Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Abstract

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-Derythritol 4-phosphate pathway in E. coli to enhance squalene production (Fig. 1).Bact...

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum

“…Ten milliliters of the CM culturegrown mycelia were transferred to 50 ml of minimal medium (MM) (22) and grown for 96 h at 28°C and 250 rpm. Mycelia were used for extraction and quantification of ergosterol-squalene as previously reported (23,24). All measurements were made in triplicate.…”

Squalene epoxidase as a target for manipulation of squalene levels in the yeastSaccharomyces cerevisiae

“…Specific physicochemical and antioxidant properties of squalene imply also its applications in food industry, pharmacology, and cosmetics (for review, see Spanova & Daum, 2011). Another promising approach in the search for new squalene resources is the use of microorganisms as squalene producers on industrial scale (Ghimire et al, 2009;Nakazawa et al, 2012). Shark liver as the traditional source of squalene is currently being substituted by plant sources such as olive or amaranth oil (He & Corke, 2003;Garc ıa-González et al, 2008).…”

Knowns and unknowns of membrane lipid synthesis in streptomycetes

“…4). These sequential reactions are catalysed by the FPP synthase, putatively encoded by hopD in S. peucetius [51, 52] and by SCO6763 in S. coelicolor [49, 53]. …”

“…However, homologues to HpnC and HpnD are also present in Streptomyces peucetius – named there HopA and HopB, respectively – and each was reported to induce a small SQ accumulation when expressed in E. coli , suggesting that both are capable of independent synthesis of SQ, similar to the bifunctional eukaryotic SQ synthases [52]. This stands in contrast to the results by Pan et al [56] where it is reported that coexpression of HpnD, HpnC and HpnE from Zymomonas mobilis is required for SQ biosynthesis in E. coli .…”