Mordaka, Pawel Mateusz
(2014)
Reductions using Clostridium sporogenes.
PhD thesis, University of Nottingham.
Abstract
Clostridium sporogenes was previously shown to be an extraordinary source for unusual reductases. It can catalyze reduction of wide a range of substrates such as nitroalkenes, enoates and nitro compounds, and can be used to generate chiral products.
In preliminary studies, the ClosTron gene knock-out system for Clostridia was used to inactivate the fldZ gene assumed to encode the enzyme responsible for reduction of cinnamic acid in the reductive branch of L-phenylalanine fermentation via the Stickland reaction.
Biotransformations with the fldZ mutant showed that C. sporogenes possesses multiple enzymatic activities, reducing enoates, β,β- and α,β-disubstituted nitroalkenes with different yields and enantioselectivities. The fldZ reductase was found to be responsible for reduction of cinnamic acid, (E)-1-nitro-2-phenylpropene, (E)-2-nitro-1-phenylpropene and β-nitrostyrene. However, the mutant could still reduce (E)-2-nitro-1-phenylpropene, β-nitrostyrene and cinnamic acid confirming the presence of other C=C double bond reductases in C. sporogenes.
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 and OYE-like reductase, which 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 (>99% and 16% 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 mutant in complex and minimal media showed that the fldZ reductase in 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.
Biocatalytic reduction of aromatic nitro compounds to amines can be used as alternative to chemo-reductive routes in preparation of pharmaceutical and agrochemical products. Protein extracts of C. sporogenes were found to reduce aromatic nitro compounds with different yields depending on the substrate structure and electron donor used in the reaction. The genome of C. sporogenes was screened and that allowed identification of six genes encoding hypothetical nitroreductases, which were subsequently overexpressed in E. coli. However, biotransformations using the recombinant nitroreductases did not show amine product formation.
A novel Nylon 6 biosynthesis pathway was designed starting from biorenewable feedstocks. The crucial step in this pathway, reductive cleavage of pipecolic acid to 6-aminocaproic acid was proposed to be catalysed by C. sporogenes D-proline reductase. Thus, the activity of this enzyme was tested towards L- and D-pipecolic acid. Biotransformations showed that pipecolic acid was not accepted as a substrate. In the future, the idea of using D-proline reductase for Nylon biosynthesis may be exploited by improving the reductase activity using protein engineering techniques.
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