Development of hybrid strains for the mineralization of chloroaromatics by patchwork assembly

The persistence of chloroaromatic compounds can be caused by various bottlenecks, such as incomplete degradative pathways or inappropriate regulation of these pathways. Patchwork assembly of existing pathways in novel combinations provides a general route for the development of strains degrading chloroaromatics. The recruitment of known complementary enzyme sequences in a suitable host organism by conjugative transfer of genes might generate a functioning hybrid pathway for the mineralization of some chloroaromatics not degraded by the parent organisms. The rational combination uses (a) peripheral, funneling degradation sequences originating from aromatics-degrading strains to fulfill the conversion of the respective analogous chloroaromatic compound to chlorocatechols as the central intermediates; (b) a central chlorocatechol degradation sequence, the so-called modified ortho pathway, which brings about elimination of chlorine substituents; and (c) steps of the 3-oxoadipate pathway to reach the tricarboxylic acid cycle. The genetic organization of these pathway segments has been well characterized. The specificity of enzymes of the xylene, benzene, biphenyl, and chlorocatechol pathways and the specificity of the induction systems for the chlorinated substrates are analyzed in various organisms to illustrate eventual bottlenecks and to provide alternatives that are effective in the conversion of the "new" substrate. Hybrid pathways are investigated in "new" strains degrading chlorinated benzoates, toluenes, benzenes, and biphenyls. Problems occurring after the conjugative DNA transfer and the "natural" solution of these are examined, such as the prevention of misrouting into the meta pathway, to give a functioning hybrid pathway. Some examples clearly indicate that patchwork assembly also happens in nature.