S32: Novel alkene reductases from Clostridium sporogenes

Biocatalytic reduction of nitroalkenes is a promising reaction in chiral synthesis, as it gives up to two new asymmetric carbons in the structure and the product can be easily converted into corresponding amines, aldehydes, carboxylic acids or denitrated compounds. Clostridium sporogenes, a Gram positive obligately anaerobic bacterium, was previously shown to be an extraordinary source for unusual reductases as it can catalyze biocatalytic reduction of wide range of substrates such as nitroalkenes, enoates and nitro compounds, and can be used to produce chiral products.

The C. sporogenes genome sequencing project allowed identification of the gene encoding fldZ enoate reductase assumed to be responsible for enantioselective reduction of unsaturated carboxylic acids. Therefore, we used the ClosTron gene knock out system for Clostridia to inactivate the fldZ gene. The analysis of the fldZ mutants showed that C. sporogenes possesses multiple enzymatic activities, reducing enoates, β,β-disubstituted and α,β-disubstituted nitroalkenes with different yields and enantioselectivities.

Further analysis of the mutants and the sequence of C. sporogenes genome resulted in identification of a novel reductase that might be involved in reduction of cinnamoyl-CoA to phenylpropanoyl-CoA. In addition, putative genes with homology to the ‘old yellow enzyme’ reductase and five potential nitroreductases were identified.

Therefore, these genes were all cloned and the products of their overexpression were tested for reduction of wide range of alkenes, carboxylic acids and nitro compounds. In conclusion, C. sporogenes contains at least two nitroalkene reductases with unusual properties, which may provide new opportunities for application in industrial biocatalysis.