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