Team:Uppsala/Description
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Saffron (Crocus sativus, L.) has been used traditionally as a dye, spice and a herbal remedy in many cultures. It contains four bioactive constituents in the form of crocin, crocetin, picrocrocin and safranal. Crocin is a water soluble apocarotenoid (organic pigment) commonly found in plants of the crocus, and gardenia species (and is most present in Saffron). It is a diester formed from gentiobiose, crocetin, and a powerful antioxidant (1) with free scavenger properties. As such, saffron (and by extension crocin) has been used as a medicine in many cultures. It has a deep red color, and forms an orange solution when dissolved in water, meaning it is also a potent dye for industrial and biotechnical purposes. Recent studies suggest that when isolated as a pure compound, crocin has several potent medicinal properties such as being a neuroprotective agent (2), tumor-suppressing agent (3), anti-inflammatory substance (4), antidepressant (5), cognitive enhancer in the form of improved memory (6), and also reducing obesity in rats (7) to name a few. Due to the price caused by the tedious work required to gather sufficient amount of Saffron to extract the compound from, we instead aimed to express crocin recombinantly in Escherichia Coli. For the purpose of making it readily available to extract and purify, hopefully making it cheaper to obtain, increasing our understanding of the compound, and potentially saving/helping people with the results. No team has previously dedicated themselves to producing only α-crocin before. The Uppsala team of 2013 failed to express the compound even though it should have worked theoretically. In light of recent studies, we identified a likely culprit in the form of a faulty enzyme, and our goal was therefore to attempt recombinant expression using a different enzymatic approach in the existing pathway.
Recent studies suggest that when isolated as a pure compound, crocin has several potent medicinal properties such as being a neuroprotective agent (2), tumor-suppressing agent (3), anti-inflammatory substance (4), antidepressant (5), cognitive enhancer in the form of improved memory (6), and also reducing obesity in rats (7) to name a few. Due to the price caused by the tedious work required to gather sufficient amount of Saffron to extract the compound from, we instead aimed to express crocin recombinantly in Escherichia Coli. For the purpose of making it readily available to extract and purify, hopefully making it cheaper to obtain, increasing our understanding of the compound, and potentially saving/helping people with the results. No team has previously dedicated themselves to producing only α-crocin before. The Uppsala team of 2013 failed to express the compound even though it should have worked theoretically. In light of recent studies, we identified a likely culprit in the form of a faulty enzyme, and our goal was therefore to attempt recombinant expression using a different enzymatic approach in the existing pathway.
The pathway attempted to express crocin from Zeaxanthin involves three steps. Zeaxanthin is first transformed into crocetin dialdehyde through catalysis by the carotenoid cleavage dioxygenase CCD2. The enzyme CaCCD2 was proven by Ahrazem et. al (9) to parallel crocin accumulation and it has previously been shown that CCD2 is the key enzyme responsible for the production of saffron apocarotenoids, cleaving the 7, 8 and 70, 80 double bonds adjacent to a 3-OHb-ionone ring converting zeaxanthin to crocetin dialdehyde (9).
The now formed Crocetin Dialdehyde then reacts with the enzyme CsADH-2946 to form Crocetin. Aldehyde dehydrogenases are a group of enzymes that catalyse the oxidation of aldehydes to carboxylic acids, meaning that through the reaction, the Crocetin dialdehyde obtains two OH-groups, effectively transforming it into Crocetin. The last step involved in the enzymatic pathway is the transformation of Crocetin to Crocin catalysed by UGTCs2.Glucuronosyltransferases are enzymes that catalyze the glucuronidation reaction on a wide range of structurally diverse endogenous and exogenous chemicals. The glucuronidation reaction consists of the transfer of the glucuronosyl group to substrate molecules. The resulting glucuronide is more polar, and by extension water soluble (10). UDP-glycosyltransferases (UGTs), converts Crocetin to Crocin through glucuronidation of the two OH-molecules bound to the newly added OH-groups by two R-groups. Read more here...
