From 2007.igem.org

What metabolite should we use for the feeding of the germline by the soma ?

In order to choose our auxotrophy, we need to take several constraints into account:

• No simple bypass or reversion: The auxotrophy phenotype of the germline must be stable. There must be no simple metabolic bypass or reversion possibilities.
• Overproduction and excretion: Prototroph cells must be able to overproduce and excrete the metabolite, or at least to release it when dying.
• Absorption: auxotroph cells should be able to live in very low concentration of this metabolite. Indeed we should expect only little metabolite to be excreted.
• Survival: auxotroph cells should be able to live as long as possible when deprived of the metabolism.
• No growth on LB: We would like our synthetic organism to be able to grow on rich medium. This would facilitate our lab work, and more generally allow our synthetic organism to grow in a variety of conditions.
• Feedback: For purposes described below, we would like to have a sensor device sensitive to our metabolite concentration.

Candidate metabolite

DAP:

Diaminopimelate is a precursor both for lysine and for cell wall components. It is synthesized from aspartate-semialdehyde, through 5 reactions catalysed by (DapA, DapB, DapD, ArgD and DapE). All this five genes are essential, that is to say, when any of these genes is deleted the strain is DAP auxotroph and cannot grow on rich medium.

It as been found that mutants of the gene catalysing the step from DAP to lysine (LysA) are excreting DAP (1). This is supposedly because of the negative feedback loop of the lysine concentration on the DapA activity. This provides a proof that DAP can be excreted by the cell when overproduced. In order to reproduce this effect, we can try to find DapA mutants which would be insensitive to feedback. The lysine pathway is pretty well conserved across all kingdoms. In plants, DAP is only an intermediated in the lysine biosynthesis. There is no constitutive need for DAP, and there is a strong negative feedback of lysine on DapA. On the other side, gram+ bacteria which have a thick cell wall, present a strong need for DAP without respect to the lysine concentration. Gram+ DapA genes are thus insensitive to lysine. Gram– bacteria which have a smaller cell wall, also have a smaller need for DAP, which is consistent with the fact that there is a small negative feedback from lysine on gram- DapA.

Soy DapA feedback insensitive mutants have been identified and expressed in Coli, showing complementation of DapA mutants and DAP overproduction (2). We can also think of using gram + DapA genes.

Last point, DapA expression has been shown to depend on DAP concentration (3). The mechanism is unknown, but there is great chance that any gene placed under the control of the DapA promoter will have its expression controlled by DAP concentration.

(1) Relation Between Excreted Lipopolysaccharide Complexes and Surface Structures of a Lysine-Limited Culture of Escherichia coli K. W. KNOX,' MARET VESK,2 AND ELIZABETH WORK

(2) Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase Gregg W. Silk and Benjamin F. Matthews

(3) Acord04: Acord J, Masters M (2004). "Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid." FEMS Microbiol Lett 235(1);131-7. PMID: 15158272