VITAMIN PRODUCTION

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/Vitamin_A

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

5. Beta-carotene & lycopene production • 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 Smolke et al. Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization. Metabolic Engineering 3, 313-321 (2001)

6. Plasmids • 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. Smolke et al. Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization. Metabolic Engineering 3, 313-321 (2001)

7. Regulating relative concentrations of lycopene vs beta-carotene • The length of the hairpin (HPx) affects mRNA stability, which leads to different relative levels of phytoene, lycopene, and 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)

8. Getting the vitamins out • 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 http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b208010a&JournalCode=LC

9. Low Glucose concentration sensor • 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? 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)_

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.