Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in <i>Saccharomyces cerevisiae</i>

<div><p>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 <i>Dianthus caryophyllus</i> is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate <i>N</i>-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (<i>Saccharomyces cerevisiae</i>) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from <i>Arabidopsis thaliana</i> (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.</p></div

Structural characteristics of the dihydrocinnamoyl anthranilates (general structure shown in Fig 1B) produced in yeast and their identification based on dominant ion masses in ESI-MS spectra.

<p>^aMass accuracy = [(theoretical mass—measured mass) / (theoretical mass)] x 1.10⁶</p><p>Values were obtained from single feeding experiments for each combination of precursors.</p

Detection of <i>N</i>-(4’-hydroxydihydrocinnamoyl)-anthranilate (DHavnD) from the recombinant yeast culture medium.

<p>Representative ESI-MS spectra were obtained after LC-TOF MS analysis of <b>(A)</b> the culture medium of recombinant yeast incubated with anthranilate and 4-hydroxydihydrocinnamate, and <b>(B)</b> a DHavnD standard solution.</p

Structure of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.

<p><b>(A)</b> Cinnamoyl anthranilates. Tranilast: R₁ = R₂ = R₃ = R₆ = H, R₄ = R₅ = OMe. <b>(B)</b> Dihydrocinnamoyl anthranilates. DHavnD: R₁ = R₂ = R₃ = R₄ = R₆ = H, R₅ = OH. <b>(C)</b> Benzoyl anthranilates. Dianthramide B from <i>D</i>. <i>caryophyllus</i>: R_1-6 = H.</p

Structural characteristics of the benzoyl anthranilates (general structure shown in Fig 1C) produced in yeast and their identification based on dominant ion masses in ESI-MS spectra.

<p>^aMass accuracy = [(theoretical mass—measured mass) / (theoretical mass)] x 1.10⁶</p><p>Values were obtained from single feeding experiments for each combination of precursors.</p

Structural characteristics of the cinnamoyl anthranilates (general structure shown in Fig 1A) produced in yeast and their identification based on dominant ion masses in ESI-MS spectra.

<p>^aMass accuracy = [(theoretical mass—measured mass) / (theoretical mass)] x 1.10⁶</p><p>Values were obtained from single feeding experiments for each combination of precursors.</p

Detection of <i>N</i>-(benzoyl)-anthranilate (dianthramide B) from the recombinant yeast culture medium.

<p>Representative ESI-MS spectra were obtained after LC-TOF MS analysis of <b>(A)</b> the culture medium of recombinant yeast incubated with anthranilate and benzoic acid, and <b>(B)</b> a dianthramide B standard solution.</p

Simplified representation of the lignin and tricin biosynthetic pathways from phenylalanine.

<p>Abbreviations are: Bmr12, Brown midrib12; OMT, <i>O</i>-methyltransferase; SbCOMT, <i>Sorghum bicolor</i> caffeate <i>O</i>-methyltransferase.</p

Lignin monomeric composition in wild-type (WT) and <i>bmr12</i> sorghum biomass.

<p>For each genotype, cellulolytic lignin was isolated and analyzed by 2D ¹³C–¹H HSQC NMR spectroscopy. Regions of partial short-range ¹³C–¹H HSQC spectra are shown. Lignin monomer ratios including tricin (T) are provided on the figures. S: syringyl, G: guaiacyl, 5OH-G: 5-hydroxyguaiacyl, H: <i>p</i>-hydroxyphenyl, <i>p</i>CA: <i>p</i>-coumarate, FA: ferulate.</p

Quantification of methanol-soluble luteolin, chrysoeriol, selgin, and tricin extracted from the biomass of wild-type (WT) and <i>bmr12</i> sorghum lines.

<p>Values in <i>bmr12</i> are expressed as a percentage of the values measured in wild-type extracts which correspond to 317 ± 4 µg/g dry weight (DW) for luteolin, 7.8 ± 0.0 µg/g DW for chrysoeriol, 2.0 ± 0.2 µg/g DW for selgin, and 274 ± 3 µg/g DW for tricin. Error bars represent the standard deviation from five experimental replicates. Asterisks indicate significant differences from the wild-type using the unpaired Student’s t-test (*<i>P</i> < 0.05).</p