VICTORIA Folate Production and Lysis. System Design Lysis Overproduction of folate in e coli Folate...
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Transcript of VICTORIA Folate Production and Lysis. System Design Lysis Overproduction of folate in e coli Folate...
VICTORIA
Folate Production and Lysis
System Design
Lysis
Overproduction of folate in e coli
Folate is released into large intestine where it is eventually absorbed
Folate Production
Biosynthetic pathway of folatefolE
folB
folB
folK
folADHFR
folP
Bermingham et al.
6 enzymatic steps + final reduction step
GTPCH1 - GTP Cyclohydrolase I (folE)DHPP -7,8-Dihydroneopterin triphosphate
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
Time
pop
ula
tio
n
Pulse release
Continuous release
pLysT – T4 gene t (Morita)
Morita et al. Programmed Escherichia coli Cell Lysis by Expression of Cloned T4 Phage Lysis Genes. Biotechnol. Prog. 2001, 17, 573-576
•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.
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