1. VICTORIA Folate Production and Lysis

2. System Design Lysis Overproduction of folate in e coli Folate is released into large intestine where it is eventually absorbed

3. Folate Production

4. Biosynthetic pathway of folate folE folB folK folA DHFR folP Bermingham et al.

5. 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

6. PABA & glutamate For B.Subtilis – pabA, pabB, pabC were used to make PABA from chorismate Zhu et al

7. L.Lactis folate gene cluster Sybesma et al

8. Controlled Lysis Prophage the T4 holin + the T7 lysozyme the ldcA deletion

9. Folate Release Time population Pulse release Continuous release

10. 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.

11. 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