Design of a PoxB mutant that can be targeted by the SuMMV protease. The structure of PoxB is shown (PDB: 3ey9; Neumann
et al,
2008), highlighting the amino acid sites selected for degron insertion (in red). The impact on acetate production of different acetate pathway mutants is shown on the left of the graph. On the right of the graph, a double knockout mutant (
ΔptaΔpoxB) contains variants of
poxB expressed from a BAC. Note that complementation of unmodified
poxB (WT) from the BAC does not fully reconstitute acetate production. N‐ter and E170 refer to the location of the SuMMV degron tag in
poxB. A plasmid expressing protease SuMMV from a DAPG‐inducible promoter was also introduced into strains containing
poxB variants WT and E170 complemented on a BAC and a strain
poxB with an E170 site replacement on the genome (poxB::E170). The graph shows the acetate produced in these strains when either no inducer (white) or 25 μM DAPG (grey) is added to the culture.