Construction of novel mutant microorganisms

A process for the construction of a microorganism strain which comprises (1) culturing Pseudomonas sp. to provide strain A1 which metabolizes an aromatic substrate selected from the group consisting of toluene and alkyl-substituted toluene by the meta pathway via 2-hydroxymuconic semialdehyde or alkyl-substituted 2-hydroxymuconic semialdehyde to biomass and carbon dioxide;

• (2) culturing strain A1 in a medium containing benzoate as the sole source of carbon to provide strain A2 which metabolizes benzoate via the ortho pathway and has lost phenotypic expression of TOL plasmid functions;
• (3) culturing strain A2 in a medium containing benzoate as the sole source of carbon and containing an antibiotic which kills only growing cells to provide strain A3 which is unable to metabolize benzoate via the ortho pathway;
• (4) culturing a mixture of strain A3 and a TOL plasmid-containing strain in a growth medium, and selecting for transconjugate strain A4 having a coinheritance of TOL plasmid encoded functions;
• (5) culturing strain A4 in a medium containing m-toluate as the sole source of carbon, and containing an antibiotic which kills only growing cells, to provide strain A5 which is capable of growing on benzoate and on p-toluate but not on m-toluate;
• and (6) culturing strain A5 in a medium containing benzoate or p-toluate as the sole source of carbon, and containing an antibiotic which kills only growing cells, to provide strain A6 which metabolizes an aromatic substrate selected from the group consisting of toluene and alkyl-substituted toluene, producing 2-hydroxymuconic semialdehyde of alkyl-substituted 2-hydroxymuconic semialdehyde, and exhibits no enzymatic activity that metabolizes 2-hydroxymuconic semialdehyde or alkyl-substituted 2-hydroxymuconic semialdehyde.