Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

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

  • Aymerick Eudes,

    Affiliations 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

  • Veronica Teixeira Benites,

    Affiliations 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, Graduate Program, San Francisco State University, San Francisco, California, 94132, United States of America

  • George Wang,

    Affiliations 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

  • Edward E. K. Baidoo,

    Affiliations 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

  • Taek Soon Lee,

    Affiliations 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

  • Jay D. Keasling,

    Affiliations 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, Department of Bioengineering & Department of Chemical & Biomolecular Engineering, University of California, Berkeley, California, 94720, United States of America

  • Dominique Loqué

    dloque@lbl.gov

    Affiliations 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

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

  • Aymerick Eudes, 
  • Veronica Teixeira Benites, 
  • George Wang, 
  • Edward E. K. Baidoo, 
  • Taek Soon Lee, 
  • Jay D. Keasling, 
  • Dominique Loqué
PLOS
x

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.

Introduction

Cinnamoyl and benzoyl anthranilates are bipartite molecules consisting of cinnamate or benzoate moieties amide-linked to anthranilic acids (Fig 1). The beneficial pharmacological effects of these molecules on human health have been well-documented over the past few years. For example, avenanthramides are natural cinnamoyl anthranilates found in oats and possess antioxidant, anti-inflammatory, and antiproliferative bioactivities [1,2]. Tranilast ([N-(3’,4’-dimethoxycinnamoyl)-anthranilic acid], Fig 1A) is a synthetic cinnamoyl anthranilate marketed in Japan for the treatment of allergic diseases, scleroderma, and hypertrophic scars associated with excessive fibrotic response [3]. In particular, tranilast is an antifibrotic agent that inhibits several profibrotic growth factors [46]. Recent efforts have been made for the development of tranilast analogs to optimize the antifibrotic effects and reduce toxicity at higher doses [7]. For instance, modification of functional groups on the cinnamoyl ring and the introduction of halogens resulted in cinnamoyl anthranilates with higher bioavailability and enhanced inhibitory effects on fibrosis [812]. Other structure optimizations have included double bond saturation resulting in dihydrocinnamoyl anthranilates such as dihydroavenanthramide D (DHavnD, Fig 1B), which is an anti-inflammatory used for the treatment of skin disorders and is currently evaluated for its antidiabetic and anticancer effects [1315]. Benzoyl anthranilates (Fig 1C) are found in some plant species such as D. caryophyllus [16]; and several analogs were shown to inhibit human aldo-keto reductases involved in different pathophysiological conditions such as prostate cancer [17], as well as to possess cytotoxic activity toward cancer cell lines [18]. Moreover, certain halogenated benzoyl anthranilates are candidates for the treatment of infectious diseases because of their inhibitory effects on the malaria agent Plasmodium falciparum [19], the human African trypanosomiasis agent Trypanosoma brucei [20,21], and the opportunistic pathogenic bacterium Pseudomonas aeruginosa [22,23].

thumbnail
Fig 1. Structure of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates.

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

https://doi.org/10.1371/journal.pone.0138972.g001

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.

thumbnail
Fig 2. 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.

https://doi.org/10.1371/journal.pone.0138972.g002

Altogether, our data demonstrate that the substrate promiscuity of both 4CL5 and HCBT can be exploited for biological synthesis of structurally diverse cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates of potential pharmaceutical value.

Materials and Methods

Chemicals

The cinnamates, dihydrocinnamates (or 3-phenylpropionates), and benzoates used for the yeast feeding experiments are listed in S1, S2 and S3 Tables and were purchased from VWR International (Radnor, PA, USA). DHavnD and dianthramide B were obtained from Enamine Ltd (Monmouth Jct., NJ) and Sigma-Aldrich (Saint-Louis, MO), respectively.

Expression of 4CL5 and HCBT in yeast

The pDRf1-4CL5-HCBT1, pDRf1-HCBT1, and pDRf1-4CL5 vectors [27] were used for the expression of At4CL5 (At3g21230, also named At4CL4 in original studies [31]) and a codon-optimized HCBT (GenBank: Z84385.1) under the control of the constitutive promoters PHXT7 and PPMA1, respectively. The S. cerevisiae pad1 knockout (MATa his31 leu20 met150 ura30pad1, ATCC 4005833) [32] was transformed using the Frozen-EZ Yeast Transformation II Kit™ (Zymo Research Corporation, Irvine, CA) and selected on solid medium containing Yeast Nitrogen Base (YNB) without amino acids (Difco 291940; Difco, Detroit, MI) supplemented with 3% glucose and 1X dropout-uracil (CSM-ura; Sunrise Science Products, San Diego, CA). A pad1 knockout was chosen because PAD1 is a known phenylacrylic acid decarboxylase whose deletion in yeast prevents the degradation of exogenously supplied cinnamates [33, 34].

Production of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates

An overnight culture from a single colony of the pDRf1-4CL5-HCBT1 recombinant yeast grown on 2X YNB medium without amino acids, supplemented with 6% glucose and 2X CSM-Ura, was used to inoculated 4 mL of fresh minimal medium at an OD600 = 0.15 and shaken at 200 rpm at 30°C. All precursors were prepared in DMSO and added 5 hours post inoculation at the concentrations indicated in S1, S2 and S3 Tables. The anthranilate acceptors were added to the medium at a final concentration of 300 μM (for anthranilate, 3-hydroxyanthranilate, 3-methylanthranilate, and 5-nitroanthranilate) or 50 μM (for 3-chloroanthranilate, 5-methylanthranilate, 3-methoxyanthranilate, 5-fluoroanthranilate, 5-iodoanthranilate, and 5-chloroanthranilate). These concentrations were selected to limit toxicity and growth inhibition due to either the supplied precursors or the metabolites produced. The cultures were shaken at 200 rpm at 30°C for 24 h in the presence of the precursors for the production of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates. Yeast colonies harboring the pDRf1-HCBT1 or pDRf1-4CL5 control vectors were grown under similar conditions. For the detection of metabolites, an aliquot of the culture medium was collected and cleared by centrifugation (21,000xg for 5 min at 4°C), mixed with an equal volume of cold methanol:water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to LC-TOF MS analysis. The separation and identification of the metabolites were performed using high-performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS) as previously described [35]. For each compound, the measured masses agreed with the expected theoretical masses within less than 5 ppm mass error. Standard solutions of DHavnD and dianthramide B were prepared in methanol:water (1:1, v/v). Values obtained for the production of DHavnD and dianthramide B are the average of four replicates (n = 4). ESI-MS spectra of other cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates were obtained from single feeding experiments for each combination of precursors.

Results

Production of halogenated cinnamoyl anthranilates

A yeast strain that co-expresses 4CL5 and HCBT was used as a catalyst for the production of non-natural halogenated cinnamoyl anthranilates. We showed previously that HCBT can accept anthranilate or 3-hydroxyanthranilate as substrates for the production of cinnamoyl anthranilates [27]. We further investigated the substrate promiscuity of HCBT and the possibility of producing additional cinnamoyl conjugates by feeding the yeast strain with new anthranilates in combination with p-coumarate. Of 10 anthranilates individually supplied to the culture medium, five novel p-coumaroyl anthranilates were conclusively produced upon feeding with 3-methylanthranilate, 3-methoxyanthranilate, 3-chloroanthranilate, 5-methylanthranilate, and 5-fluoroanthranilate—indicating that HCBT can also accept these anthranilate analogs (Table 1, S1 Fig). Based on their expected masses, these compounds were identified by LC-MS analysis of the culture medium but could not be detected in control yeast cultures grown with only anthranilates (without p-coumarate). Next, to assess the capacity of the yeast strain to produce non-natural cinnamoyl anthranilates, we fed the 4CL5- and HCBT-expressing yeast strain several halogenated cinnamates in combination with the seven different anthranilates identified as HCBT acceptors. As a result, 45 novel halogenated cinnamoyl anthranilates were biosynthesized out of 98 combinations tested using a series of 14 fluorinated, chlorinated, and brominated cinnamates (Table 1, S1 Fig). These results demonstrate the coenzyme A-ligase activity of 4CL5 toward these non-natural cinnamates and the capacity of HCBT to couple the corresponding CoA-thioesters to various anthranilates.

thumbnail
Table 1. 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.

Values were obtained from single feeding experiments for each combination of precursors.

https://doi.org/10.1371/journal.pone.0138972.t001

Production of dihydrocinnamoyl anthranilates

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.

thumbnail
Fig 3. 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.

https://doi.org/10.1371/journal.pone.0138972.g003

thumbnail
Table 2. 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.

Values were obtained from single feeding experiments for each combination of precursors.

https://doi.org/10.1371/journal.pone.0138972.t002

Production of benzoyl anthranilates

The production of benzoyl anthranilates by the 4CL5-HCBT yeast strain was tested because of the capacity of HCBT to use benzoyl-CoA as a donor in addition to coumaroyl-CoA [30]. We first successfully produced a benzoyl anthranilate named dianthramide B (1.20 ± 0.12 μM), by feeding the 4CL5- and HCBT-expressing yeast strain with benzoic acid and anthranilate. The identity of this new compound, which was detected directly from the culture medium, was confirmed with the authentic standard that exhibits the same LC-MS elution profile and mass (Fig 4), and by its absence in control cultures of strains expressing either 4CL5 or HCBT alone. Considering this unexpected substrate affinity of 4CL5 for benzoic acid, we fed 75 benzoate derivatives in combination with anthranilate for the synthesis of the corresponding benzoyl conjugates. This preliminary screening resulted in the production of 34 individual benzoyl anthranilates, including halogenated benzyol anthranilates, which were detected directly from the culture medium by LC-MS analysis (Table 3, S3 Fig). A second round of production using 3-hydroxyanthranilate or 3-methylanthranilate instead of anthranilate in the culture medium led to the production of 50 additional benzoyl anthranilates (Table 3, S3 Fig), which were absent from the culture medium of the yeast strain fed only with the anthranilates. These results demonstrate the capacity for 4CL5 to ligate coenzyme A onto at least 34 benzoate analogs; and the capacity for HCBT to conjugate the corresponding benzoyl-CoAs with various anthranilates.

thumbnail
Fig 4. Detection of N-(benzoyl)-anthranilate (dianthramide B) 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 benzoic acid, and (B) a dianthramide B standard solution.

https://doi.org/10.1371/journal.pone.0138972.g004

thumbnail
Table 3. 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.

Values were obtained from single feeding experiments for each combination of precursors.

https://doi.org/10.1371/journal.pone.0138972.t003

Discussion

With an emphasis on the class of cinnamyol, dihydrocinnamoyl, and benzoyl anthranilates, we illustrate in this study the possibility of producing numerous chemically diverse molecules using biological synthesis rather than conventional chemical synthesis. Our data imply that the promiscuity of 4CL5 allows the catalytic conversion of a great diversity of dihydrocinnamates, benzoates, and various cinnamates into the corresponding acyl-CoA-thioesters. To our knowledge, this is the first description of a bona fide 4-coumaroyl:CoA ligase (EC 6.2.1.12) showing benzoyl:CoA (EC 6.2.1.25), 3-hydroxybenzoyl:CoA (EC 6.2.1.37), 4-hydroxybenzoyl:CoA (EC 6.2.1.27), and 4-chlorobenzoyl:CoA (EC 6.2.1.33) ligase activities. Our original attempts to co-express HCBT with known bacterial benzoyl:CoA ligases for the production of benzoyl anthranilates in yeast were unsuccessful, possibly due to the high pH optima (pH > 8.5) of these enzymes [36,37]. Nevertheless, using the 4CL5 enzyme, we demonstrate the feasibility of producing a substantial diversity of benzoyl-CoA thioesters and benzoate conjugate molecules in yeast. This discovery opens new possibilities for the heterologous combinatorial production of valuable benzoylated metabolites such as benzylbenzoates; benzophenones; the anticancer drug taxol; polyketides with antimicrobial activities (e.g., wailupemycin, enterocin, soraphen A); and unnatural polyketides using engineered benzoyl-CoA-dependent polyketide synthases [38]. Furthermore, heterologously synthesized benzoyl anthranilates can be used as scaffolds for the synthesis of related anti-adenoviral compounds and oncogene inhibitors [39,40].

We observed the activity of 4CL5 towards various dihydrocinnamates and non-natural halogenated cinnamates and exploited its catalytic property to biosynthesize libraries of non-natural and structurally diverse cinnamoyl and dihydrocinnamoyl anthranilates using HCBT. For example, the drug DHavnD was synthesized, and utilization of alternate precursors resulted in the rapid production of 27 additional DHavnD analogs. These results point towards the eventual design of more biologically active drugs through the addition of halogens. They also illustrate the advantage of biological synthesis to achieve bifunctionalization, as exemplified by several of our bi-halogenated compounds. Finally, through co-expression with the adequate synthases, the capacity of 4CL5 to activate dihydrocinnamates creates the potential for biomanufacture of valuable natural products, such as the antibacterial dihydrocinnamoyl forms of flavans and chalcones [41,42].

The HCBT enzyme used in this study belongs to the BAHD enzyme family, which contains multiple members that catalyze the transfer of cinnamoyl- and benzoyl-CoAs into a great diversity of distinct acceptors [43]. Although HCBT offers flexibility for a wide range of acyl-CoA donors, its affinity towards acceptors seems limited to anthranilates. Therefore, engineering yeast strains that co-express 4CL5 with various BAHD transferases would considerably expand the type and number of molecules that can be biosynthesized heterologously.

Ultimately, biosynthesis of particular cinnamoyl or benzoyl anthranilates from renewable and inexpensive carbon sources could be desirable for cost-effective manufacturing. For this purpose, we recently demonstrated a de novo pathway for the production of p-coumarate and two avenanthramides from glucose in E. coli [35]. In this pathway, additional expression of hydroxycinnamoyl-CoA double-bond reductase could be used for the synthesis of dihydrocinnamates [44], whereas benzoate biosynthesis can be achieved from the aromatic amino acid phenylalanine [45]. Finally, the recent discovery of halogenases from bacteria and fungi has already proven to be useful for de novo synthesis of halogenated bioactive metabolites in microorganisms [46,47].

As a conclusion, the use of two promiscuous enzymes, 4CL5 and HCBT, demonstrates the potential to develop a platform for the precursor-directed combinatorial biosynthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates. In this study and in our previous work [27], this system using a single engineered yeast strain supported the production of more than 180 target metabolites belonging to cinnamoyl, dihydrocinnamoyl, or benzoyl anthranilate families. Moreover, we believe that testing our system with more substituted cinnamates and benzoates could result in the production of several additional metabolites.

Supporting Information

S1 Fig. LC-MS elution profiles of 50 novel cinnamoyl anthranilates produced by the recombinant 4CL5-HCBT yeast strain.

ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of the yeast strain fed with the precursors indicated in Table 1.

https://doi.org/10.1371/journal.pone.0138972.s001

(PPTX)

S2 Fig. LC-MS elution profiles of 27 dihydrocinnamoyl anthranilates produced by the recombinant 4CL5-HCBT yeast strain.

ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of the yeast strain fed with the precursors indicated in Table 2.

https://doi.org/10.1371/journal.pone.0138972.s002

(PPTX)

S3 Fig. LC-MS elution profiles of 84 benzoyl anthranilates produced by the recombinant 4CL5-HCBT yeast strain.

ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of the yeast strain fed with the precursors indicated in Table 3.

https://doi.org/10.1371/journal.pone.0138972.s003

(PPTX)

S1 Table. Structures and concentrations of the cinnamates used for the yeast feedings.

https://doi.org/10.1371/journal.pone.0138972.s004

(DOCX)

S2 Table. Structures and concentrations of the dihydrocinnamates used for the yeast feedings.

https://doi.org/10.1371/journal.pone.0138972.s005

(DOCX)

S3 Table. Structures and concentrations of the benzoates used for the yeast feedings.

https://doi.org/10.1371/journal.pone.0138972.s006

(DOCX)

Acknowledgments

Authors are grateful to Sabin Russell for editing this manuscript.

Author Contributions

Conceived and designed the experiments: AE DL. Performed the experiments: AE VTB GW EB. Analyzed the data: AE VTB GW EB DL. Contributed reagents/materials/analysis tools: AE EB JK DL TSL. Wrote the paper: AE EB JK DL.

References

  1. 1. Meydani M. Potential health benefits of avenanthramides of oats. Nutr Rev 2009;67:731–725. pmid:19941618
  2. 2. Singh R, De S, Belkheir A. Avena sativa (Oat), a potential neutraceutical and therapeutic agent: an overview. Crit Rev Food Sci Nutr 2013;53:126–144. pmid:23072529
  3. 3. Yamada H, Ide A, Sugiura M, Tajima S. Treatment of cutaneous sarcoidosis with tranilast. J Dermatol 1995;22:149–152. pmid:7536763
  4. 4. Pinto YM, Pinto-Sietsma SJ, Philipp T, Engler S, Kossamehl P, Hocher B, et al. Reduction in left ventricular messenger RNA for transforming growth factor beta(1) attenuates left ventricular fibrosis and improves survival without lowering blood pressure in the hypertensive TGR(mRen2)27 Rat. Hypertension 2000;36:747–754. pmid:11082138
  5. 5. Qi W, Chen X, Twigg S, Polhill TS, Gilbert RE, Pollock CA. Tranilast attenuates connective tissue growth factor-induced extracellular matrix accumulation in renal cells. Kidney Int 2006;69:989–995. pmid:16528248
  6. 6. Ward MR, Sasahara T, Agrotis A, Dilley RJ, Jennings GL, Bobik A. Inhibitory effects of tranilast on expression of transforming growth factor-beta isoforms and receptors in injured arteries. Atherosclerosis 1998;137:267–275. pmid:9622270
  7. 7. Zammit SC, Cox AJ, Gow RM, Zhang Y, Gilbert RE, Krum H, et al. Evaluation and optimization of antifibrotic activity of cinnamoyl anthranilates. Bioorg Med Chem Lett 2009;19:7003–7006. pmid:19879136
  8. 8. Gilbert RE, Zhang Y, Williams SJ, Zammit SC, Stapleton DI, Cox AJ, et al. A purpose-synthesised anti-fibrotic agent attenuates experimental kidney diseases in the rat. PLoS One 2012;7:e47160. pmid:23071743
  9. 9. Tan SM, Zhang Y, Wang B, Tan CY, Zammit SC, Williams SJ, et al. An orally active anti-fibrotic compound, FT23, attenuates structural and functional abnormalities in an experimental model of diabetic cardiomyopathy. Clin Exp Pharmacol Physiol 2012;39:650–656. pmid:22612418
  10. 10. Williams SJ, Zammit SC, Cox AJ, Shackleford DM, Morizzi J, Zhang Y, et al. 3',4'-Bis-difluoromethoxycinnamoylanthranilate (FT061): an orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathy. Bioorg Med Chem Lett 2013;23:6868–6873. pmid:24169234
  11. 11. Zhang Y, Edgley AJ, Cox AJ, Powell AK, Wang B, Kompa AR, et al. FT011, a new anti-fibrotic drug, attenuates fibrosis and chronic heart failure in experimental diabetic cardiomyopathy. Eur J Heart Fail. 2012;14:549–562. pmid:22417655
  12. 12. Zhang Y, Elsik M, Edgley AJ, Cox AJ, Kompa AR, Wang B, et al. A new anti-fibrotic drug attenuates cardiac remodeling and systolic dysfunction following experimental myocardial infarction. Int J Cardiol 2013;168:1174–1185. pmid:23219315
  13. 13. Heuschkel S, Wohlrab J, Neubert RH. Dermal and transdermal targeting of dihydroavenanthramide D using enhancer molecules and novel microemulsions. Eur J Pharm Biopharm 2009;72:552–560. pmid:19233266
  14. 14. Lee YR, Noh EM, Oh HJ, Hur H, Kim JM, Han JH, et al. Dihydroavenanthramide D inhibits human breast cancer cell invasion through suppression of MMP-9 expression. Biochem Biophys Res Commun 2011;405:552–557. pmid:21262201
  15. 15. Lv N, Song MY, Lee YR, Choi HN, Kwon KB, Park JW, et al. Dihydroavenanthramide D protects pancreatic beta-cells from cytokine and streptozotocin toxicity. Biochem Biophys Res Commun 2009;387:97–102. pmid:19576175
  16. 16. Ponchet M, Martin-Tanguy J, Marais A, Poupet A. Dianthramides A and B, two N-benzoylanthranilic acid derivatives from elicited tissues of Dianthus caryophyllus Phytochemistry 1984;23:1901–1903.
  17. 17. Sinreih M, Sosič I, Beranič N, Turk S, Adeniji AO, Penning TM, et al. N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo-keto reductase AKR1C3. Bioorg Med Chem Lett 2012;22:5948–5951. pmid:22897946
  18. 18. Hsieh PW, Chang FR, Wu CC, Wu KY, Li CM, Chen SL, et al. New cytotoxic cyclic peptides and dianthramide from Dianthus superbus. J Nat Prod 2004;67:1522–1527. pmid:15387653
  19. 19. Harris MT, Walker DM, Drew ME, Mitchell WG, Dao K, Schroeder CE, et al. (2013) Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase. Antimicrob Agents Chemother 2013;57:3731–3737. pmid:23716053
  20. 20. Sharlow E, Golden JE, Dodson H, Morris M, Hesser M, Lyda T, et al. Identification of inhibitors of Trypanosoma brucei hexokinases. In: Probe Reports from the NIH Molecular Libraries Program. National Center for Biotechnology Information, Bethesda, MD. 2010. Available at http://www.ncbi.nlm.nih.gov/books/NBK63599/
  21. 21. Sharlow ER, Lyda TA, Dodson HC, Mustata G, Morris MT, Leimgruber SS, et al. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity. PLoS Negl Trop Dis 2010;4:e659. pmid:20405000
  22. 22. Hinsberger S, de Jong JC, Groh M, Haupenthal J, Hartmann RW. Benzamidobenzoic acids as potent PqsD inhibitors for the treatment of Pseudomonas aeruginosa infections. Eur J Med Chem 2014;76:343–351. pmid:24589489
  23. 23. Weidel E, de Jong JC, Brengel C, Storz MP, Braunshausen A, Negri M, et al. Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking. J Med Chem. 2013;56:6146–6155. pmid:23834469
  24. 24. Keasling JD. Manufacturing molecules through metabolic engineering. Science 2010;330:1355–1358. pmid:21127247
  25. 25. Sun H, Liu Z, Zhao H, Ang EL. Recent advances in combinatorial biosynthesis for drug discovery. Drug Des Devel Ther 2015;9:823–833. pmid:25709407
  26. 26. Siddiqui MS, Thodey K, Trenchard I, Smolke CD. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res 2010;12:144–170.
  27. 27. Eudes A, Baidoo EE, Yang F, Burd H, Hadi MZ, Collins FW, et al. Production of tranilast N-(3',4'-dimethoxycinnamoyl)-anthranilic acid. and its analogs in yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2011;89:989–1000. pmid:20972784
  28. 28. Chan KKJ , O'Hagan D. The rare fluorinated natural products and biotechnological prospects for fluorine enzymology. Methods Enzymol 2012;516:219–235. pmid:23034231
  29. 29. Kosjek T, Heath E. Halogenated Heterocycles as Pharmaceuticals. Top Heterocycl Chem 2012;27:219–246.
  30. 30. Yang Q, Reinhard K, Schiltz E, Matern U. Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L. Plant Mol Biol 1997;35:777–789. pmid:9426598
  31. 31. Hamberger B, Hahlbrock K. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc Natl Acad Sci U S A 2004;101:2209–2214. pmid:14769935
  32. 32. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, et al. Functional characterization of the S. cerevisiae genome by deletion and parallel analysis. Science 1999;285:901–906. pmid:10436161
  33. 33. Clausen M, Lamb CJ, Megnet R, Doerner PW. PAD1 encodes phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in Saccharomyces cerevisiae. Gene 1994;142:107–112. pmid:8181743
  34. 34. Jiang H, Wood KV, Morgan JA. Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 2005;71:2962–2969. pmid:15932991
  35. 35. Eudes A, Juminaga D, Baidoo EE, Collins FW, Keasling JD, Loqué D. Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli. Microb Cell Fact 2013;12:62. pmid:23806124
  36. 36. Geissler JF, Harwood CS, Gibson J. Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate. J Bacteriol 1988;170:1709–1714. pmid:3350788
  37. 37. Gibson J, Dispensa M, Fogg GC, Evans DT, Harwood CS. 4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation. J Bacteriol 1994;176:634–641. pmid:8300518
  38. 38. Abe I. Engineered biosynthesis of plant polyketides: structure-based and precursor-directed approach. Top Curr Chem 2010;297:45–66. pmid:21495256
  39. 39. Huth JR, Yu L, Collins I, Mack J, Mendoza R, Isaac B, et al. NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter. J Med Chem 2004;47:4851–4857. pmid:15369388
  40. 40. Öberg CT, Strand M, Andersson EK, Edlund K, Tran NP, Mei YF, et al. Synthesis, biological evaluation, and structure-activity relationships of 2-2-(benzoylamino)benzoylamino]benzoic acid analogues as inhibitors of adenovirus replication. J Med Chem 2012;55:3170–3181. pmid:22369233
  41. 41. Lavoie S, Legault J, Simard F, Chiasson É, Pichette A. New antibacterial dihydrochalcone derivatives from buds of Populus balsamifera Tetrahedron Lett 2013;54:1631–1633.
  42. 42. Simard F, Legault J, Lavoie S, Pichette A. Balsacones D-I, dihydrocinnamoyl flavans from Populus balsamifera buds. Phytochemistry 2014;100:141–149. pmid:24485585
  43. 43. D’Auria JC. Acyltransferases in plants: a good time to be BAHD. Curr Opin Plant Biol 2006;9:331–340. pmid:16616872
  44. 44. Ibdah M, Berim A, Martens S, Valderrama AL, Palmieri L, Lewinsohn E, et al. Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh. Phytochemistry 2014;107:24–31. pmid:25152451
  45. 45. Widhalm JR, Dudareva N. A familiar ring to it: biosynthesis of plant benzoic acids. Mol Plant 2015;8:83–97. pmid:25578274
  46. 46. van Pée KH, Patallo EP. Flavin-dependant halogenases involved in secondary metabolism in bacteria. Appl Microbiol Biotechnol 2006;70:631–641. pmid:16544142
  47. 47. Zeng J, Zhan J. A novel fungal Flavin-dependant halogenase for natural product biosynthesis. ChemBioChem 2010;11:2119–2123. pmid:20827793