(15a) Identification and Reconstruction of Pathways for Lignin Catabolism

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Cellulosic biofuel production yields a substantial lignin byproduct stream that currently has few applications. Biological conversion of lignin compounds into chemicals and fuels has the potential to improve the economics of cellulosic biofuels. Microbial strains have been isolated with the ability to specifically degrade challenging lignin dimers, and the associated pathways could substantially improve the conversion of lignin into valuable biochemicals. However, no dimer degradation pathway has been characterized to the level necessary for reconstruction in a heterologous host, largely due to the combined challenges of fully identifying the relevant enzymes and then optimizing the resulting pathways for heterologous function. We have used a barcoded transposon library to rapidly identify entire catabolic pathways in genetically-intractable microbes such as the lignin-degrading strain Novosphingobium aromaticivorans. Simultaneously, we are reconstructing pathways for lignin catabolism in Escherichia coli to enable pathway characterization in a tractable and well-defined host. Evolutionary optimization of these heterologous pathways identified factors limiting pathway activity and will allow the design of characterized, optimized synthetic operons for lignin dimer degradation.