Appl Environ Microbiol 69(5): 2699-2706

Production of Plant-Specific Flavanones by <em>Escherichia coli</em> Containing an Artificial Gene Cluster

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

^{Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81 3 5841 5123. Fax: 81 3 5841 8021. E-mail:}pj.ca.oykot-u.cce.liam@irohusa.

^{Present address: Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea.}

Received 2002 Dec 11; Accepted 2003 Feb 5.

Abstract

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins—PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata—were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P_T7) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P_T7 in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P_T7 and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.

Abstract

In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, and flavonoids. In addition to their roles in the structure and protection of the plant, phenylpropanoid compounds have an important effect on plant qualities, such as texture, flavor, color, and other characteristics (29). Recently, the flavonoid-derived plant natural products have drawn much attention because of their benefits to human health, such as antimicrobial, cancer chemopreventive, antioxidant, and antiasthmatic activities (6, 14). These phenylpropanoid and flavonoid biosynthetic enzymes are therefore attractive targets for metabolic engineering to provide enhancement or initiation of the production of economically desirable traits or compounds. The phenylpropanoid and flavonoid biosynthetic pathways and their regulation have been the subject of many studies (4, 5, 25, 29). Recent advances in the regulation of the pathways and the biochemistry of specific enzymes and enzyme complexes have opened up strategies for the enhancement of flavonoid compounds by modifying the pathways (4).

The phenylpropanoid pathway in plants converts phenylalanine into naringenin chalcone (Fig. (Fig.1).1). As the first step, phenylalanine is deaminated to yield cinnamic acid by the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is hydroxylated by cinnamate-4-hydroxylase (C4H) to 4-coumaric acid, which is then activated to 4-coumaroyl-coenzyme A (CoA) by the action of 4-coumarate:CoA ligase (4CL). Chalcone synthase (CHS) catalyzes the stepwise condensation of three acetate units from malonyl-CoA with 4-coumaroyl-CoA to yield naringenin chalcone, the precursor for a large number of flavonoids (29). Naringenin chalcone is converted to naringenin by chalcone isomerase or nonenzymatically in vitro (5, 19, 24, 25). Because some of the PALs show tyrosine ammonia lyase activity, tyrosine is also used as the precursor (2, 15, 22, 26).

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FIG. 1.

Flavanone biosynthetic pathway in plants. The dashed arrows represent the expected flavanone biosynthetic pathway in E. coli containing the artificial gene cluster including PAL, 4CL, and CHS. TAL, tyrosine ammonia-lyase; CHI, chalcone isomerase.

Production of flavanones or flavonoids by genetically engineered bacteria has not been reported before, although the heterologous expression of phenylpropanoid biosynthetic enzymes in bacteria has been reported (1, 8, 16, 30). One of the barriers for the production of these compounds is the difficulty in expression of active C4H, which would not be efficiently expressed due to its instability and the lack of its specific cytochrome P-450 reductase in bacteria (8, 20). We have recently discovered a 4CL in the gram-positive filamentous bacterium Streptomyces coelicolor A3(2) that can activate cinnamic acid to cinnamoyl-CoA, as well as 4-coumaric acid to 4-coumaroyl-CoA (12). The use of the bacterial 4CL enzyme would bypass the C4H step for the production of pinocembrin chalcone from phenylalanine via the phenylpropanoid pathway (Fig. (Fig.1).1). On the basis of this idea, we constructed an artificial gene cluster containing PAL from a yeast, Rhodotorula rubra; 4CL from an actinomycete, S. coelicolor A3(2); and CHS from a licorice plant, Glycyrrhiza echinata, to produce a plant-specific flavanone, pinocembrin. As expected, the Escherichia coli cells containing the gene cluster produced pinocembrin. In addition, the E. coli cells produced naringenin, because the yeast PAL enzyme also used tyrosine as a substrate and yielded 4-coumaric acid, as is found for many PALs (2, 22, 26), and the 4CL enzyme used cinnamic acid and 4-coumaric acid to yield the corresponding CoA thioester compounds. This paper describes successful production of plant-specific flavanones, pinocembrin and naringenin, in E. coli. Furthermore, we compared the yields of the flavanones by E. coli cells that contained the gene clusters in different organizations.

Acknowledgments

E. I. Hwang was supported by the Japan Society for the Promotion of Science. This work was supported by a research grant from the Noda Institute for Scientific Research, by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-03-VI-2-1), and by a grant from the Industrial Technology Research Grant Program 2000 of the New Energy and Industrial Technology Development Organization of Japan (00A03004).

Acknowledgments

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