Understanding butanol tolerance and assimilation in P seudomonas putida   BIRD ‐1: an integrated omics approach

Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost compounds. However, the strain has two major limitations: it assimilates butanol as sole carbon source and butanol concentrations above 1% (v/v) are toxic. With the aim of facilitating BIRD-1 strain design for industrial use, a genome-wide mini-Tn5 transposon mutant library was screened for clones exhibiting increased butanol sensitivity or deficiency in butanol assimilation. Twenty-one mutants were selected that were affected in one or both of the processes. These mutants exhibited insertions in various genes, including those involved in the TCA cycle, fatty acid metabolism, transcription, cofactor synthesis and membrane integrity. An omics-based analysis revealed key genes involved in the butanol response. Transcriptomic and proteomic studies were carried out to compare short and long-term tolerance and assimilation traits. Pseudomonas putida initiates various butanol assimilation pathways via alcohol and aldehyde dehydrogenases that channel the compound to central metabolism through the glyoxylate shunt pathway. Accordingly, isocitrate lyase - a key enzyme of the pathway - was the most abundant protein when butanol was used as the sole carbon source. Upregulation of two genes encoding proteins PPUBIRD1₂240 and PPUBIRD1₂241 (acyl-CoA dehydrogenase and acyl-CoA synthetase respectively) linked butanol assimilation with acyl-CoA metabolism. Butanol tolerance was found to be primarily linked to classic solvent defense mechanisms, such as efflux pumps, membrane modifications and control of redox state. Our results also highlight the intensive energy requirements for butanol production and tolerance; thus, enhancing TCA cycle operation may represent a promising strategy for enhanced butanol production.