Nucleic acid isolated from Tetrahymena which codes for a triterpenoid cyclase, its production, and use

#The present invention relates to a triterpenoid cyclase (tetrahymanol cyclase) isolated from Tetrahymena, its coding nucleic acid, its production, and use.

#The inventive triterpenoid cyclase catalyzes the formation of tetrahymanol from squalene by a direct cyclization of squalene (Capsi et al. (1968) J. AM. CHEM. SOC. 90:3563-3564; Abe et al. (1993) CHEM. REV. 93:2189-2206). The triterpenoid cyclase also recognizes oxidosqualene as a substrate (Abe & Rohmer (1994) J. CHEM. SOC. PERKIN TRANS. 1:783-791). In addition to pentacyclic triterpenoids, the squalene tetrahymanol cyclase also catalyzes the formation of tetracyclic triterpenoids (Abe & Rohmer (1991) J. CHEM. SOC. CHEM. COMMUN. 902-903). Tetrahymanol (or gammaceran-3-ol), which is derived from isoprene, is a member of the isoprenoid class. Isoprenoids play an important role as phytohormones and carotenoids, and as components of chlorophyll, ubiquinone, plant resins, oils, and latex. As steroid hormones, isoprenoids effect important functions in animals. The formation of tetrahymanol can be reprimed in Tetrahymena by adding sterols, such as cholesterol (Conner et al. (1968) J. PROTOZOOL. 15:600-605; Conner et al. (1969) J. BIOL. CHEM. 244:2325-2333).

#Isoprenoids are also important components of bacterial and eukaryotic membranes. Similar to hopanoids and sterols (such as cholesterol), pentacyclic triterpenoid has tetrahymanol membrane-stabilizing properties (Conner et al. (1968; 1969); Poralla et al. (1980) FEBS LETT. 113:107-110). By restricting the fluidity of the lipid acid residues of membrane lipids, a condensed (membrane-solidifying) effect is achieved above the phase transition temperature; while below the phase transition temperature, the fluidity of the membrane is increased, thus preventing the optimal close packing of fatty acid residues. In addition, the membrane fluidity depends on the fatty acid composition of the membrane lipids. The fluidity of membranes increases in proportion to the levels of unsaturated fatty acids. With temperature changes, organisms are able to regulate the fluidity of their membranes, for example, via the fatty acid composition. Below the phase transition temperature, isoprenoids and unsaturated fatty acids increase the membrane fluidity via a synergistic effect. The inhibition of the synthesis of the cyclic triterpenoids alters membrane stability. This reduced membrane fluidity can be compensated by an increased proportion of polyunsaturated fatty acids (PUFAs) in the membrane, i.e., the content of PUFAs can be increased by inhibition of the triterpenoid cyclase.

#FIG. 1. Results of a BLASTP database comparison of the protein sequence according to SEQ ID No. 12 with protein databases.

#FIG. 2A. Alignment of the protein sequence according to SEQ ID No. 12 with known pentacyclic triterpenoid cyclases, lanosterol synthesis isolated from Alicyclobacillus acidoterrestris (SEQ ID No. 14).

#FIG. 2B. Alignment of the protein sequence according to SEQ ID No. 12 with known pentacyclic triterpenoid cyclases, squalene-hopene cyclase isolated from Alicyclobacillus acidocaldarius (SEQ ID No. 15).

#The present invention relates to nucleic acids isolated from Tetrahymena which code for a ciliate-specific triterpenoid cyclase. The inventive nucleotide sequences and the polypeptide sequences derived therefrom demonstrate a surprisingly low sequence identity to known isoprenoid cyclases. Another aspect of the invention is the genomic nucleotide sequence of the triterpenoid cyclase, which in addition to the coding sequence, contains non-coding nucleotide sequences, such as introns, promoters, and flanking sequences. The invention also relates to the use of nucleic acids for regulating expression, targeted knockout, or repriming of this gene. By regulating the expression of the triterpenoid cyclase, it is possible to modify the levels of multiple unsaturated fatty PUFAs in an organism. Moreover, as a result of the targeted knockout of triterpenoid cyclase, an enrichment of squalene, an intermediate in the synthesis of various triterpenoids, can be achieved. Squalene serves as a synthetic module for terpenes. Partially modified squalene (e.g., in hydrogenated form) is used in dermatology products and cosmetics, as well as in various derivatives found in skin and hair care products. In a further embodiment, a targeted overexpression of the inventive nucleic acids may result in the production of cyclic triterpenoids such as pentacyclic triterpenoids (e.g., tetrahymanol or hopane), and tetracyclic triterpenoids (e.g., lanosterol or cycloartenol). Cyclic triterpenoids are used in the synthesis of steroid hormones and saponins. These compounds display good skin penetration and diffusion properties, and therefore, they are also used in cosmetics and dermatology products.

#The inventive nucleic acids can be isolated from ciliates, preferably Tetrahymena, and most preferred from Tetrahymena thermophila.

#One aspect of the present invention is to provide a nucleic acid isolated from Tetrahymena, which codes for a polypeptide with the activity of a triterpenoid cyclase. Another aspect of the invention is the regulation of gene expression or genetic knockout (adequate inhibition) of the triterpenoid cyclase in a host organism, preferably in Tetrahymena, in order to increase the level of PUFAs produced in the organism. In particular, a further aspect is to enrich the levels of gamma-linolenic acid (GLA) by means of the host's own production of PUFA for restoration of membrane stability.