Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach

Recently, the program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. We explored the utility of this program further and applied it for engineering of the aldoxime dehydratase from Bacillus sp. OxB-1. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. In part, this activity increase can be rationalised by an improved heme incorporation of the variants. For example, a single mutation gave a 1.8 fold increased enzymatic activity and 30% improved absolute heme incorporation.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Synthesis of Bifunctional Molecules for the Production of Polymers Based on Unsaturated Fatty Acids as Bioderived Raw Materials

Currently, investigations of polymer-building blocks made from biorenewable feedstocks such as, for example, fatty acids, are of high interest for the chemical industry. An alternative synthesis of nitrile-substituted aliphatic carboxylic acids as precursors for ω-amino acids, which are useful to produce polymers, was investigated starting from biorenewable fatty acids. By hydroformylation of unsaturated fatty acids or unsaturated acids being accessible from unsaturated fatty acids by cross-metathesis reactions, aldehydes are formed. In this work, the hydroformylation of such unsaturated acids led to the formation of the corresponding aldehydes, which were afterwards converted with hydroxylamine to aldoximes. Subsequent dehydration by an aldoxime dehydratase as a biocatalyst or by CuII acetate led to the desired nitriles. Within this work, C7-, C9- and C11-carboxylic acids with a terminal nitrile functionality as well as a branched nitrile-functionalized stearate derivative were synthesized by means of this approach. As these nitriles serve as precursors for amino acids being suitable for polymerization, this work represents an alternative synthetic access to polyamide precursors, which starts directly from unsaturated fatty acids as biorenewable resources and avoids harsh reaction conditions as well as and by-product formation.

Aldoxime Dehydratase Mutants as Improved Biocatalysts for a Sustainable Synthesis of Biorenewables-Based 2-Furonitrile

2-Furonitrile is an interesting nitrile product for the chemical industry due to its use as intermediate in the field of fine chemicals and pharmaceuticals or as a potential sweetener, as well as due to its access from biorenewables. As an alternative to current processes based on, e.g., the ammoxidation of furfural with ammonia as a gas phase reaction running at > 400 °C, we recently reported an enzymatic dehydration of 2-furfuryl aldoxime being obtained easily from furfural and hydroxylamine. However, improving the catalytic properties of the aldoxime dehydratase biocatalyst from Rhodococcus sp. YH3-3 (OxdYH3-3) in terms of activity and stability remained a challenge. In this contribution, the successful development of aldoxime dehydratase OxdYH3-3 mutants that were generated by directed evolution and its enhanced activity toward 2-furfuryl aldoxime is reported. The mutant OxdYH3-3 N266S showed an improved activity of up to six times higher than the wild type when utilizing a substrate concentration of 50–100 mM of 2-furfuryl aldoxime.