Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression.

Effects of mutation of 2,3-butanediol formation pathway on glycerol metabolism and 1,3-propanediol production by Klebsiella pneumoniae J2B.

The current study investigates the impact of mutation of 2,3-butanediol (BDO) formation pathway on glycerol metabolism and 1,3-propanediol (PDO) production by lactate dehydrogenase deficient mutant of Klebsiella pneumoniae J2B. To this end, BDO pathway genes, budA, budB, budC and budO (whole-bud operon), were deleted from K. pneumoniae J2B ΔldhA and the mutants were studied for glycerol metabolism and alcohols (PDO, BDO) production. ΔbudO-mutant-only could completely abolish BDO production, but with reductions in cell growth and PDO production. By modifying the culture medium, the ΔbudO mutant could recover its performance on the flask scale. However, in bioreactor experiments, the ΔbudO mutant accumulated a significant amount of pyruvate (>73mM) in the late phase and PDO production stopped concomitantly. Glycolytic intermediates of glycerol, especially glyceraldehyde-3-phosphate (G3P) was highly inhibitory to glycerol dehydratase (GDHt); its accumulation, followed by pyruvate accumulation, was assumed to be responsible for the ΔbudO mutant's low PDO production.

Complete Genome Sequence of Pseudomonas denitrificans ATCC 13867.

Pseudomonas denitrificans ATCC 13867, a Gram-negative facultative anaerobic bacterium, is known to produce vitamin B12 under aerobic conditions. This paper reports the annotated whole-genome sequence of the circular chromosome of this organism.

Production of 3-hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans.

3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.