Elimination of by-product formation during production of 1,3-propanediol in Klebsiella pneumoniae by inactivation of glycerol oxidative pathway

Abstract

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).

Engineering Escherichia coli for Microbial Production of Butanone

“…The increased PEP pool potentially enhances propionyl-CoA production, as more carbon flux is diverted toward oxaloacetate (OAA) via the anaplerotic reactions catalyzed by the endogenous PEP carboxylase or PEP carboxykinase (encoded by ppc or pckA, respectively) (24,35,36). Similar to other Gram-negative bacteria that utilize glycerol (e.g., Klebsiella pneumoniae), inactivating the fermentative GldA-DhaK pathway in E. coli enhances acetate formation (24,37). Inactivating dhaK is known to decrease the NADH/NAD ϩ ratio, thus reducing the biosynthesis of reduced products such as ethanol and 1-propanol.…”

“…Inactivating dhaK is known to decrease the NADH/NAD ϩ ratio, thus reducing the biosynthesis of reduced products such as ethanol and 1-propanol. The cell potentially compensates for the NADH deficiency by enhancing acetate formation for ATP production (24,37). An increase in the PEP pool can also result in the decarboxylation of pyruvate to acetate catalyzed by PoxB (38).…”

Use of Glycerol in Biotechnological Applications

“…The regeneration of reduction equivalents for 1,3-PDO production from glycerol is ensured in K. pneumoniae by simultaneous operation of the oxidative pathway of glycerol utilization. Here, NADH is generated by glycerol dehydrogenase during oxidation of glycerol to dihydroxyacetone and during catabolism of dihydroxyacetone phosphate, which is formed from dihydroxyacetone by dihydroxyacetone kinase (Seo et al, 2009). Thus, the demand of NADH limits the theoretical yield of 1,3-PDO production from glycerol.…”

“…Furthermore, the byproducts 3-HPA, succinic acid, lactic acid, acetic acid and ethanol were significantly reduced . When analyzing a K. pneumoniae mutant strain defective in the genes for NADH-dependent 1,3-PDO dehydrogenase and the oxydative glycerol utilization pathway Seo et al (2009) reported that the mutant surprisingly retained the ability of 1,3-PDO production. 1,3-PDO yields were low but a strongly reduced byproduct formation was reported (Seo et al, 2009).…”

“…When analyzing a K. pneumoniae mutant strain defective in the genes for NADH-dependent 1,3-PDO dehydrogenase and the oxydative glycerol utilization pathway Seo et al (2009) reported that the mutant surprisingly retained the ability of 1,3-PDO production. 1,3-PDO yields were low but a strongly reduced byproduct formation was reported (Seo et al, 2009). Later, Seo et al (2010 published the identification of a second 1,3-PDO dehydrogenase, which possesses high homology to E. coli YqhD and is, in contrast to the dhaT encoded enzyme, NADPH-dependent.…”