Engineered acetaldehyde dehydrogenase for the efficient degradation of acetaldehyde

Acetaldehyde is highly cytotoxic and widely presents in food and the environment. Aldehyde dehydrogenase (ALDH) can degrade acetaldehyde to non-toxic acetic acid, showing potential for acetaldehyde elimination. However, a lack of high-throughput methods for screening efficient variants is a significant obstacle to ALDH design. Here, we established a visualized high-throughput method to screen recombinantly expressed ALDH variants in Bacillus subtilis by fluorescent probes of dual-acceptor cyanine-based in response to NADH, the acetaldehyde degradation product. Molecular docking revealed key amino acids in the binding region of acetaldehyde to ALDH. Combined with saturation mutagenesis and visualization high-throughput methods, a variant ALDH(S273N) with an activity of 119.82 U center dot mL(-1) was screened. The optimal reaction temperature and pH of ALDH(S273N) were 60 degrees C and 9.0, respectively. ALDH(S273N) showed stability at 30-50 degrees C and pH 5.0-9.0. The activity of ALDH(S273N) was increased to 263.52 U center dot mL(-1) by fermentation optimization, which was 5.58 times that of ALDH(WT). The degradation rate of ALDH(S273N) to 100 mmol L-1 acetaldehyde was 87.34% within 2 h, which was 4.2 times that of the wild enzyme (20.81%). As far as we know, this is the ALDH with the highest activity reported so far, and it is also the first time that ALDH has been used for the efficient degradation of acetaldehyde. Overall, the reported high-throughput screening method and developed mutants represent a significant advance in green bio-elimination technologies of acetaldehyde.

Automated summary

A visualized high-throughput method was developed to screen ALDH variants, and a variant ALDH(S273N) with the highest activity was identified. This variant showed efficient degradation of acetaldehyde, making it a promising green bio-elimination technology.