Folate Production and Lysis VICTORIA System Design 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

fol. E Biosynthetic pathway of folate fol. B fol. K fol. P fol. B fol. A DHFR Bermingham et al.

6 enzymatic steps + final reduction step GTPCH 1 - GTP Cyclohydrolase I (fol. E) DHPP -7, 8 -Dihydroneopterin triphosphate pyrophosphohydrolase DHNA - 7, 8 -Dihydroneopterin aldolase (fol. B) HPPK - 6 -Hydroxymethyl-7, 8 -dihydropterin pyrophosphokinase (fol. K) DHPS - Dihydropteroate synthase (fol. P) FPGS - Folylpoly-g-glutamate synthetase (fol. C) DHFR – dihydrofolate reductase (fol. A) Reduces 7, 8 -dihydrofolate 5, 6, 7, 8 -tetrahydrofolate

PABA & glutamate For B. Subtilis – pab. A, pab. B, pab. C were used to make PABA from chorismate Zhu et al

L. Lactis folate gene cluster Sybesma et al

Controlled Lysis • Prophage • the T 4 holin + the T 7 lysozyme • the ldc. A deletion

Folate Release population Pulse release Time Continuous release

p. Lys. T – T 4 gene t (Morita) • Produces T 7 lysozyme which degrades petidoglycan, but won’t lyse the cell until the expression of T 4 holin (expressed by gene t) which creates a break in the membrane, allowing the T 7 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 T 4 Phage Lysis Genes. Biotechnol. Prog. 2001, 17, 573 -576

ldc. A deletion ldc. A 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 ldc. A 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