Multiplexed fitness profiling by RB-TnSeq elucidates pathways for lignin-related aromatic catabolism in Sphingobium sp. SYK-6.

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin. [Display omitted] • An RB-TnSeq library is generated in the aromatic catabolic bacterium, Sphingobium sp. SYK-6 • The library is enriched under dozens of growth conditions to determine gene fitness • Fitness profiling identifies details of lignin-related aromatic catabolism and tolerance • This RB-TnSeq library is a resource for future fitness profiling in additional conditions Bleem et al. develop a randomly barcoded transposon insertion (RB-TnSeq) library in Sphingobium sp. SYK-6 and then apply it to quantify gene fitness in aromatic catabolism and stress tolerance conditions. The library identifies details of SYK-6 metabolism, provides metabolic engineering targets for biological lignin valorization, and enables future fitness profiling. [ABSTRACT FROM AUTHOR]