The analysis of the C. sporogenes genome sequence allowed identification of two hypothetical genes encoding proteins with homology to flavin containing C=C double bond reductases, fldZ 2-enoate reductase, assumed to be responsible for reduction of cinnamate in the reductive branch of L-phenylalanine fermentation via the Stickland reaction, and an OYE-like reductase. Both reductases were subsequently cloned, overexpressed in E. coli under anaerobic conditions and tested for reduction of unsaturated compounds. The activity tests showed that fldZ possesses a narrow substrate range and can reduce only aromatic enoates such as cinnamic acid or p-coumaric acid. FldZ also reduced (E)-1-nitro-2-phenylpropene and (E)-2-nitro-1-phenylpropene with excellent and poor enantioselectivities respectively. On the other hand, the OYE-like reductase did not show activity towards unsaturated substrates in the activity assays and the substrate range of this reductase is unknown.
Growth experiments comparing wild type C. sporogenes and the fldZ genomic knock out mutant created with the ClosTron system showed that the fldZ reductase is not involved in the L-phenylalanine fermentation. Further analysis of the C. sporogenes genome resulted in identification of a novel reductase that might be involved in reduction of cinnamoyl-CoA to 3-phenylpropionyl-CoA in the Stickland reaction.
C. sporogenes was found to be a source for C=C double bond reductases, which now can be fully exploited in industrial applications.