3-Hydroxypropionic acid (3-HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3-HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an "imbalance between the two enzymes" and the "instability of the first enzyme DhaB" were the major factors limiting 3-HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl-thio-beta-galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH-BGK1 showed the highest level of 3-HP production (2.8 g/L) under shake-flask conditions. When an aerobic fed-batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH-BGK1 produced 38.7 g 3-HP/L with an average yield of 35%. This article reports the highest level of 3-HP production from glycerol thus far.
3-Hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, serves as a precursor to 3-Hydroxypropionic acid (3-HP), a commercially valuable platform chemical. To achieve the effective conversion of 3-HPA to 3-HP, an aldH gene encoding an aldehyde dehydrogenase in Escherichia coli K-12 (AldH) was cloned, expressed, and characterized for its properties. The recombinant AldH exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. AldH preferred NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes tested. Among the aldehydes used, the specific activity was highest (38.1 U mg(-1) protein) for 3-HPA at pH 8.0 and 37 degrees C. The catalytic efficiency (kcat) and the specificity constant (kcat/Km) for 3-HPA in the presence of NAD+ were 28.5 s(-1) and 58.6x10(3) M(-1) s(-1), respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol (DTT) or 2-mercaptoethanol, while several metal ions, particularly Hg2+, Ag+, Cu2+, and Zn2+, inhibited AldH activity. This study illustrates that AldH is a potentially useful enzyme in converting 3-HPA to 3-HP.
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