure 5B) that has the same retention time as standard umbelliferone. The molecular mass of this reaction product was 162 Da (g/mol), which is the predicted molecular mass of umbelliferone (data not shown). Therefore, umbelliferone was successfully synthesized by strain B-CM6.Figure 5


Biological synthesis of coumarins in Escherichia coli.

Yang SM, Shim GY, Kim BG, Ahn JH - Microb. Cell Fact. (2015)

Production of umbelliferone from glucose using B-CM6. A, standard umbelliferone; B, reaction product from B-CM6 (P1, 2-hydroxy p-coumaric acid; P2, p-coumaric acid; P3, umbelliferone).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419511&req=5

Fig5: Production of umbelliferone from glucose using B-CM6. A, standard umbelliferone; B, reaction product from B-CM6 (P1, 2-hydroxy p-coumaric acid; P2, p-coumaric acid; P3, umbelliferone).
Mentions: Umbelliferone and esculetin are synthesized from p-coumaric acid, and caffeic acid, respectively. p-Coumaric acid is synthesized from tyrosine by the action of tyrosine ammonia lyase (TAL). Subsequently, 3′-hydroxylation of p-coumaric acid leads to the synthesis of caffeic acid. Therefore, for the synthesis of umbelliferone, an additional gene, TAL is needed, which was previously cloned in our lab [26]. E. coli BL21 (DE3) was transformed with the TAL gene along with pG-pIbF6′H2-Os4CL. The resulting strain, B-CM6, was used to synthesize umbelliferone. The analysis of the culture filtrate by HPLC showed a peak (peak 3 in Figure 5B) that has the same retention time as standard umbelliferone. The molecular mass of this reaction product was 162 Da (g/mol), which is the predicted molecular mass of umbelliferone (data not shown). Therefore, umbelliferone was successfully synthesized by strain B-CM6.Figure 5

Bottom Line: The solubility of F6'H was critical for the final yield.Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains.The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143-701, Korea. lcanldyl@nate.com.

ABSTRACT

Background: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.

Results: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.

Conclusions: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

No MeSH data available.


Related in: MedlinePlus