How about finding the actual number of copies of particular genes or any other genes possesing functional domain of your gene(s) of interest present in E. coli genome using HMMER profile based approach ? So that you can know what are the genes having the functional domain than you can classify whether they are isozymes or novel genes involved in synthesis of metabolite.

First of all try to find out the most probable metabolic route of synthesis of that metabolite using databank like KEGG. Then try to put in the most probable enzymes involved in the different stages and try to locate the key enzyme(s) from this map. Now accordingly, use the protein database to find out the genes that were responsible for making these enzymes. You may be surprised to see that inspite of taking all precaution of deletion of the genes, you have left behind certain genes which can carry out the metabolism.