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Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae.

Eudes A, Teixeira Benites V, Wang G, Baidoo EE, Lee TS, Keasling JD, Loqué D - PLoS ONE (2015)

Bottom Line: We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT.Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules.This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute, Emery Station East, 5885 Hollis St, 4th Floor, Emeryville, California, 94608, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States of America.

ABSTRACT
Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.

No MeSH data available.


Related in: MedlinePlus

Strategy used for the biological synthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.Diagram of the reactions catalyzed by 4CL5 and HCBT in the yeast strain engineered for the production of various cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates upon feeding with cinnamates, dihydrocinnamates, or benzoates (donors); and with anthranilates (acceptors). HSCoA, Coenzyme A.
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pone.0138972.g002: Strategy used for the biological synthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.Diagram of the reactions catalyzed by 4CL5 and HCBT in the yeast strain engineered for the production of various cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates upon feeding with cinnamates, dihydrocinnamates, or benzoates (donors); and with anthranilates (acceptors). HSCoA, Coenzyme A.

Mentions: The chemical synthesis of pharmaceuticals such as cinnamoyl and benzoyl anthranilates—or their purification from source organisms—consumes nonrenewable petroleum-based chemicals, generates toxic byproducts that require downstream waste-processing, and increases production costs. By contrast, biological synthesis is an eco-friendly production method with reduced requirements for toxic chemicals and natural resources. It offers consistent quality, scalability, simple extraction, and potential for higher synthesis efficiency [24]. In addition, biological synthesis could expand the chemical diversity of natural products, the structural complexity of which is sometimes challenging to achieve using multistep chemical synthesis [25]. In this area, the industrial microorganism yeast (Saccharomyces cerevisiae) has emerged as a powerful tool for the biosynthesis of secondary metabolites considering its advantages for the expression of complex metabolic pathways [26]. We previously reported on a yeast strain engineered for the production of tranilast and several analogs [27]. Cinnamates supplied to this strain are converted into coumaroyl-CoAs by 4-coumaroyl:CoA ligase 5 (4CL5) from Arabidopsis thaliana and coupled to anthranilate or 3-hydroxyanthranilate by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT) from D. caryophyllus (Fig 2). In an earlier study, 13 methoxylated and hydroxylated cinnamates were successfully used as precursors for the production of the corresponding hydroxy/methoxycinnamoyl anthranilates [27]. Here, we show how we extended our yeast production platform by screening several new cinnamate derivatives that could potentially be converted by our yeast strain into cinnamoyl anthranilates and explored benzoates as precursors for the production of benzoyl anthranilates (Fig 2). First, a series of halogenated cinnamates were tested because of the importance of halogen groups—particularly fluoride—in drug development [28,29]. Second, several dihydrocinnamates, which correspond to cinnamates with a saturated double bond on the propanoid tail, were tested and successfully converted into dihydrocinnamoyl anthranilates—including those that were halogenated. Third, since HCBT is known to use benzoyl-CoA in addition to coumaroyl-CoA [30], we attempted to feed the yeast strain with benzoic acid derivatives and confirmed production of a series of halogenated benzoyl anthranilates.


Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae.

Eudes A, Teixeira Benites V, Wang G, Baidoo EE, Lee TS, Keasling JD, Loqué D - PLoS ONE (2015)

Strategy used for the biological synthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.Diagram of the reactions catalyzed by 4CL5 and HCBT in the yeast strain engineered for the production of various cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates upon feeding with cinnamates, dihydrocinnamates, or benzoates (donors); and with anthranilates (acceptors). HSCoA, Coenzyme A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591981&req=5

pone.0138972.g002: Strategy used for the biological synthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.Diagram of the reactions catalyzed by 4CL5 and HCBT in the yeast strain engineered for the production of various cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates upon feeding with cinnamates, dihydrocinnamates, or benzoates (donors); and with anthranilates (acceptors). HSCoA, Coenzyme A.
Mentions: The chemical synthesis of pharmaceuticals such as cinnamoyl and benzoyl anthranilates—or their purification from source organisms—consumes nonrenewable petroleum-based chemicals, generates toxic byproducts that require downstream waste-processing, and increases production costs. By contrast, biological synthesis is an eco-friendly production method with reduced requirements for toxic chemicals and natural resources. It offers consistent quality, scalability, simple extraction, and potential for higher synthesis efficiency [24]. In addition, biological synthesis could expand the chemical diversity of natural products, the structural complexity of which is sometimes challenging to achieve using multistep chemical synthesis [25]. In this area, the industrial microorganism yeast (Saccharomyces cerevisiae) has emerged as a powerful tool for the biosynthesis of secondary metabolites considering its advantages for the expression of complex metabolic pathways [26]. We previously reported on a yeast strain engineered for the production of tranilast and several analogs [27]. Cinnamates supplied to this strain are converted into coumaroyl-CoAs by 4-coumaroyl:CoA ligase 5 (4CL5) from Arabidopsis thaliana and coupled to anthranilate or 3-hydroxyanthranilate by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT) from D. caryophyllus (Fig 2). In an earlier study, 13 methoxylated and hydroxylated cinnamates were successfully used as precursors for the production of the corresponding hydroxy/methoxycinnamoyl anthranilates [27]. Here, we show how we extended our yeast production platform by screening several new cinnamate derivatives that could potentially be converted by our yeast strain into cinnamoyl anthranilates and explored benzoates as precursors for the production of benzoyl anthranilates (Fig 2). First, a series of halogenated cinnamates were tested because of the importance of halogen groups—particularly fluoride—in drug development [28,29]. Second, several dihydrocinnamates, which correspond to cinnamates with a saturated double bond on the propanoid tail, were tested and successfully converted into dihydrocinnamoyl anthranilates—including those that were halogenated. Third, since HCBT is known to use benzoyl-CoA in addition to coumaroyl-CoA [30], we attempted to feed the yeast strain with benzoic acid derivatives and confirmed production of a series of halogenated benzoyl anthranilates.

Bottom Line: We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT.Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules.This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute, Emery Station East, 5885 Hollis St, 4th Floor, Emeryville, California, 94608, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States of America.

ABSTRACT
Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.

No MeSH data available.


Related in: MedlinePlus