This review summarizes how ultrahigh-throughput screening methods employ cells and biomimetic compartments to access the vast, unexplored diversity of biocatalysts with novel functions derived from directed evolution and metagenomics libraries.
Incorporating artificial metal‐cofactors into protein scaffolds results in a new class of catalysts, termed biohybrid catalysts or artificial metalloenzymes. Biohybrid catalysts can be modified chemically at the first coordination sphere of the metal complex, as well as at the second coordination sphere provided by the protein scaffold. Protein‐scaffold reengineering by directed evolution exploits the full power of nature's diversity, but requires validated screening and sophisticated metal cofactor conjugation to evolve biohybrid catalysts. In this Minireview, we summarize the recent efforts in this field to establish high‐throughput screening methods for biohybrid catalysts and we show how non‐chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we shed light on the potential of this field and challenges that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.
The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD ؉ -oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a cooxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed. G enome sequencing of organisms provides information regarding the distribution of genes encoding biotechnologically important metabolic pathways. This is true for the clostridial butyrogenic pathway, which converts acetyl-conenzyme A (CoA)-the terminal oxidation product of glucose via glycolysis-to butyrate. Genes encoding enzymes from this pathway are widespread in genome-sequenced clostridia and related species (1-8). In spite of the central importance of butyrate-forming genes in these organisms, only individual enzymes from a comparably small selection of organisms have been purified and carefully studied in the past (9-15). In particular, Clostridium acetobutylicum enzymes were of great interest due to the organism's capability of producing acetone and butanol (16)(17)(18). Driven by the urgent need to replace oil-derived fossil fuels, genetic engineering of microbes for production of butan-1-ol, a promising alternative transportation fuel, is proceeding worldwide (16,(18)(19)(20)(21)(22)(23)(24).A long-known metabolic route such as the butyrate pathway (Fig. 1A) can suddenly gain fundamentally new meanings when hitherto-unknown properties of individual enzymes within this pathway become known. While for decades the role of butyrate formation was generally a...
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