Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster

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.

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.

FIG.2.

Schematic representation of the strategies used for construction of pET26b-3GS, pET26b-rbs-3GS, and pET26b-P_T7-3GS. The following abbreviations are used for restriction enzymes: S, SplI; Bs, BstPI; H, HindIII; N, NdeI; E, EcoRI; and B, BamHI. A HindIII site (H∗) was created within the 4CL coding sequence without changing the amino acid sequence. By DNA manipulation, including several cycles of fragment-primed PCR, PAL encoding a 713-amino-acid (aa) protein, 4CL encoding a 522-aa protein, and CHS encoding a 388-aa protein are placed under the control of the T7 promoter and an RBS in a high-copy-number vector, pET26b. In pET26b-3GS, the start codons of 4CL and CHS are overlapped with the stop codons of the preceding genes. In pET26b-rbs-3GS, the RBS is placed in front of all three genes. In pET26b-P_T7-3GS, both the RBS and the T7 promoter are placed in front of the three genes.

FIG. 3.

HPLC analysis of the culture broth of E. coli harboring pET26b-3GS or pET26b. For detection of peaks 1, 2, and 3 (4-coumaric acid, cinnamic acid, and naringenin, respectively) and of peaks 1, 2, and 4 (4-coumaric acid, cinnamic acid, and pinocembrin, respectively), different elutions were used. See Materials and Methods for the details of the HPLC conditions. Ab, absorbance.

FIG. 4.

Selected ion chromatograms by LC-APCIMS of the compounds produced by E. coli BL21(DE3) harboring pET26b-3GS (top) and pET26b (bottom). Peaks 1 to 4 were measured on the basis of m/z 162.5-163.5, 146.5-147.5, 270.5-271.5, and 254.5-255.5[M − H]⁻, respectively, obtained from the authentic samples. Peak identities: 1, 4-coumaric acid; 2, cinnamic acid; 3, naringenin; 4, pinocembrin. See Materials and Methods for the details of HPLC and LC-APCIMS protocols.

FIG. 5.

SDS-polyacrylamide gel electrophoresis of the soluble fractions of E. coli harboring the expression plasmids. The soluble fractions of E. coli BL21(DE3) harboring pET26b as a control (lane 1), pET26b-3GS (lane 2), pET26b-rbs-3GS (lane 3), and pET26b-P_T7-3GS (lane 4) were run, together with molecular mass markers (lane M).