Effects of Pyruvate Formate Lyase Inactivation in Klebsiella Pneumoniae and Its Application to Diol Production

The fermentative production of 2,3-butanediol and 1,3-propanediol has been intensively studied because of the potential of these products as platform chemicals, such as polymers, fuels, and plastics. Among several natural diol producers, Klebsiella pneumoniae can effectively synthesize both 2,3-butanediol and 1,3-propanediol from a wide range of carbon sources. In this study, three genes, including wabG, ldhA, and pflB, were removed in K. pneumoniae genome to reduce both its pathogenicity and the production of byproducts. In flask cultivation with minimal medium, the yield of 2,3-butanediol and 1,3-propanediol from rationally engineered K. pneumoniae (¢wabG ¢ldhA ¢pflB) increased significantly. In particular, the inactivation of pflB significantly decreased the synthesis of byproducts and improved the yield of 2,3-butanediol from glucose to 92.2% of the theoretical maximum. However, the growth rate of the pflB mutant was slightly reduced compared to its parental strain. Intracellular acetyl-CoA and redox balance was measured in pflB deficient K. pneumoniae to find the reason of reduced cell mass production. Transcriptomic analysis with RNAseq was also conducted and the pflB mutation in K. pneumoniae significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system. Our results indicate that the growth defect of pflB deficient K. pneumoniae is primarily due to the reduction in the size of the acetyl-CoA pool that redistributes the carbon flux to diol production.