VITAMIN PRODUCTION

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VITAMIN PRODUCTION

• Victoria Hsiao

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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.

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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.

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Beta-carotene synthesis pathway
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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

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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.

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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.

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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

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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?

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Without a concentration sensor

• Somehow control the relative rates of lysozyme
  and vitamin production such that the lysozyme
  concentration doesnt 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.
• Wed have to time each separately and make lysis
  gene expression much slower than vitamin
  production.