Limits...
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

Analysis of reaction product from B-CM12. A, standard esculetin; B, reaction product from B-CM12 (P1, esculetin; P2, caffeic acid; P3, p-coumaric acid; P4, umbelliferone).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Analysis of reaction product from B-CM12. A, standard esculetin; B, reaction product from B-CM12 (P1, esculetin; P2, caffeic acid; P3, p-coumaric acid; P4, umbelliferone).

Mentions: p-Coumaric acid is synthesized from tyrosine. Therefore, the intracellular tyrosine concentration is likely to have an effect on the yield of umbelliferone. In order to increase tyrosine production in E. coli, different combinations of four genes (aroG, tyrA, ppsA, and tktA) were used. These four genes have been used previously [26]. B-CM7, in which aroG and tyrA were overexpressed, produced more umbelliferone than other strains (23 mg/L) (Figure 6). The strains B-CM8 and B-CM9, in which feedback-free aroG (aroGfbr) and tyrA (tyrAfbr) [27] were overexpressed, produced less umbelliferone than the strain B-CM6, in which neither aroG nor tyrA was overexpressed. Although it is known that the feedback-free version of aroG and tyrA increase the production of tyrosine [28], this result indicates that there might be a certain optimal concentration of tyrosine beyond which umbelliferone yield does not increase, and even decrease probably due to the metabolic load of producing too much tyrosine.Figure 6


Biological synthesis of coumarins in Escherichia coli.

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

Analysis of reaction product from B-CM12. A, standard esculetin; B, reaction product from B-CM12 (P1, esculetin; P2, caffeic acid; P3, p-coumaric acid; P4, umbelliferone).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Analysis of reaction product from B-CM12. A, standard esculetin; B, reaction product from B-CM12 (P1, esculetin; P2, caffeic acid; P3, p-coumaric acid; P4, umbelliferone).
Mentions: p-Coumaric acid is synthesized from tyrosine. Therefore, the intracellular tyrosine concentration is likely to have an effect on the yield of umbelliferone. In order to increase tyrosine production in E. coli, different combinations of four genes (aroG, tyrA, ppsA, and tktA) were used. These four genes have been used previously [26]. B-CM7, in which aroG and tyrA were overexpressed, produced more umbelliferone than other strains (23 mg/L) (Figure 6). The strains B-CM8 and B-CM9, in which feedback-free aroG (aroGfbr) and tyrA (tyrAfbr) [27] were overexpressed, produced less umbelliferone than the strain B-CM6, in which neither aroG nor tyrA was overexpressed. Although it is known that the feedback-free version of aroG and tyrA increase the production of tyrosine [28], this result indicates that there might be a certain optimal concentration of tyrosine beyond which umbelliferone yield does not increase, and even decrease probably due to the metabolic load of producing too much tyrosine.Figure 6

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