1. VITAMIN PRODUCTION Victoria Hsiao

2. Carotenoids – Background  Carotenoids are pigments (C40) that naturally occur in chloroplasts and other photosynthetic organisms and absorb light for photosynthesis.  There are over 600 carotenoids including beta- carotene.  Carotenoids have been found to have antioxidative properties, reducing the risk of mortality from chronic illnesses.  Animals are incapable of producing carotenoids and must obtain them from their diet. E.g the pink in flamingos & red in lobsters are from carotenoids in their diets. http://en.wikipedia.org/wiki/Carotenoid

3. Beta-carotene & Lycopene  Most commonly known as the pigment that makes carrots orange, beta-carotene is a precursor of Vitamin A.  Vitamin A, important for vision and as an antioxidant, is made from beta-carotene via beta-carotene dioxygenase.  Lycopene is an intermediate in the production of beta- carotene, and is a bright red carotenoid.  Lycopene is found in tomatoes, pink grapefruit, red bell peppers, etc., and has been found to have antioxidant activity. http://en.wikipedia.org/wiki/Lycopene http://en.wikipedia.org/wiki/Lycopene http://en.wikipedia.org/wiki/Vitamin_A

4. Beta-carotene synthesis pathway http://en.wikipedia.org/wiki/Lycopene

5. Beta-carotene & lycopene production Smolke et al. Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization. Metabolic Engineering 3, 313-321 (2001) IPP is naturally produced by e coli IPP – isopentyl-pyrophosphate FPP – farnesyl-pyrophosphate GGPP- geranylgeranyldiphosphate CrtE – GGPP synthase CrtB – phytoene synthase CrtI – phytoene desaturase CrtY – lycopene cyclase

6. Plasmids Smolke et al. Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization. Metabolic Engineering 3, 313-321 (2001) pAC-PHYT & pAC-PHYT+ enables the cell to produce a constant level of phytoene. The p70 plasmids are used to regulate the relative concentration levels of beta-carotene vs lycopene.

7. Regulating relative concentrations of lycopene vs beta-carotene Smolke et al. Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization. Metabolic Engineering 3, 313-321 (2001) The length of the hairpin (HPx) affects mRNA stability, which leads to different relative levels of phytoene, lycopene, and beta carotene.

8. Getting the vitamins out http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b208010a&JournalCode=LC  Lysis:  E coli have a peptidoglycan layer between the inner and outer membranes which prevents bursting due to osmotic pressure.  Prinz et al treated E coli with lysozymes, then controlled the osmotic conditions to get the cells to lyse. (first they had the cells in a high sucrose solution when they then diluted with DI water)  Vitamin production  high concentration of vitamins inside the cell  triggers lysozyme production which breaks down peptidoglycan layer  lysis via osmosis

9. Low Glucose concentration sensor Yun et al. Development of a Novel Vector System for Programmed Cell Lysis in Escherichia coli. Journal of Microbiology and Biotechnology 17(7) 1162-1168 (2007)_  Yun et al developed the pBlueLysis+ plasmid which detects low levels of glucose and automatically expresses a lysis gene.  Is pBlueLysis+ accessible?  Would high vitamin concentration eventually lead to low glucose concentrations?

10. Without a concentration sensor  Somehow control the relative rates of lysozyme and vitamin production such that the lysozyme concentration doesn’t reach the critical level until a significant amount of vitamin has already been produced.  So both the lysis gene and the vitamin gene are constantly being expressed independently.  We’d have to time each separately and make lysis gene expression much slower than vitamin production.