BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 126.96.36.199). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in S. cerevisiae. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.
The successful ligation of santalene synthase was confirmed by a diagnostic digest with SpeI and ApaI (Santalene diagnostic digest confirmation). While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form.
The results of a gas-chromatography mass-spectrometry assay for the presence of santalene will be posted here shortly. Preliminary assays looking for bands of the size of the synthase gene and organic products in cell lysate hint that expression may be occurring but were not definitive.
Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117.
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8.
Rodriguez S, Kirby J, Denby CM, Keasling JD. Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites. Nat Protoc. 2014;9(8):1980-96.
Sequence and Features
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