Limits...
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.


Detection of N-(4’-hydroxydihydrocinnamoyl)-anthranilate (DHavnD) from the recombinant yeast culture medium.Representative ESI-MS spectra were obtained after LC-TOF MS analysis of (A) the culture medium of recombinant yeast incubated with anthranilate and 4-hydroxydihydrocinnamate, and (B) a DHavnD standard solution.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591981&req=5

pone.0138972.g003: Detection of N-(4’-hydroxydihydrocinnamoyl)-anthranilate (DHavnD) from the recombinant yeast culture medium.Representative ESI-MS spectra were obtained after LC-TOF MS analysis of (A) the culture medium of recombinant yeast incubated with anthranilate and 4-hydroxydihydrocinnamate, and (B) a DHavnD standard solution.

Mentions: We attempted to produce dihydrocinnamoyl anthranilates by feeding the yeast strain with various dihydrocinnamates (i.e., 3-phenylpropionate derivatives) and anthranilates. First, by comparison with the LC-MS elution profile of an authentic standard, the production of DHavnD (4.03 ± 0.08 μM) was successfully achieved by feeding 4-hydroxydihydrocinnamate and anthranilate (Fig 3), which indicated the promiscuity of 4CL5 and HCBT to use as substrates the saturated propanoid tail of cinnamate and cinnamoyl-CoA, respectively. No DHavnD was detected from the culture medium of control strains, fed with the same precursors and expressing either 4CL5 or HCBT alone. Next, as a preliminary round of screening, the medium of the engineered yeast was supplied with a series of 22 dihydrocinnamates (including halogenated dihydrocinnamates) in combination with anthranilate, which led to the production of 14 individual dihydrocinnamoyl anthranilates, according to the LC-MS analysis of the medium (Table 2, S2 Fig). The dihydrocinnamates that yielded a detectable product in the first round of screening were then co-fed with 3-hydroxyanthranilate or 3-methylanthranilate, which resulted in the production of 13 additional dihydrocinnamoyl anthranilates (Table 2, S2 Fig). The new compounds identified were not produced in the control yeast cultures fed only with anthranilates, demonstrating again the substrate promiscuity of both 4CL5 and HCBT enzymes in our in vivo production system.


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)

Detection of N-(4’-hydroxydihydrocinnamoyl)-anthranilate (DHavnD) from the recombinant yeast culture medium.Representative ESI-MS spectra were obtained after LC-TOF MS analysis of (A) the culture medium of recombinant yeast incubated with anthranilate and 4-hydroxydihydrocinnamate, and (B) a DHavnD standard solution.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138972.g003: Detection of N-(4’-hydroxydihydrocinnamoyl)-anthranilate (DHavnD) from the recombinant yeast culture medium.Representative ESI-MS spectra were obtained after LC-TOF MS analysis of (A) the culture medium of recombinant yeast incubated with anthranilate and 4-hydroxydihydrocinnamate, and (B) a DHavnD standard solution.
Mentions: We attempted to produce dihydrocinnamoyl anthranilates by feeding the yeast strain with various dihydrocinnamates (i.e., 3-phenylpropionate derivatives) and anthranilates. First, by comparison with the LC-MS elution profile of an authentic standard, the production of DHavnD (4.03 ± 0.08 μM) was successfully achieved by feeding 4-hydroxydihydrocinnamate and anthranilate (Fig 3), which indicated the promiscuity of 4CL5 and HCBT to use as substrates the saturated propanoid tail of cinnamate and cinnamoyl-CoA, respectively. No DHavnD was detected from the culture medium of control strains, fed with the same precursors and expressing either 4CL5 or HCBT alone. Next, as a preliminary round of screening, the medium of the engineered yeast was supplied with a series of 22 dihydrocinnamates (including halogenated dihydrocinnamates) in combination with anthranilate, which led to the production of 14 individual dihydrocinnamoyl anthranilates, according to the LC-MS analysis of the medium (Table 2, S2 Fig). The dihydrocinnamates that yielded a detectable product in the first round of screening were then co-fed with 3-hydroxyanthranilate or 3-methylanthranilate, which resulted in the production of 13 additional dihydrocinnamoyl anthranilates (Table 2, S2 Fig). The new compounds identified were not produced in the control yeast cultures fed only with anthranilates, demonstrating again the substrate promiscuity of both 4CL5 and HCBT enzymes in our in vivo production system.

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.