CRISPRi based system for enhancing 1-butanol production in engineered Klebsiella pneumoniae

The introduction of a new metabolic pathway will affect the host's other metabolic pathways. Differential metabolomics is a useful method to reveal the relationship between the different metabolic pathways inside a microorganism. Three Klebsiella pneumoniae strains with different 1-butanol production capability were chosen to study the differential metabolomics to reveal the relationship between 1-butanol synthesis and other metabolic pathways. The biosynthesis of Val, Leu, Ile, Met, Gly and Ala were all found to have close relationships with the 1-butanol synthesis. To tuning the synthesis of these amino acids, CRISPRi (Clustered regularly interspaced short palindromic repeats interference) system was adopted. The resulting transcription levels, intracellular amino acid content, and 1-butanol production were significantly affected. In comparison with KLA, the concentrations of intracellular Ile, Leu, Val, Met, and Ala in all the repressed strains was reduced, and the resulting content of intracellular Thr and 1-butanol production was increased. The largest increase in 1-butanol production was obtained with the strain KLA-ilvB3 with a yield increase 154% higher than that of KLA. The results show that CRISPRi is a feasible method to manipulate genes in Klebsiella pneumoniae.