A consortium-based approach to adaptive laboratory evolution of Acinetobacter baylyi ADP1 for lignin valorization

ABSTRACT The utility of Acinetobacter baylyi ADP1 (ADP1) for lignin valorization has yet to be sufficiently investigated compared to other organisms such as Pseudomonas , Rhodococcus, etc. In this study, a two-step Adaptive Laboratory Evolution (ALE) process was used to evolve a unique ADP1 strain ( A. baylyi SAG_185). Initially, several ADP1 strains were evolved for substrate tolerance to specific lignin-related aromatics (LRAs). Subsequently, a consortium of these strains was adaptively evolved in a mixture of LRAs, which resulted in the evolution of SAG_185. This strain was capable of simultaneous utilization of multiple LRAs at higher concentrations as well as grow on a depolymerized lignin-rich residue obtained from enzymatic hydrolysis of pre-treated corncob. This is the first report on such an evolutionary strategy. Whole-genome sequence analysis of all the evolved strains revealed large-scale mutations involving insertion sequences (IS). In particular, SAG_185 revealed a critical mutation in the vanR repressor gene, resulting in the up-regulation of vanAB genes required to convert vanillate to the key intermediate, protocatechuate (PCA). Additionally, there were two large deletions of 9kb and 38kb DNA segments, including genes for putative transcriptional regulators of LysR, MarR and AraC family. The evolved strains also showed mutations in the hcaE gene, responsible for the uptake of LRAs. The vast number of mutations in hypothetical proteins, transporter and regulatory sequences indicate the underlying effects of these regions on the uptake of multiple LRAs. Overall, our findings provide potential targets for reverse engineering of A. baylyi ADP1 for lignin valorization. IMPORTANCE This study shows a novel strategy for adaptive laboratory evolution, which can be generically adopted to evolve bacterial strains for taking up multiple substrates which are toxic at higher concentrations. We developed a two-step evolutionary strategy to evolve a unique strain, A. baylyi SAG_185, which could take up multiple lignin-related aromatic monomers at higher concentrations as well as grow on depolymerized lignin. Initially, individual strains were adapted to utilize single aromatic monomers at higher concentrations. After many unsuccessful attempts to adapt these strains for utilizing multiple monomers, a consortium of the five evolved strains were grown on a mixture of aromatics and adapted to utilize all the monomers at high concentrations. The adapted consortia resulted in the evolution of SAG_185. Whole-genome sequence analysis of all these strains gave rise to many interesting insights on potential genetic targets for reverse engineering of A. baylyi ADP1 for lignin valorization.