Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64

ABSTRACT Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription–polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s−1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli

Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, are useful for simplifying the recombinant protein purification process, for screening molecules that fail to remain soluble after tag removal, and to promote higher yields of soluble target protein. Herein, we review controlled intracellular processing (CIP) systems, tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular protein processing regarding system design features, significant advantages and limitations of the various strategies.

Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression

Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a “shuttle” vector containing a removable selective marker, which allows feasible cloning steps in E. coli and subsequent protein expression in LAB. In fact, the cassette can be easily excised from the selected recombinant plasmid, and the resulting marker-free vector transformed into the final LAB host. Further useful elements, as improved MCS, 6xHis-Tag, and thrombin cleavage site sequences were introduced. The resulting vector allows easy cloning in E. coli, can be quickly converted in a food-grade expression vector and harbors additional elements for improved recombinant protein purification. Overall, such features make the new vector an improved tool for food-grade expression.