https://video.igem.org/videos/watch/8bf3fb7f-c2d5-4262-aa76-b436691ee205 Title: Croteen: Turning Over Before Dawn-Improving Crocetin production in Escherichia coli through Semi-Rational Engineering of CsCCD2 Description: High school student Lifei lies awake restlessly before dawn. Seeking relief at a traditional Chinese medicine clinic, she discovers saffron — a prized sterile triploid spice used to soothe the mind for over 1,200 years, and the world’s most costly spice crop. Saffron’s calming effects stem from crocetin, the bioavailable aglycone released from crocin by gut enzymes. To bypass the high cost and climate risks of manual stigma harvesting, we engineered Escherichia coli BL21(DE3) for direct de novo crocetin biosynthesis. Our pathway leverages native MEP precursors to produce zeaxanthin via the CrtEBIYZ cascade, which CsCCD2 and NcALD then convert to crocetin. The initial two-plasmid system produced crocetin but accumulated zeaxanthin; we therefore consolidated eight genes into the single vector pET-Ds-Cro, added the idi gene, and balanced expression with two T7/lac promoters. HPLC confirmed a titer of 2.37 μg/g dry cell weight (DCW), with CCD identified as the rate-limiting enzyme. Using structure-guided engineering combining AlphaFold prediction, molecular docking, and PyMOL analysis, we generated two beneficial CsCCD2 variants: N366G, boosting yield by ~13% at 20 °C, and D253G, improving enzyme thermostability. Guided by human practices — a 207-person survey and consultations with clinicians, growers, and industry experts — we focused on anxiety relief and sleep improvement, developing two prototypes: oral soft capsules and topical essential oil. A patent application has been accepted, a B2B commercial model designed, and two minimum viable product (MVP) prototypes completed. Remaining challenges include low absolute titer, CCD thermal lability, and batch-to-batch variability. Our next step is to combine the N366G and D253G mutations to enhance both catalytic efficiency and thermostability, enabling robust crocetin production at higher induction temperatures. We do not aim to replace traditional saffron agriculture, but to open a new industrial path for crocetin biosynthesis — so that people like Lifei need no longer toss and turn waiting for dawn. Wiki | Official Page Thu, 29 Oct 2026 21:23:35 GMT https://validator.w3.org/feed/docs/rss2.html PeerTube - https://video.igem.org https://video.igem.org/client/assets/images/icons/icon-1500x1500.png https://video.igem.org/videos/watch/8bf3fb7f-c2d5-4262-aa76-b436691ee205 All rights reserved, unless otherwise specified in the terms specified at https://video.igem.org/about and potential licenses granted by each content's rightholder.