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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

Biosynthesis pathway of coumarins starting with glucose. ppsA, phosphoenolpyruvate synthetase; tktA, transketolase; tyrR, phenylalanine DNA-binding transcription repressor; aroG, deoxyphosphoheptonate aldolase; tyrA, prephenate dehydrogenase; pheA, prephenate dehydratase; tyrB, phenylalanine aminotransferase; TAL, tyrosine amino lyase; 4CL, 4-coumaroyl-CoA ligase; C3H, coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; F6′H, feruloyl CoA 6′-hydroxylase. Tyrosine inhibits tyrR, and tyrA.
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Fig1: Biosynthesis pathway of coumarins starting with glucose. ppsA, phosphoenolpyruvate synthetase; tktA, transketolase; tyrR, phenylalanine DNA-binding transcription repressor; aroG, deoxyphosphoheptonate aldolase; tyrA, prephenate dehydrogenase; pheA, prephenate dehydratase; tyrB, phenylalanine aminotransferase; TAL, tyrosine amino lyase; 4CL, 4-coumaroyl-CoA ligase; C3H, coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; F6′H, feruloyl CoA 6′-hydroxylase. Tyrosine inhibits tyrR, and tyrA.

Mentions: The biosynthetic pathway from cinnamic acid to coumarin was first elucidated in Arabidopsis thaliana [10]. The hydroxylation at the 6′-carbon of cinnamoyl-CoA by a 2-oxoglutarate-dependent dioxygenase (feruloyl CoA 6′-hydroxylase [F6′H], also known as p-coumaryol CoA 2′-hydroxylase [C2′H]) is a key step for the biosynthesis of coumarin [11]. In sweet potato (Ipomoea batatas), genes homologous to F6′H from A. thaliana (IbF6′H1 and IbF6′H2) have been cloned and characterized [12]. These studies opened possibilities that three coumarins (umbelliferone, esculetin, and scopoletin) are synthesized from p-coumaric acid, caffeic acid, and ferulic acid, respectively, by a combination of p-cinnamic acid:CoA ligase (4CL) and F6′H [10,12] (Figure 1).Figure 1


Biological synthesis of coumarins in Escherichia coli.

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

Biosynthesis pathway of coumarins starting with glucose. ppsA, phosphoenolpyruvate synthetase; tktA, transketolase; tyrR, phenylalanine DNA-binding transcription repressor; aroG, deoxyphosphoheptonate aldolase; tyrA, prephenate dehydrogenase; pheA, prephenate dehydratase; tyrB, phenylalanine aminotransferase; TAL, tyrosine amino lyase; 4CL, 4-coumaroyl-CoA ligase; C3H, coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; F6′H, feruloyl CoA 6′-hydroxylase. Tyrosine inhibits tyrR, and tyrA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Biosynthesis pathway of coumarins starting with glucose. ppsA, phosphoenolpyruvate synthetase; tktA, transketolase; tyrR, phenylalanine DNA-binding transcription repressor; aroG, deoxyphosphoheptonate aldolase; tyrA, prephenate dehydrogenase; pheA, prephenate dehydratase; tyrB, phenylalanine aminotransferase; TAL, tyrosine amino lyase; 4CL, 4-coumaroyl-CoA ligase; C3H, coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; F6′H, feruloyl CoA 6′-hydroxylase. Tyrosine inhibits tyrR, and tyrA.
Mentions: The biosynthetic pathway from cinnamic acid to coumarin was first elucidated in Arabidopsis thaliana [10]. The hydroxylation at the 6′-carbon of cinnamoyl-CoA by a 2-oxoglutarate-dependent dioxygenase (feruloyl CoA 6′-hydroxylase [F6′H], also known as p-coumaryol CoA 2′-hydroxylase [C2′H]) is a key step for the biosynthesis of coumarin [11]. In sweet potato (Ipomoea batatas), genes homologous to F6′H from A. thaliana (IbF6′H1 and IbF6′H2) have been cloned and characterized [12]. These studies opened possibilities that three coumarins (umbelliferone, esculetin, and scopoletin) are synthesized from p-coumaric acid, caffeic acid, and ferulic acid, respectively, by a combination of p-cinnamic acid:CoA ligase (4CL) and F6′H [10,12] (Figure 1).Figure 1

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