Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD(+), for enhancing the production of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD(+) regeneration. We engineered a-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD(+), were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD(+), less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM)

Selectivity criteria for hydrogenation of citral in the liquid phase over unsupported bimetallic nickel-chromium and nickel-molybdenum catalysts

The hydrogenation of citral has been investigated in cyclohexane with unsupported bimetallic Ni1-x Crx and Ni1-x Mo catalysts, prepared by co-reduction of mixtures of dry iodides of appropriate composition with sodium naphthalene as the reducing agent. The reaction was carried out in a static reactor and its progress was followed by measuring the concentrations of the components in the liquid phase. The measured product distributions versus citral conversion thus obtained allowed the calculation of the ratios kibM/kibN on which depend the theoretical equations for the concentration of the products deduced from a LANGMUIR-HINSELWOOD mechanism. The ratios kibM/kibN where ki, kj are the rate constants and bM, bN are the adsorption equilibrium constants for two competitive parallel reactions or two consecutive reactions, thus computed have been used as selectivity criteria. Whereas Ni0,88 Cr0,12 was the most active and the most selective catalyst in the hydrogenation of citral to citronellal, the molybdenum doped catalysts showed better selectivity in the hydrogenation of citral to citronellol. The initial hydrogenation rate increased with the molybdenum or the chromium content, but the selectivity was almost independent of the atomic composition