VITAMIN PRODUCTION
Victoria Hsiao
Carotenoids – Background
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Carotenoids are pigments (C40) that naturally occur in
chloroplasts and other photosynthetic organisms and
absorb light for photosynthesis.
There are over 600 carotenoids including betacarotene.
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
Beta-carotene & Lycopene
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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 betacarotene, 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
Beta-carotene synthesis pathway
http://en.wikipedia.org/wiki/Lycopene
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)
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)
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)
Getting the vitamins out
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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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http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b208010a&JournalCode=LC
Low Glucose concentration sensor
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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)_
Without a concentration sensor
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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.