Novel enzymes for lignin degradation generated with an easy chromosomal gene amplification method

Gene duplication and divergence underlie evolutionary processes. To accelerate the emergence of new enzymes in the laboratory, a method was devised to create chromosomal arrays of duplicated genes. Gene amplification should increase the chance for beneficial mutations to accrue in a targeted region and allow rapid selection of mutants during continuous culture. Acinetobacter baylyi ADP1, a non-pathogenic, genetically malleable soil bacterium was ideal for developing this technique, called Evolution by Amplification and Synthetic Biology (EASy). With selection, 10 to 300 copies of specific genomic regions could be obtained. When key foreign genes were inserted into the ADP1 chromosome and then amplified, they expanded the ability of ADP1 to consume aromatic carbon sources. Growth depended on multiple copies of the foreign DNA, presumably because gene function and/or expression were not optimal. In fewer than 1,000 generations, mutants arose in in which a single copy of modified foreign DNA was sufficient for improved growth. Improvements included the ability to grow on higher concentrations of the substrate, faster growth, and higher growth yields. The copy number of the amplified DNA decreased as new alleles evolved under selection. In one example, a chimeric enzyme emerged that fused a cytochrome P450 family member to a ring-cleavage dioxygenase. There are no known examples of similar natural chimeric enzymes. These studies were designed to expand and modify bacterial aromatic compound catabolism to improve lignin degradation. Currently, lignin is a vastly underused biomass feedstock with an exciting potential for future use in bioenergy and biotechnology.