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

Squalene is a linear triterpene formed via the MVA or MEP biosynthetic pathway and is widely distributed in bacteria, fungi, algae, plants, and animals. Metabolically, squalene is used not only as a precursor in the synthesis of complex secondary metabolites such as sterols, hormones, and vitamins, but also as a carbon source in aerobic and anaerobic fermentation in microorganisms. Owing to the increasing roles of squalene as an antioxidant, anticancer, and anti-inflammatory agent, the demand for this chemical is highly urgent. As a result, with the exception of traditional methods of the isolation of squalene from animals (shark liver oil) and plants, biotechnological methods using microorganisms as producers have afforded increased yield and productivity, but a reduction in progress. In this paper, we first review the biosynthetic routes of squalene and its typical derivatives, particularly the squalene synthase route. Second, typical biotechnological methods for the enhanced production of squalene using microbial cell factories are summarized and classified. Finally, the outline and discussion of the novel trend in the production of squalene with several updated events to 2015 are presented.

Isoflavonoid representatives such as genistein, daidzein are high potent anti-cancer, anti-bacterial, anti-oxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12-derived metK encoding S-adenosylmethionine (SAM) synthase (accession number: K02129) for enhancement of SAM as precursor and a Streptomyces avermitilis-originated SaOMT2 (O-methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 7-O-methyl-genistein and 7-O-methyl-daidzein were confirmed individually by chromatographical data such as HPLC, LC-TOF-MS and NMR ( H and C) as well. Furthermore, substrates concentration, incubation time and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed-batch fermentation with scale up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 μM (46.81 mg/L) and 382.50 μM (102.88 mg/L) for 7-O-methyl genistein and 7-O-methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS and HepG2) and endothelical (HUVEC) cells were investigated and demonstrated. Taken together this research work described the production of isoflavonoid-7-O-methoxides by E. coli engineering, improvement of production, characterization of produced compounds and preliminary in vitro biological activities of the these flavonoids being manufactured. This article is protected by copyright. All rights reserved.

Glycosyltransferase from Bacillus licheniformis DSM13 (YjiC) was used for enzymatic modification of emodin and aloe-emodin in vitro and in vivo. In order to increase the availability of UDP-glucose, three genes involved in the production of precursors of NDP-sugar in Escherichia coli BL21 (DE3) viz. D-glucose phosphate isomerase (pgi), D-glucose-6-phosphate dehydrogenase (zwf), and UDP-sugar hydrolase (ushA) were deleted and glucose-1-phosphate urididyltransferase (galU) gene was over expressed. To improve the yield of the products; substrate, time and media parameters were optimized, and the production was scaled up using a 3 L fermentor. The maximum yield of glycosylated products of emodin (emodin-O-β-D-glucoside) and aloe-emodin (aloe-emodin-O-β-D-glucoside) were approximately 144 µM (38 mg/L) and 168 µM (45 mg/L) respectively, representing almost 72 % and 84 % bioconversion of emodin and aloe-emodin when 200 µM of emodin and aloe-emodin were supplemented in the culture. Additionally, the emodin and aloe emodin major glycosylated products exhibited the highest stability at pH 8.0 and the stability of products was up to 70 °C and 60 °C respectively. Furthermore, the biological activities of emodin and its major glucoside (P1) were compared and their anti-cancer activities were assayed in several cancer cell lines. The results demonstrate that YjiC has the capacity to catalyze the glycosylation of these aromatic compounds and that glycosylation of anthraquinones enhances their aqueous solubility while retaining their biological activities.