Bacterial strains, plasmids, and culture conditions. E. coli
JM109 and pUC18, purchased from Takara Biochemicals, were used for DNA manipulation (23
). E. coli
BL21(DE3) and pET26b, purchased from Novagen, were used for the production of flavanones.
Restriction enzymes, T4 DNA ligase, and Taq
DNA polymerase were purchased from Takara Biochemicals. Recombinant DNA techniques were described previously (23
cDNA from R. rubra
(4a) was obtained from S. Kawai, Tokyo University of Agriculture and Technology. CHS
cDNA from G. echinata
L. was from T. Akashi and S. Ayabe, Nihon University (T. Akashi, unpublished data). 4CL
from S. coelicolor
A3(2) was cloned and characterized in our laboratory (12
). After the DNA manipulation, the absence of undesired alterations during PCR was checked by nucleotide sequencing on an automated nucleotide sequencer.
Construction of pET26b-3GS.
Plasmid pET26b-3GS was constructed by standard DNA manipulation, including several cycles of fragment-primed PCR as follows (Fig. ). Using PAL, 4CL, or CHS cDNA as a template, seven DNA fragments were amplified by PCR with appropriate pairs of primers (Table ). Fragments 1, 4, and 7 were cloned into pUC18 using EcoRI plus HindIII, BamHI plus HindIII, and HindIII plus BamHI, resulting in 1-pUC18, 4-pUC18, and 7-pUC18, respectively. Fragments 2 and 3 were connected by a fragment-primed PCR procedure and cloned between the BamHI and HindIII sites of pUC18, resulting in 23-pUC18. Fragments 5 and 6 were also connected by a fragment-primed PCR procedure and cloned between the HindIII and BamHI sites of pUC18, resulting in 56-pUC18. The absence of undesired alterations in PCR was confirmed by nucleotide sequencing. A 0.5-kb BstPI-HindIII fragment from 4-pUC18 was cloned between the corresponding sites of 23-pUC18, resulting in 234-pUC18. A 1.8-kb EcoRI-SplI fragment from 1-pUC18 was cloned between the corresponding sites of 234-pUC18, resulting in 1234-pUC18. A 0.7-kb NdeI-BamHI fragment from 7-pUC18 was cloned between the corresponding sites of 56-pUC18, resulting in 567-pUC18. Finally, both a 3.1-kb NdeI-HindIII fragment from 1234-pUC18 and a 1.8-kb HindIII-BamHI fragment from 567-pUC18 were cloned between the NdeI and BamHI sites of pET26b by three-fragment ligation, resulting in pET26b-3GS. In this construction, the start codons of 4CL and CHS overlapped the termination codons of the preceding genes.
FIG.2. Schematic representation of the strategies used for construction of pET26b-3GS, pET26b-rbs-3GS, and pET26b-PT7-3GS. The following abbreviations are used for restriction enzymes: S, SplI; Bs, BstPI; H, HindIII; N, NdeI; E, EcoRI; and B, BamHI. A HindIII (more ...) Construction of pET26b-rbs-3GS and pET26b-PT7-3GS.
An ~0.4-kb 3′ fragment (nucleotide positions 587 to 713, taking the first nucleotide of the translational start codon of PAL as 1) of PAL was amplified by PCR using two primers, PAL-F-EcoRI and PAL-R-BamHI (Table ), and pET-3GS as a template. The amplified fragment was cloned into pUC18 using the EcoRI and BamHI sites, resulting in pUC18-3′-fragment-PAL. An EcoRI-SplI fragment of 1234-pUC18 and an SplI-BamHI fragment of pUC18-3′-fragment-PAL was cloned between the EcoRI and BamHI sites of pUC18 by three-fragment ligation to create pUC18-PAL. PAL was excised from pUC18-PAL by using NdeI plus BamHI and cloned into pET26b, resulting in pET26b-PAL (Fig. ). For construction of pET16b-4CL, 4CL was amplified by PCR from pUC18-4CL as a template with the two primers 4CL-F-NcoI and 4CL-R-BamHI. The amplified fragment was cloned into previously prepared pUC18N, which had an NcoI site instead of the original SmaI site of pUC18. 4CL was cloned into pET16b by using NcoI and BamHI, resulting in pET16b-4CL. For construction of pET16b-CHS, CHS was amplified by PCR from pUC18-CHS as a template with the two primers CHS-F-NcoI and CHS-R-BamHI. The amplified fragment was cloned into pUC18N and recombined into pET16b, resulting in pET16b-CHS. 4CL with the T7 promoter (PT7) and/or the ribosome-binding sequence (RBS) was then amplified by PCR from pET16b-4CL as a template with two primer sets, rbs-4CL-F-BamHI plus 4CL-R-EcoRI and PT7-4CL-F-BamHI plus 4CL-R-EcoRI, respectively. Both amplified fragments were cloned into pUC18. CHS with PT7 and/or RBS was similarly amplified (primer sets rbs-CHS-F-EcoRI plus CHS-R-HindIII and PT7-CHS-F-EcoRI plus CHS-R-HindIII, respectively) and cloned into pUC18. Finally, rbs-4CL and rbs-CHS or PT7-4CL and PT7-CHS were introduced into pET26b-PAL stepwise to construct pET26b-rbs-3GS and pET26b-PT7-3GS, respectively.
Expression and fermentation.
E. coli BL21(DE3) harboring pET26b-3GS, pET26b-rbs-3GS, or pET26b-PT7-3GS was precultured in Luria-Bertani liquid medium containing 5 μg of kanamycin/ml at 30°C for 16 h with reciprocal shaking. Isopropyl β-d-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM. After an incubation period of 2 h at 26°C, the cells were harvested by centrifugation and disrupted by sonication to analyze the total proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Soluble fractions were obtained by ultracentrifugation of the cell lysates.
For identification of compounds produced by E. coli harboring the expression plasmids or pET26b, a portion (20 ml) of the preculture was inoculated into 200 ml of M9 medium with 5 μg of kanamycin/ml and cultured for 5 h at 26°C in the presence of 1 mM IPTG. The cells were then harvested and washed once with M9 medium. A portion of the cells (500 mg [wet weight]) was transferred to 200 ml of fresh M9 medium with 5 μg of kanamycin/ml and 1 mM IPTG and cultivated at 26°C for 65 h with reciprocal shaking.
Extraction and analysis of flavanones.
The culture broth was prepared, and the pH was adjusted to pH 9.0 with NaOH. After the broth had stood at room temperature for 1 h, the materials in the broth were extracted with the same volume of ethyl acetate. The organic layer was evaporated to dryness, and the residue was dissolved in 100 ml of acetonitrile containing 0.1% acetic acid for high-performance liquid chromatography (HPLC) analysis on a Waters 600E chromatograph. The compounds produced were separated on a reversed-phase DOCOSIL-B column (C22; Senshu Scientific Co.), maintained at 40°C, by elution with an acetonitrile-water gradient, both containing 0.1% acetic acid, at a flow rate of 1.0 ml/min. The HPLC conditions were as follows: for detection of 4-coumaric acid, cinnamic acid, and naringenin, 20 to 30% CH3CN for 55 min and 30 to 100% CH3CN for 5 min; for detection of pinocembrin, 30 to 40% CH3CN for 10 min, 40% CH3CN for 30 min, and 40 to 100% CH3CN for 5 min. Absorbances at 309, 277, and 290 nm were monitored for 4-coumaric acid, cinnamic acid, and flavanones, respectively. The retention times of the authentic samples under these HPLC conditions were 8.67 (4-coumaric acid), 34.29 (cinnamic acid), 45.56 (naringenin), and 31.89 (pinocembrin) min. These authentic compounds, except for pinocembrin, were purchased from Sigma-Aldrich. Pinocembrin was a gift from H. Kuroda, Kyoto University.
Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) was performed on a Thermo Quest LCQ apparatus equipped with a reversed-phase DOCOSIL-B column with detection at 254 and 290 nm under the same conditions as for HPLC. The negative ion values by LC-APCIMS and the retention times of the authentic compounds were as follows: 4-coumaric acid, m/z 163.3[M − H]−, 9.22 min; cinnamic acid, m/z 147.5[M − H]−, 33.76 min; naringenin, m/z 271.3[M − H]−, 44.67 min; pinocembrin, m/z 255.4[M − H]−, 31.01 min. The m/z values and retention times of the four compounds produced by E. coli harboring pET26b-3GS were identical to those of the respective authentic samples.