Appl Environ Microbiol 71(6): 2962-2969

Metabolic Engineering of the Phenylpropanoid Pathway in <em>Saccharomyces cerevisiae</em>

School of Chemical Engineering,^{Department of Chemistry, Purdue University, West Lafayette, Indiana 47907}²

^{Corresponding author. Mailing address: School of Chemical Engineering, Purdue University, FRNY Hall, West Lafayette, IN 47907-2050. Phone: (765) 494-4088. Fax: (765) 494-0805. E-mail:}ude.eudrup@nagromaj.

Received 2004 Jul 7; Accepted 2004 Dec 18.

Abstract

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter^{of naringenin and 0.8 mg liter}^{of pinocembrin. Several by-products, such as 2′,4′,6′-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor}l-tyrosine.

Abstract

Flavonoids are a class of widely distributed water-soluble plant pigments derived from the phenylpropanoid pathway, with more than 6,000 identified so far (13). In addition to their in planta function of protecting plants from UV irradiation and attacks by fungi and animals (13), they have also been shown to possess anti-inflammatory, antiallergenic, and antioxidant activities in humans (3, 6, 13, 24, 26). Many flavonoids are reported to possess activities against certain cancer types, such as skin cancer, breast cancer, and colon cancer (4, 8, 11, 22, 29). Studying the effects of specific flavonoids requires their purification from plant tissue, which is often difficult because of the low concentrations of certain flavonoids and numerous similar natural products. The productivity of flavonoids is also limited by the low growth rates of plants. The chemical synthesis of flavonoids can be achieved from simple starting materials. However, extreme reaction conditions and toxic chemicals are required (12). Therefore, the transfer of plant metabolic pathways into heterologous hosts such as bacteria or Saccharomyces cerevisiae is an attractive alternative source of flavonoids.

Several groups have recently begun to reconstitute the early steps of the phenylpropanoid pathway in microbes such as Escherichia coli (17, 37) and Saccharomyces cerevisiae (30). In plants, the biosynthesis of naringenin, the central precursor of most flavonoids, involves the following five enzymes in the phenylpropanoid pathway: phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:coenzyme A (CoA) ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) (Fig. (Fig.1).1). PALs from some plants, for example, Zea mays L., also have tyrosine ammonia lyase (TAL) activity, converting tyrosine to p-coumaric acid (Fig. (Fig.1),1), the substrate of 4CL (31). Since tyrosine already has a 4-hydroxyl group, this pathway bypasses C4H. In addition, naringenin chalcone cyclizes in acidic solution without requiring the enzyme CHI (14, 27). The production of flavanones in genetically engineered E. coli was first reported by Hwang et al. (17). In their study, an artificial gene cluster containing PAL, 4CL, and CHS was constructed, and E. coli cells expressing these three enzymes produced two flavanones, pinocembrin and naringenin. Recently, Watts and coworkers cloned a bacterial TAL gene which was coexpressed with 4CL and CHS in E. coli. Their study showed that naringenin production could reach levels as high as 20.8 mg liter⁽37). Ro and Douglas connected the first two enzymes in S. cerevisiae by coexpressing Populus PAL, C4H, and cytochrome P450 reductase. They evaluated the carbon flux through the multienzyme system from phenylalanine to p-coumaric acid in yeast (30).

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FIG. 1.

Proposed reactions catalyzed by S. cerevisiae AH22 coexpressing PAL, 4CL, and CHS. PAL (TAL), phenylalanine (tyrosine) ammonia lyase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; Padp1, phenylacrylic acid decarboxylase. Overexpressed proteins are shown in bold. The biosynthesis of naringenin in plants is shown in the box.

In this study, we describe the production of naringenin and pinocembrin by construction of the phenylpropanoid pathway in the yeast S. cerevisiae. We chose S. cerevisiae as the host because it has some advantages over E. coli for expressing certain eukaryotic heterologous proteins. We hypothesized that plant enzymes would be better expressed in a eukaryotic host since yeast is capable of performing posttranslational modifications of the eukaryotic proteins. In addition, yeast has similar intracellular compartments to those of plant cells. Furthermore, several cytochrome P450 (CYP) enzymes are involved in flavonoid biosynthesis, and yeast has been shown by several groups to be an excellent host for in vivo CYP activity (16, 19, 28, 33, 36). One reason for this is the presence of an endoplasmic reticulum, which is where CYP and CYP reductase are targeted in plants.

We inserted PAL from the red yeast Rhodosporidium toruloides, 4CL from the plant Arabidopsis thaliana, and CHS from the plant Hypericum androsaemum into a yeast expression vector. Each gene was under the control of its own galactose-inducible promoter. Yeast harboring this vector produced naringenin and pinocembrin through the phenylpropanoid pathway, as well as four by-products, two of which were identified as phloretin and 2′,4′,6′-trihydroxydihydrochalcone through a sequential side reaction (Fig. (Fig.1).1). To our knowledge, this is the first study with a successful synthesis of flavonoids in a heterologous eukaryotic system.

Acknowledgments

We thank BioMarin Pharmaceutical Inc. (Novato, Calif.) for the phenylalanine ammonia lyase gene, C. Chapple (Biochemistry, Purdue University) for the 4-coumarate:CoA ligase gene, L. Beerhues (Institute of Pharmaceutical Biology, Germany) for the chalcone synthase gene, and N. Ho (LORRE, Purdue University) for the GAL10 promoter sequence, the XKS1 termination sequence, the pKS2μHyg plasmid, and S. cerevisiae AH22. We thank M. Sedlak for his helpful suggestions and technical assistance and G. Shaner for his help with revising the manuscript. We also thank D. Winski and D. Miles for their help with sample analysis.

Acknowledgments

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