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Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.
Squalene, an industrially important compound obtained primarily from the liver oil of deep-sea sharks and whales, is an important ingredient in skin cosmetics due to its photoprotective role (2, 7). The decreased cancer risk associated with high olive oil consumption could result from high squalene content (12, 16). Squalene has a chemopreventive effect on colon cancer (14). Moreover, squalene has wide applications in fine chemicals, magnetic tape, and low-temperature lubricants and as an additive in animal feed (1).
The use of shark liver oil is limited, due to the presence of environmental pollutants, such as polychlorinated biphenyls, heavy metals, and methylmercury residues, as well as an unpleasant fishy odor and taste (17, 19). Moreover, the presence of similar compounds, such as cholesterol, in the oils from marine animal liver can make squalene purification difficult. In addition, squalene production is limited by uncertain availability because of international concern for the protection of marine animals. Squalene has also been obtained from plant sources (4, 10, 11, 18), but very few methods can produce sufficient quantities at the desired purity level for pharmaceutical and industrial applications (6). The use of engineered microbial cell factories for the biosynthesis of squalene may be a suitable alternative to address these issues.
In the genome project for Streptomyces peucetius ATCC 27952, a cluster of genes which comprises five open reading frames, encoding hopanoid biosynthesis, has been detected and annotated. Even though these open reading frames share sequence homology with genes involved in hopanoid biosynthesis, no plausible hopanoid products have been isolated from S. peucetius in all laboratory cultures. Therefore, the hopanoid biosynthetic gene cluster of S. peucetius was considered “cryptic” in the present study. We were interested in activating the so-called “cryptic” hopanoid biosynthetic gene cluster of S. peucetius to produce pharmaceutically important compounds by using genetic engineering tools. Isoprenoid production in Escherichia coli has been extensively studied and reviewed (5, 8, 9, 15, 20, 21), but very few reports detail squalene formation in E. coli by the use of exogenous genes (13). In the present study, we introduced three cryptic genes (hopABD) from the hopanoid biosynthesis gene cluster from S. peucetius that catalyzed squalene production and also modulated the 2-C-methyl-d-erythritol 4-phosphate pathway in E. coli to enhance squalene production (Fig. (Fig.11).
The bacterial strains and plasmids used in this study are listed in Table Table1,1, and the oligonucleotides used in this study are summarized in Table Table2.2. S. peucetius was cultured in R2YE liquid medium at 28°C for 5 days on a rotary shaker (230 × g) to isolate genomic DNA. E. coli strains were grown in LB medium at 37°C or plated onto agar plates for subcloning and DNA manipulation and supplemented with ampicillin (100 μg/ml), kanamycin (100 μg/ml), and spectinomycin (35 μg/ml) (all purchased from Sigma, St. Louis, MO) wherever required for plasmid maintenance. E. coli strains were grown in 2× YT medium (Difco Bacto tryptone, Difco Bacto yeast extract, Nacl, and 5 N NaOH) at 20°C for squalene production on a rotary shaker (220 × g), and the cells were induced with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), with 0.5% (wt/vol) glycerol used as a carbon source plus 0.5% (wt/vol) MgSO4-7H2O.
To characterize the putative genes encoding squalene synthases, hopA and hopB, and the putative gene encoding farnesyl diphosphate synthase, hopD, and their involvement in squalene biosynthesis, the recombinants pHOP-A and pHOP-B were separately transformed into E. coli BL21(DE3). The transformants were grown in 2× YT medium at 20°C for 48 h after 0.1 mM IPTG induction with 0.5% (wt/vol) glycerol as a carbon source, and lipids were isolated. The process of squalene extraction and analytical methods (thin-layer chromatography [TLC], high-pressure liquid chromatography [HPLC], and nuclear magnetic resonance [NMR]) are given in the supplemental material. The formation of squalene was detected by TLC (Rf value of ~0.9) (see Fig. S1 in the supplemental material), gas chromatography/mass spectrometry (retention time of 43.5 min) (see Fig. S2 in the supplemental material), and HPLC (retention time of ~25.4 min) (Fig. (Fig.2,2, lines II and III) and compared with authentic squalene. Thus, hopA and hopB genes encode squalene synthases. Transformation of recombinant pHOP-D in E. coli along with hopA or hopB enhanced squalene production (data not shown), but along with hopAB, it markedly increased squalene production (4.1 mg/liter), indicating that the hopD gene might encode the FPP synthase (Fig. (Fig.2,2, line IV).
Squalene is synthesized in E. coli by the expression of the hopA or hopB gene, and its production is further increased by incorporation of the hopD gene, as mentioned above. 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI) are two enzymes involved in isoprenoid biosynthesis in the methylerythritol phosphate (MEP) pathway that can limit isoprenoid production yields in E. coli (3). To further improve squalene production, we amplified idi and dxs genes from the genomic DNA of E. coli and generated plasmid pIDXS and transformed pHOP-AB and pHOP-D plasmids into BL21(DE3) to overexpress hopA, hopB, hopD, idi, and dxs genes. The transformant was grown in 2× YT medium as described above, and the lipid was isolated. Squalene production increased considerably (11.8 mg/liter), as verified by HPLC (Fig. (Fig.2,2, line V) and TLC.
We purified squalene by the use of a simple preparative TLC as shown in the supplemental material. Several fractions rich in squalene, detected by preparative TLC with the Sigma standard, were scraped from the plates. The lipid was collected and dissolved in chloroform-methanol (1:1 [vol/vol]), and combined supernatants were brought to dryness by using a rotary evaporator to yield a colorless liquid. The purified squalene was dissolved in CDCl3 and subjected to NMR. The structure of purified squalene was verified by NMR spectra (see Fig. S3 in the supplemental material). The NMR spectra were consistent with the literature (6).
We heterologously expressed three cryptic genes (hopABD) from the hopanoid biosynthesis gene cluster of S. peucetius that catalyzed squalene production and also overexpressed the idi and dxs genes involved in providing additional isoprenoid precursors in the MEP pathway in E. coli to improve squalene production. We succeeded in obtaining 11.8 mg/liter of pure squalene under optimized conditions in the present study by shaking flask cultures. We believe that the use of genetically engineered E. coli could be an excellent alternative for producing commercial squalene.
The nucleotide sequences reported in this paper have been deposited into the GenBank database under the accession numbers FJ529811 (hopA), FJ529812 (hopB), and FJ529814 (hopD).
This study was supported by the 21C Frontier Microbial Genomics and Application Center Program, the Ministry of Science and Technology (grant 11-2008-14-007-00), Republic of Korea.
Published ahead of print on 18 September 2009.
†Supplemental material for this article may be found at http://aem.asm.org/.