ABSTRACT

Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ- guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engi- neered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.

Keywords:

Engineered Escherichia coli, δ-Guaiene Production, Isoprenoids, Mevalonate Pathway, Secondary Metabolism, Sesquiterpene

1. Introduction

It is well known that Aquilaria plants very occasionally form dark resinous heartwood called agarwood upon mechanical wounding or microbial infection [1] . These tissues produce a variety of sesquiterpene compounds with aroma such as δ-guaiene, α-guaiene and α-humulene, and, therefore, they have been used as a scent, perfume and traditional medicines. It has been assumed [2] that guaianolide sesquiterpenes are synthesized via two steps of cyclization reactions catalyzed by δ-guaiene synthase (GS) with farnesyl diphosphate (FPP) as the substrate (Figure 1). Although commercial value of agarwood is very high, this material is not formed under normal environmental conditions [1] , and the artificial transformation of Aquilaria plants to agarwood has been quite difficult.

We showed previously [3] [4] that treatment of cell cultures of A. microcarpa with either a plant hormone methyl jasmonate or yeast extract resulted in a marked tran-

scriptional activation of GS, and this enabled us to isolate several homologous GS genes. To our knowledge, δ-guaiene cannot be purchased from commercial venders, and, therefore, we attempted to construct the production system of this compound employing bacterial cells. A GS gene, together with a farnesyl diphosphate synthase gene (FPS) isolated from A. microcarpa [5] , was inserted into pRSFDuet-1, and Escherichia coli cells transformed with this expression vector were found to generate δ-guaiene at the concentration of approximately 0.6 μg/ml culture in a nutrient-enriched Terrific broth [6] .

In E. coli, isoprenoids are biosynthesized not through mevalonate (MVA) but through non-mevalonate pathway [7] . However, it was reported [8] [9] [10] [11] that E. coli transformed with MVA pathway-related genes obtained from heterogeneous sources showed appreciable terpenoid-producing activities by coexpression with appropriate biosynthetic enzyme genes. Recently, Harada et al. [12] constructed MVA pathway-engineered E. coli cells by the transformation with a gene cluster (Figure 2) isolated from Streptomyces sp. [13] [14] , and demonstrated [15] that efficient carotenoid production was achieved in this strain by overexpression of several genes involved in the biosynthetic processes of the tetraterpene pigments. In the present experiments, we have attempted to construct the efficient producing system of δ-guaiene, which is biosynthesized in Aquilaria plants only under very limited condition, employing MVA pathway-engineered E. coli cells.

2. Materials and Methods

2.1. Construction of Expression Vectors

The translatable regions of FPS and GS isolated from cultured cells of A. microcarpa [3] [5] were amplified by PCR, and subcloned into pRSFDuet-1 (Novagen) according to the method described previously in detail [6] . This expression vector was designated as pRSF-FPS/GS. A gene cluster encoding six MVA pathway enzymes, 3-hydroxy-3- methylglutaryl CoA (HMGCoA) synthase, HMG-CoA reductase, MVA kinase, phosphomevalonate (PMVA) kinase, diphosphomevalonate (DPMVA) decarboxylase and type 2 isopentenyl diphosphate (IPP) isomerase (Figure 2), isolated from Streptomyces sp. strain CL190 [13] [14] was PCR-amplified, subcloned into pACYC184, and designated pAC-Mev (Figure 3). The successive insertion of type 1 IPP isomerase gene isolated from Saccharomyces cerevisiae (Scidi) and acetoacetate-CoA ligase gene from Rattusnorvegicus (Aacl) into pAC-Mev was performed as described previously in detail [12] , and the constructed vectors were designated pAC-Mev/Scidi and pAC-Mev/Scidi/ Aacl, respectively (Figure 2 and Figure 3).

2.2. Transformation and Cultivation of E. coli Cells

Transformation and cultivation of E. coli was performed according to the method described previously [6] with several modifications. E. coli BL21 cells were transformed with four expression vectors, pRSF-FPS/GS, pAC-Mev, pAC-Mev/Scidi and pAC-Mev/ Scidi/Aacl, and the transformants were grown in LB medium overnight at 37˚C. The

cultures were transferred into fresh LB medium, and isopropyl β-D-1-thiogalactopy- ranoside (IPTG, final concentration 0.1 mM) was added to the cultures at an optical density of 0.4 - 0.5 at 600 nm. In parallel experiments, overnight culture of E. coli was transferred into a nutrient-enriched Terrific broth instead of LB medium, and, then, IPTG was added in a similar manner. Immediately after the addition of IPTG, 100 μl-aliquots were transferred into screw-capped 4 ml vials, and 0.2 μg limonene (NacalaiTesque, 1 μl ethanol solution) was added to the cultures as an internal standard. If necessary, 100 μg mevalonolactone (MVL, Tokyo Kasei, 1 μl-aliquot) and/or 100 μg lithium acetoacetate (LAA, Sigma-Aldrich, 1 μl-aliquot) were also added to the 100 μl E.

coli cultures, and the mixtures were further incubated at 25˚C overnight.

2.3. GC-MS Analysis of δ-Guaiene

The sesquiterpene compounds accumulated in the head space of the cultures were extracted with a solid phase microextraction assembly for 30 min at room temperature (Supelco, 100 µm polydimethylsiloxane fiber), and identification of the products was performed by GC-MS analysis (Shimadzu, GCMS-QP2010 Ultra; Agilent, CAJ&W DB-5MS 0.32 mm × 30 m column) according to the method described previously in detail [6] . The reaction products were separated to calculate retention indices, and the identification of the compounds was based on the comparison of reported retention indices and mass spectra with those in databases as described previously [3] [16] [17] .

2.4. Quantitative Determination of δ-Guaiene

Determination of δ-guaiene concentration was carried out by GC analysis (GL Sciences, GC-353; Supelco, SPB-1 0.32 mm × 30 m column; equipped with flame ionization detector and Hitachi D-2500 Chromatointegrator). The column pressure was at 260 kPa (carrier N2), and the injection port and the interface temperature were adjusted at 250˚C. The oven temperature was started at 50˚C for 2 min and was increased to 130˚C at the rate of 4˚C/min, then raised to 250˚C at 30˚C/min and kept for 10 min. The reaction products extracted with the microextraction assembly were injected in the splitless mode, and were separated under the conditions described above. δ-Guaiene concentration was determined by a calibration curve plotted the ratio of the peak areas of authentic sample at various concentrations to the internal standard [6] .

3. Results

3.1. Effect of Overexpression of MVA Pathway Genes on δ-Guaiene Production

We have previously reported [3] that sesquiterpene compounds produced by the pRSF-FPS/GS-transformed E. coli cells were detected neither in medium nor cells, but were recovered only from the head space of the cultures. In the total ion chromatogram of GC-MS analysis (Figure 4(a)), two peaks, together with the internal standard limonene, were found to be released from the transformed E. coli cultures, and they were identified as α-guaiene and δ-guaiene by the retention indices and mass spectra (Figure 4(b)) in the data base, respectively [3] [16] [17] . E. coli cells transformed with solely pRSF-FPS/GS liberated low concentration of δ-guaiene when they were cultivated in LB medium (0.4 μg/ml culture). However, the sesquiterpene-producing activity was significantly enhanced by the incubation of the transformant in a nutrient-enriched Terrific broth, and it increased to the level of 1.8 μg δ-guaiene/ml culture (Figure 5). An appreciable increase in sesquiterpene-producing activity was observed by the transformation of E. coli with pRSF-FPS/GS plus pAC-Mev, and the sesquiterpene contents were elevated to 1.0 μg/ml culture in LB medium and 4.8 μg/ml culture in Terrific broth, respectively. Since MVA pathway was expected to be constructed in the pAC-Mev- transformed E.coli cells, we examined the possible effect of the addition of an isoprene precursor, MVL, on the sesquiterpene production in this transformant. As shown in

Figure 5, δ-guaiene accumulation in pAC-Mev-transformed cells grown in LB medium appeared to be slightly enhanced by the addition of MVL, and approximately 1.6-fold increase in the productivity was observed. In contrast, pAC-Mev-transformant cultivated in Terrific broth showed a marked elevation of the biosynthetic activity upon the coincubation with MVL, and the concentration of δ-guaiene was found to be 31.4 μg/ml culture.

3.2. Effect of Overexpression of Scidi Gene on δ-Guaiene Production

Harada et al. reported [12] that FPP content was estimated to increase several times when type 1 IPP isomerase gene derived from S. cerevisiae (Scidi) was introduced and overexpressed in E. coli, together with type 2 IPP isomerase (Figure 2). Therefore, E. coli cells were transformed with pRSF-FPS/GS plus pAC-Mev/Scidi (Figure 3), and the sesquiterpene-producing activity of the transformant was examined in the absence and presence of MVL (Figure 5). E. coli overexpressing FPS, GS, six MVA cluster genes plus Scidi liberated 1.2 and 7.2 μg δ-guaiene/ml culture in the absence and presence of MVL, respectively, when the cells were grown in LB medium. On the other hand, pAC-Mev/Scidi-transformed cells in Terrific broth generated the compound at the concentration of 5.0 and 35.3 μg/ml culture in the absence and presence of MVL.

3.3. Effect of Overexpression of Aacl Gene on δ-Guaiene Production

It was reported [15] that an efficient biosynthetic system of isoprenoids from acetoacetate could be established by the transformation of E. coli by a rat acetoacetate-CoA ligase gene (Aacl), together with the MVA pathway gene cluster, although Aacl translate is an important enzyme of butanoate metabolism rather than isoprenoid pathway (Figure 2 and Figure 3). In this case, it was also demonstrated that, LAA could be added to the culture medium as a convenient substrate of Aacl to form MVA (Figure 2). Therefore, we prepared E. coli cells transformed with pRSF-FPS/GS plus pAC-Mev/ Scidi/Aacl, and examined the δ-guaiene productivity of the cells overexpressing these genes. As shown in Figure 6, the transformed E. coli in LB medium liberated 1.6 μg δ-guaiene/ ml culture, and the content increased to 12.0 and 17.3 μg/ml by the addition of MVL and LAA to the cultures, respectively. On the other hand, the transformed cells cultivated in Terrific broth generated 5.8 μg/ml culture of δ-guaiene in the absence of the precursors, while the concentration appreciably increased to 37.9 and 42.3 μg/ml culture by the addition of MVL and LAA.

We expected the possibility that some synergic effect of MVL and LAA on the sesquiterpene production might be observed upon the coincubation of these two precursors with the transformed cells. Therefore, MVL and LAA were concomitantly added to the transformant cultures, and δ-guaiene concentrations were determined (Figure 6). However, no enhancement of δ-guaiene production was observed in the transformed cells even in the presence of the two isoprene precursors, and the contents of the com-

pound decreased to 14.5 and 29.3 μg δ-guaiene/ml culture in LB medium and in Terrific broth, respectively. In repeated experiments, notable activation of δ-guaiene biosynthesis was not observed by the coincubation of the transformed cells with these two precursors, and the concentrations of the compound were almost similar or rather decreased though the values were varied in some extent.

4. Discussion

We showed previously [6] that δ-guaiene biosynthesis was markedly activated when GS and FPS were coexpressed in E. coli cells (Figure 1), and the content of the compound was approximately 1 - 2 μg/ml culture even in the nutrient-enriched Terrific broth. In the present study, we have demonstrated that FPS- and GS-overexpressing E. coli cells transformed with a Streptomyces MVA pathway gene cluster exhibited an appreciable biosynthetic activity of δ-guaiene, especially in the nutrient-enriched Terrific broth (Figure 5). Accumulation of the compound was increased to 31.4 μg/ml culture when the transformed cells were incubated in the presence of MVL. Therefore, 17.4-fold increase in δ-guaiene-producing activity was observed when MVA pathway-engineered E. coli cells were incubated in the enriched medium with the precursor supplement. Similarly, the sesquiterpene contents increased to 35.3 and 42.3 μg/ml culture in pAC-Mev/ Scidi- and pAC-Mev/Scidi/Aacl-transformants in the presence of MVL and LAA (Figure 5 and Figure 6), and the productivity was elevated to 19.6- and 23.5-fold levels, respectively, compared with a control E. coli strain expressing solely FPS plus GS. These results clearly indicate that the introduction of MVA pathway in E.coli is a powerful tool to construct δ-guaiene-producing system in the bacterial cells, as well as some other terpenoid compounds [8] [9] [10] [11] .

Although addition of MVL or LAA to MVA pathway-engineered E. coli cell culture resulted in the marked enhancement of δ-guaiene biosynthesis, no synergic or somewhat inhibitory effect was observed when pAC-Mev/Scidi/Aacl-transformant was incubated in the presence of both of the precursors (Figure 6). At present, no direct evidence is available to explain this apparent discrepancy, however, it might be possible that intracellular concentration of the isoprene units and/or their precursors might be almost saturated in pAC-Mev/Scidi/Aacl-transformed E. coli culture supplemented with both MVL and LAA, and certain inhibitory mechanism might function under these “overdose” conditions.

Further improvement of E. coli cell culture system as the δ-guaiene-producing apparatus is in progress in our laboratory.

Acknowledgements

This work was supported, in part, by a Grant-in-Aid (No. 15K07990 to FK) from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

Cite this paper

Kurosaki, F., Kato, T., Misawa, N. and Taura, F. (2016) Efficient Production of δ-Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by Mevalonate Pathway-Engi- neered Escherichia coli Cells. Advances in Bioscience and Biotechnology, 7, 435-445. http://dx.doi.org/10.4236/abb.2016.711042

References