Transcript Folate Production and Lysis

Folate Production and Lysis
VICTORIA
System Design
Lysis
Overproduction of
folate in e coli
Folate is released
into large intestine
where it is eventually
absorbed
Folate Production
folE
Biosynthetic pathway of folate
folB
folK
folP
folB
folA
DHFR
Bermingham et al.
6 enzymatic steps + final reduction step
 GTPCH1 - GTP Cyclohydrolase I (folE)
 DHPP -7,8-Dihydroneopterin triphosphate
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pyrophosphohydrolase
DHNA - 7,8-Dihydroneopterin aldolase (folB)
HPPK - 6-Hydroxymethyl-7,8-dihydropterin
pyrophosphokinase (folK)
DHPS - Dihydropteroate synthase (folP)
FPGS - Folylpoly-g-glutamate synthetase (folC)
DHFR – dihydrofolate reductase (folA)

Reduces 7,8-dihydrofolate  5,6,7,8-tetrahydrofolate
PABA & glutamate
 For B.Subtilis – pabA, pabB, pabC were used to
make PABA from chorismate
Zhu et al
L.Lactis folate gene cluster
Sybesma et al
Controlled Lysis
•Prophage
•the T4 holin + the T7 lysozyme
•the ldcA deletion
Folate Release
population
Pulse release
Time
Continuous release
pLysT – T4 gene t (Morita)
•Produces T7
lysozyme which
degrades
petidoglycan, but
won’t lyse the cell
until the expression of
T4 holin (expressed
by gene t) which
creates a break in the
membrane, allowing
the T7 to reach the
peptidoglycan layer.
•Requires IPTG to
trigger expression of
the plasmid.
Morita et al. Programmed Escherichia coli Cell Lysis by Expression of Cloned T4 Phage Lysis Genes. Biotechnol. Prog.
2001, 17, 573-576
ldcA deletion
 ldcA encodes a cytoplasmic L,D-carboxypeptidase which creates peptide bonds
in the peptidoglycan layer between the inner and outer membranes.
 Normally, the murein (peptidoglycan) is continuously recycled into a
pentapeptide and replaced.
 Deleting ldcA results in the accumulation of a tetrapeptide instead of a
pentapeptide, causing a weakened cell wall, and resulting in spontaenous
autolysis during the stationary growth phase.
 Can the E.coli reach a stationary growth phase in the intestine?
 The E.coli would become more and more fragile as time goes on since it’s still
recycling the good murein but not replacing it, so eventually it would lyse?
Templin et al. A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli. The EMBO
Journal Vol.18 No.15 pp.4108–4117, 1999