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Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Sueharu Horinouchi
asuhori{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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100 mg l–1. Furthermore, this system gave approximately 60 mg curcumin l–1 from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.
Three supplementary figures with additional spectrometric data on the curcuminoids are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Researches over the last few decades have shown that the medicinal properties of turmeric are attributable mainly to curcumin. Curcumin, which belongs chemically to a class of curcuminoids that are bis-
,β-unsaturated β-diketones (Fig. 1), possesses various pharmacological effects, including anti-inflammatory, antioxidant, anticarcinogenic and antitumor activities (Duvoix et al., 2005; Sharma et al., 2005; Maheshwari et al., 2006; Anand et al., 2007). Recent clinical trials have demonstrated the efficacy of curcumin in various diseases, such as colorectal cancer and Alzheimer's disease (Shishodia et al., 2007). In addition, curcumin possesses hepatoprotective activity on liver injury; Nanji et al. (2003), Bruck et al. (2007) and Muriel & Rivera-Espinoza (2008) reported that curcumin prevents thioacetamide-induced liver fibrosis and alcohol-induced liver diseases. Despite the strong potential of curcumin in the prevention and cure of various diseases, the only natural source of curcuminoids is Curcuma species; curcuminoids and related compounds have been found exclusively in plants of the order Zingiberales.
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; Kaneko et al., 2003; Miyahisa et al., 2005). Watts et al. (2004) and Yan et al. (2005a) also reported metabolic engineering for production of flavonoids in E. coli. Furthermore, co-incubation of two microbial hosts, E. coli expressing the phenylpropanoid pathway and producing flavanones and Saccharomyces cerevisiae expressing isoflavone synthase, led to production of the isoflavone genistein (Katsuyama et al., 2007d). Jiang et al. (2005) and Yan et al. (2005b) reported the production of flavonoids by Saccharomyces cerevisiae. Resveratrol was also produced in micro-organisms and mammalian cells (Zhang et al., 2006; Beekwilder et al., 2006; Katsuyama et al., 2007a). Recently, Beekwilder et al. (2007) reported the production of a raspberry ketone by E. coli. The successful production of flavonoids and stilbenes depends on efficient expression of type III polyketide synthases (PKSs), responsible for the key step of extension and cyclization of polyketides to yield flavanone and stilbene skeletons in the respective organisms (Schröder, 1997; Austin & Noel, 2003). We have improved the artificial pathways for the purpose of production of natural and ‘unnatural’ plant-specific polyketides, including flavonoids, isoflavones and stilbenes, and established a system in which, in principle, each of the three steps is carried on a plasmid: a substrate synthesis step for CoA ester synthesis from carboxylic acids by 4-coumarate : CoA ligase; a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthases and chalcone isomerase and into stilbenes by stilbene synthase; and a modification step for modification of the flavanones and stilbenes by various modification enzymes (Katsuyama et al., 2007c). This system is useful and convenient in production of various polyketides by employing various combinations of enzyme genes at each step. In fact, 87 polyketides, including 36 unnatural flavonoids and stilbenes, were produced by recombinant E. coli cells containing various combinations of enzyme genes at the three steps (Katsuyama et al., 2007c).
We have recently identified a type III PKS, named CUS (curcuminoid synthase), that is capable of synthesis of curcuminoids from 4-coumaroyl-CoA and malonyl-CoA in a unique manner (Katsuyama et al., 2007b). The CUS type III PKS is from rice, Oryza sativa, which is phylogenetically distinct from Zingiberales. CUS catalyses the synthesis of bisdemethoxycurcumin [3] from two molecules of p-coumaroyl-CoA and one molecule of malonyl-CoA (Fig. 1a
). CUS also accepts other phenylpropanoid-derived CoA esters, such as cinnamoyl-CoA and feruloyl-CoA, as a substrate to produce dicinnamoylmethane [4] and curcumin [1], respectively. In addition to curcuminoids, CUS produces triketide pyrones, [5] to [7], as by-products, by condensing two malonyl-CoAs to p-coumaroyl-CoA (Fig. 1a). The discovery of CUS prompted us to employ it as a type III PKS at the polyketide synthesis step in the artificial biosynthesis pathway for microbial production of plant-specific curcuminoids.
Here we describe the efficient production of curcuminoids by E. coli carrying an artificial biosynthesis pathway including CUS and enzymes of the phenylpropanoid pathway (Fig. 1b). Exogenous supplementation of tyrosine or phenylalanine, the precursors of the phenylpropanoid pathway, to the recombinant E. coli cells and incubation of them in minimal medium resulted in the production of curcuminoids, such as bisdemethoxycurcumin [3], dicinnamoylmethane [4], and cinnamoyl-p-coumaroylmethane [8]. Rice bran pitch, an industrial waste residue from production of rice edible oil, was also successfully used as a source of ferulic acid to yield curcumin.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). pCDF-PAL/LE4CL-1 (Katsuyama et al., 2007a), pCDF-LE4CL-1 (Katsuyama et al., 2007c), pRSF-ACC (Miyahisa et al., 2005), and pET-CUS (Katsuyama et al., 2007b) were described previously. For the structures and the selection markers of these plasmids, see Fig. 1(c).
Production of curcuminoids by E. coli.
Ampicillin (100 µg ml–1), kanamycin (50 µg ml–1) and streptomycin (50 µg ml–1) were used when necessary. Recombinant E. coli BLR(DE3) cells were precultured overnight at 37 °C in 2 ml Luria–Bertani (LB) medium. The preculture was transferred into 100 ml LB medium and cultured at 26 °C until the OD600 reached 0.6. IPTG was then added at a final concentration of 1 mM and the culture was continued for 5 h. The cells were harvested by centrifugation and the cells (0.5 g wet weight) were suspended in 20 ml M9 or LB medium. To the cell suspension (25 g l–1 wet weight) in M9 medium, 3 mM each of amino acids (tyrosine and phenylalanine) or 1 mM each of carboxylic acids, plus 40 g glucose l–1, 25 g CaCO3 l–1, appropriate antibiotics and 1 mM IPTG were added, and the culture was incubated at 26 °C for 60 h. Then 2 ml of the culture broth was taken and adjusted to pH 3.0 with 6 M HCl. After extraction with an equal volume of ethyl acetate, materials were concentrated by evaporation and dissolved in 100 µl DMSO for HPLC and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) analyses. LC-APCIMS in positive mode was performed on an Esquire High-capacity Trap Plus (Bruker Daltonics) equipped with a PEGASIL-B C4 column (4.6x200 mm; Senshu Scientific Co.) and the sample was eluted with a gradient of acetonitrile in water (both containing 0.1 % acetic acid) at a flow rate of 1 ml min–1. The conditions of the gradient were 10–100 % acetonitrile for 45 min. UV spectra were detected on an Agilent 1100 series UV detector. HPLC with a Senshu PEGASIL-B C4 reversed-phase HPLC column (4.6x250 mm) was carried out on a Hitachi LaChrom ELITE system and the sample was eluted with a linear acetonitrile gradient (10–100 % over 45 min) in water containing 0.1 % trifluoroacetic acid at a flow rate of 1.0 ml min–1. UV spectra were acquired on a Hitachi L-2450 diode array detector. Commercially available curcumin [1] and NMR spectroscopically pure bisdemethoxycurcumin [3], cinnamoyl-p-coumaroylmethane [8], dicinnamoylmethane [4] and a triketide pyrone [5] derived from p-coumaroyl-CoA were used to prepare calibration curves.
Preparation of rice bran pitch medium.
Rice bran pitch medium was prepared as described by Zheng et al. (2007). Briefly, 0.5 g rice bran pitch was dissolved in a solution containing 50 ml each of 0.4 M NaOH and ethanol, heated to 90 °C, hydrolysed for 5 h and cooled to 4 °C. After precipitates had been removed by filtration, the filtrate was evaporated to remove ethanol. To 40 ml of the solution, 5 ml sterilized 40 % glucose, 5 ml solution A (6 g Na2HPO4 l–1, 3 g KH2PO4 l–1, 0.5 g NaCl l–1 and 1 g NH4Cl l–1), 50 µl 1 M MgSO4 (final concentration, 1 mM), 50 µl 100 mM CaCl2 (final, 0.1 mM), 50 µl 100 mg ampicillin ml–1 (final, 100 µg ml–1), 50 µl 50 mg kanamycin ml–1 (final, 50 µg ml–1), 50 µl 50 mg streptomycin ml–1 (final, 50 µg ml–1), and 50 µl 1 M IPTG (final, 1 mM) were added. The solution prepared in this way was used as a source of ferulic acid for curcumin production.
Structural elucidation of curcuminoids.
Curcumin [1] was identified by comparing the retention time, UV spectra and MS/MS fragments with those of the authentic sample. Bisdemethoxycurcumin [3], cinnamoyl-p-coumaroylmethane [8] and dicinnamoylmethane [4] were identified by NMR spectroscopic studies and LC-APCIMS analysis. Each of the curcuminoids was prepared from a 200 ml-scale M9 culture as described above and purified by reversed-phase preparative HPLC. Bisdemethoxycurcumin [3] and cinnamoyl-p-coumaroylmethane [8] were purified by HPLC on an instrument equipped with a Senshu DOCOSIL-B C22 column (20x250 mm) by elution with 30 % and 90 % acetonitrile, respectively, in water containing 0.1 % trifluoroacetic acid. Dicinnamoylmethane [4] was purified by HPLC on an instrument equipped with a PEGASIL-B C4 column (10x250 mm) by elution with 90 % acetonitrile in water containing 0.1 % trifluoroacetic acid. Spectroscopic data of the curcuminoids were as follows.
Bisdemethoxycurcumin [3]. 1H NMR (500 MHz, CD3OD): 
=7.57 (d, 2H, J=15.5 Hz), 7.48 (d, 4H, J=8.5 Hz), 6.81 (d, 4H, J=8.5 Hz), 6.58 (d, 2H, J=15.5 Hz), 5.94 (s, 1H). HPLC: Rt=26.0. LC-APCIMS (positive): MS, m/z 309 [M+H]+, MS/MS (precursor ion at m/z 309), m/z 147 (47) [C9H7O2]+ m/z 225 (100) [M+H–C4H4O2]+. HRMS (ESI): found for [C19H16O4+H]+, 309.11593, calcd, 309.11268. UV: 
max 417 nm.
Cinnamoyl-p-coumaroylmethane [8]. 1H NMR (500 MHz, CDCl3): =7.67 (d, 1H, J=16 Hz), 7.64 (d, 1H, J=16 Hz), 7.57 (dd, 2H, J=2, 7.5 Hz), 7.49 (d, 2H, J=8.5 Hz), 7.40 (m, 3H), 6.87 (d, 2H, J=8.5 Hz), 6.63 (d, 1H, J=16 Hz), 6.52 (d, 1H, J=16 Hz), 5.83 (s, 1H). HPLC: Rt=30.3. LC-APCIMS (positive): MS, m/z 293 [M+H]+, MS/MS (precursor ion at m/z 293), m/z 131 (17) [C9H7O1]+ m/z 147 (26) [C9H7O2]+ m/z 209 (100) [M+H–C4H4O2]+. HRMS (ESI): found for [C19H16O3+H]+, 293.11578, calcd, 293.11777. UV: max 404 nm.
Dicinnamoylmethane [4]. 1H NMR (500 MHz, CDCl3): =7.68 (d, 2H, J=16 Hz), 7.57 (dd, 4H, J=2, 7 Hz), 7.39 (m, 6H), 6.65 (d, 2H, J=16 Hz), 5.87 (s, 1H). HPLC: Rt=35.5. LC-APCIMS (positive): MS, m/z 277 [M+H]+, MS/MS (precursor ion at m/z 277), m/z 131 (100) [C9H7O1]+ m/z 193 (61) [M+H–C4H4O2]+. HRMS (ESI): found for [C19H16O2+H]+, 277.11792, calcd, 277.12285. UV: max 390 nm.
Curcumin [1]. HPLC: Rt=26.8. LC-APCIMS (positive): MS, m/z 369 [M+H]+, MS/MS (precursor ion at m/z 369), m/z 175 (100) [C10H7O3]+, m/z 177 (68) [C10H9O3]+ m/z 245 (91) [C14H13O4]+ m/z 285 (91) [M+H–C4H4O2]+. UV: max 426 nm.
Demethoxycurcumin [2]. HPLC: Rt=26.4. LC-APCIMS (positive): MS, m/z 339 [M+H]+.
Cinnamoylferuloylmethane [9]. HPLC: Rt=31.1. LC-APCIMS (positive): MS, m/z 323 [M+H]+.
The details of 13C-, homonuclear correlation spectroscopy (COSY)-, and heteronuclear multiple-bond correlation (HMBC)-NMR data, MS/MS spectra, and UV spectra are also described in Supplementary Figs S1, S2 and S3, available with the online version of this paper.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Miyahisa et al., 2005). The gene cluster for the first step for the synthesis of CoA esters of tyrosine and phenylalanine consisted of PAL, encoding phenylalanine ammmonia-lyase (PAL), from the yeast Rhodotorula rubra and 4CL (ScCCL), encoding 4-coumarate : CoA ligase (4CL), from Streptomyces coelicolor A3(2). The enzymes for the second step for the extension of the carbon chains by successive condensation with malonyl-CoA and cyclization of the resultant products to form flavanones consisted of chalcone synthase from liquorice (Glycyrrhiza echinata) and chalcone isomerase from a Pueraria plant. In this case, the four enzyme genes were each under the control of the T7 promoter and a synthetic ribosome-binding sequence and assembled on a single plasmid, pET-PT7-4GS (Hwang et al., 2003; Miyahisa et al., 2005, 2006). In addition to this plasmid, we also constructed plasmid pRSF-ACC, in which the acetyl-CoA carboxylase (ACC) genes from Corynebacterium glutamicum were placed under the control of the T7 promoter and the ribosome-binding sequence to increase the intracellular pool of malonyl-CoA in E. coli. The recombinant E. coli cells harbouring the two plasmids pET-PT7-4GS and pRSF-ACC produced naringenin from tyrosine and pinocembrin from phenylalanine. In the case of stilbene production by E. coli, on the other hand, the first step for the CoA ester synthesis from tyrosine and phenylalanine by PAL and 4CL and the second step for stilbene synthesis from the CoA esters by stilbene synthase were separately placed on different plasmids. Recombinant E. coli cells harbouring pRSF-PAL/LE4CL-1 containing PAL from R. rubra and 4CL (LE4CL-1) from the plant Lithospermum erythrorhizon, pET-STS containing the stilbene synthase gene from peanut (Arachis hypogaea) and pRSF-ACC produced stilbenes (Katsuyama et al., 2007a). The successful production of flavonoid and stilbene compounds by E. coli prompted us to design an artificial gene cluster and express it for the synthesis of curcuminoids in E. coli.
Our strategy to produce curcuminoids by E. coli on the basis of the catalytic properties of CUS is depicted in Fig. 1(b). This pathway included two steps: a substrate synthesis step for CoA ester synthesis from tyrosine and phenylalanine and a polyketide synthesis step for conversion of the CoA esters into curcuminoids by CUS. At the first step, PAL converts tyrosine and phenylalanine to the corresponding phenylpropanoid acids, p-coumaric acid and cinnamic acid, respectively, which are then activated to CoA esters by 4CL. The plasmid, pCDF-PAL/LE4CL-1, for the first step contained PAL from R. rhodotorula and 4CL from L. erythrorhizon, both of which were under the control of the T7 promoter and the ribosome-binding sequence in the vector pCDFDuet-1 (Fig. 1c). At the second step, CUS condenses two molecules of the CoA ester of the phenylpropanoid acid with one molecule of malonyl-CoA to produce curcuminoids. The plasmid, pET-CUS, for the second step contained the CUS gene under the control of the T7 promoter and the ribosome-binding sequence in the vector pET16b (Fig. 1c). Both plasmids and pRSF-ACC had different replication origins and different selective markers, thus being maintained in the same E. coli cell.
Production of curcuminoids from tyrosine and phenylalanine
Recombinant E. coli BLR(DE3) cells harbouring pRSF-PAL/LE4CL-1, pRSF-ACC and pET-CUS were grown in LB medium supplemented with antibiotics to maintain the plasmids and with IPTG to induce the T7 promoter, and the cells were harvested by centrifugation. The harvested cells were suspended to give a concentration of 25 g wet weight cells per litre in LB medium supplemented with the antibiotics and IPTG and incubated at 26 °C for 60 h. Under the incubation conditions, no increase in cell mass was observed, as was found for the production of flavonoids (Miyahisa et al., 2005) and stilbenes (Katsuyama et al., 2007a). The culture broth was extracted with ethyl acetate and the extract was analysed by HPLC. The HPLC analysis revealed production of three compounds [3, 4 and 8] when detected by the absorbance at 417 nm and of two compounds [5 and 6] when detected at 360 nm (Fig. 2). The hydrophobic compounds [3, 4 and 8] were purified and identified as bisdemethoxycurcumin [3], cinnamoyl-p-coumaroylmethane [8] and dicinnamoylmethane [4] by LC-MS/MS and 1H-, 13C-, COSY- and HMBC-NMR analyses. The NMR spectra (1H, 13C, COSY and HMBC) of the bisdemethoxycurcumin produced by E. coli were identical to those of the authentic sample. Similarly, compounds [5] and [6] were both identified as a triketide pyrone. Details of the spectrometric data are described in Methods and Supplementary Figs S1–S3. These findings showed that the recombinant E. coli cells successfully produced curcuminoids, as we had expected. Most of the curcuminoids were found in the cell (data not shown).
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. As a representative, the yield of the triketide pyrone derived from p-coumaric acid was determined. The yield of this triketide pyrone [5] (22±7 mg l–1) was greater than that (6 mg l–1) of the major curcuminoid [4] in this reaction. Previous study revealed that CUS changes the product ratio between curcuminoids and pyrones depending on the substrate concentrations (Katsuyama et al., 2007b). For instance, when the reaction was started with the concentration of p-coumaroyl-CoA 10-fold higher than that of malonyl-CoA, curcuminods became predominant as a product. The low yield of curcuminoids is therefore due to low concentrations of p-coumaroyl-CoA and cinnamoyl-CoA, derived from the endogenous tyrosine and phenylalanine, respectively, in the E. coli cell by the actions of PAL and 4CL.
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). Supplementation of tyrosine and phenylalanine greatly enhanced the production of bisdemethoxycurcumin [3] and dicinnamoylmethane [4], respectively. When both phenylalanine and tyrosine were supplied, the predominant product was dicinnamoylmethane. Furthermore, the yield of dicinnamoylmethane from phenylalanine was greater than that of bisdemethoxycurcumin from tyrosine. Although CUS prefers p-coumaroyl-CoA as a substrate, approximately two times more in comparison to cinnamoyl-CoA (Katsuyama et al., 2007b), the yield of dicinnamoylmethane was greater than that of bisdemethoxycurcumin in all reactions. This may be caused by the different rates of incorporation of tyrosine and phenylalanine into the pathway, which results from the substrate preferences of PAL and 4CL.
Unexpectedly, large amounts of the triketide pyrones [5] and [6] were detected in each reaction, although we expected a decrease in the amounts of the pyrones by supplying excess amounts of the amino acids. We examined the yield of the triketide pyrone derived from p-coumaric acid in the tyrosine-supplemented reaction. The yield of the triketide pyrone [5] reached 209±18 mg l–1 and was greater than that of bisdemethoxycurcumin [3] (Table 1). This result suggests that supplementation of an excess of tyrosine increased the total amount of p-coumaroyl-CoA but that newly generated p-coumaroyl-CoA was consumed by CUS rather than being accumulated in the cell to exceed the concentration of malonyl-CoA. This idea was supported by the following observations. When the intracellular concentration of malonyl-CoA was decreased by omitting pRSF-ACC from the reaction system, the ratio of bisdemethoxycurcumin [3] (yield, 3 mg l–1) to the triketide pyrone [5] (yield, less than 0.1 mg l–1) was improved approximately 20-fold, although the yield of bisdemethoxycurcumin [3] decreased more than 10-fold.
Production of curcuminoids from carboxylic acids
We next tried to improve the yields of curcuminoids by directly supplying phenylpropanoid acids to E. coli cells carrying 4CL, CUS and ACC (Fig. 3), on the assumption that the removal of the PAL step converting the amino acids to the corresponding carboxylic acids would increase the p-coumaroyl-CoA concentration in the E. coli cell. The recombinant E. coli cells harbouring pCDF-LE4CL-1, pRSF-ACC and pET-CUS were incubated at 26 °C for 60 h in the presence of 1 mM each of the phenylpropanoid acids (p-coumaric acid, cinnamic acid or ferulic acid), glucose, antibiotics and IPTG in M9 minimal medium. The yields of curcuminoids are shown in Table 2. When p-coumaric acid was supplied, the yields of the triketide pyrone [5] and bisdemethoxycurcumin [3] were 11±4.6 mg l–1 and 91±23 mg l–1, respectively. In this reaction, the ratio of bisdemethoxycurcumin to triketide pyrone was improved: it was 200-fold higher than in the reaction starting from tyrosine, probably due to an increase of the p-coumaroyl-CoA concentration in the E. coli cells.
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). Supplementation of both p-coumaric acid and cinnamic acid to E. coli cells harbouring pCDF-LE4CL-1, pRSF-ACC and pET-CUS led to production of cinnamoyl-p-coumaroylmethane [8]. Similarly, demethoxycurcumin [2] was produced from p-coumaric acid plus cinnamic acid, and cinnamoylferuloylmethane [9] was produced from cinnamic acid plus ferulic acid (data not shown). These compounds were purified and identified by LC-APCIMS analysis. Approximately 7 mg l–1 of cinnamoyl-p-coumaroylmethane [8] was produced from an equal amount of cinnamic acid (1 mM, or 148 mg l–1) plus p-coumaric acid (1 mM, or 164 mg l–1), but with simultaneous production of 90 mg l–1 of bisdemethoxycurcumin [3]. The production of the symmetrical curcuminoid was perhaps due to the preference of p-coumaric acid to cinnamic acid as a substrate of CUS. The yield of cinnamoyl-p-coumaroylmethane [8] might be improved by changing the substrate ratio of cinnamic acid to p-coumaric acid.
Production of curcumin from rice bran pitch
Rice bran pitch is a dark and viscous oil, which is discharged in the course of production of rice edible oil from rice bran (Taniguchi et al., 1999). Some ten million tonnes of brown rice is produced every year and 10 % of its weight is polished away during production of white rice. This means that one million tonnes per year of rice bran is discharged. Approximately 40 % of the rice bran is used to produce rice edible oil. Therefore, a large amount of rice bran pitch is discharged every year. Taniguchi et al. (1999) developed a method to extract ferulic acid from rice bran pitch through hydrolysis of 
-oryzanol by alkali. We therefore planned to make use of the ferulic acid in this rice waste as a substrate for the production of curcumin [1].
We first checked whether rice bran pitch could be used as a ferulic acid source in the above-described reaction to which ferulic acid was supplied. When rice bran pitch was directly added to M9 medium, E. coli harbouring pCDF-LE4CL-1, pRSF-ACC and pET-CUS produced only a trace amount of curcumin [1] (0.68±0.37 mg l–1 from approximately 10 g l–1 of rice bran pitch). We assumed that most of the ferulic acid would be present as an esterified form, -oryzanol, that could not be used as a substrate for the system. In addition, the extraction of the products after incubation of the E. coli cells was complicated, due to the presence of oily material in the rice bran pitch. Therefore, we added a hydrolysis step to hydrolyse -oryzanol by alkali- and heat-treatments, according to the method of Taniguchi et al. (1999). Indeed, the hydrolysis process diminished the viscosity of the oily material. The resultant rice bran pitch medium contained 1.1±0.15 mM ferulic acid, as revealed by HPLC analysis, which showed that 11±1.4 mg ferulic acid was extracted from 500 mg rice bran pitch. E. coli cells (25 g wet weight l–1) harbouring pCDF-LE4CL-1, pRSF-ACC and pET-CUS and 25 g CaCO3 l–1 were added to the rice bran pitch medium, containing appropriate antibiotics and IPTG, and incubated at 26 °C for 60 h (CaCO3 was added to keep the pH of the medium slightly alkaline). In this reaction, 57±21 mg l–1 curcumin [1] was produced (Fig. 4). This means that about 60 mg curcumin was produced from 10 g rice bran pitch.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
In addition to curcuminoids, significant amounts of triketide pyrones were produced in all reactions. Efficient production of curcuminoids by CUS requires the CoA ester of a phenylpropanoid acid at a higher concentration than malonyl-CoA (Katsuyama et al., 2007b). We therefore assume that the concentration of CoA esters of phenylpropanoid acids in the reaction from the amino acids was too low to exclude the pyrone synthesis. In contrast, the yields of curcuminoids produced from the carboxylic acids reached 90–110 mg l–1, at a molar yield of approximately 60 %. These results suggested that the reaction catalysed by LE4CL-1 was not a rate-limiting step but that probably the deamination catalysed by PAL was the rate-limiting step for producing p-coumaroyl-CoA in high yield. Therefore, addition of the amino acids to the culture caused no acceleration of the reaction generating p-coumaroyl-CoA by PAL and 4CL to overcome the reaction consuming p-coumaroyl-CoA by CUS.
Rice bran pitch is an abundant by-product or waste during rice edible oil production. A previous study reported that rice bran contains an array of bioactive compounds, such as oryzanols, phytic acid, ferulic acid and inositol hecaphosphate (Renuka Devi & Arumughan, 2007). Zheng et al. (2007) reported microbial production of vanillin from ferulic acid, derived from waste residue of rice bran oil, by using Aspergillus niger and Pycnoporus cinnabarinus. Our present study also matches the trend of public requirements to make use of industrial wastes for the production of useful and valuable substances. The successful production of curcuminoids by recombinant E. coli is promising for production of various curcuminoid compounds, including ‘unnatural’ compounds, with various biological activities.
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ACKNOWLEDGEMENTS |
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Edited by: W. Quax
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Received 20 March 2008;
revised 29 May 2008;
accepted 15 June 2008.
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