{'fraction_non_alphanumeric': 0.024887800897592818, 'fraction_numerical': 0.0073439412484700125, 'mean_word_length': 5.503978779840849, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Significance Enzymes rely on cofactors, both organic molecules and metal ions, for reactivity beyond what can be performed with the 20 standard amino acids. One way to expand the reactivity of heme-dependent proteins is to swap the native iron atom in the heme cofactor for a different metal. Because the resulting metal-containing cofactor is new to nature, methods for producing these nonnative hemoproteins are difficult and often limited to the test tube. Here, we report a method for subverting the heme biosynthetic pathway in E. coli to produce cobalt protoporphyrin IX (CoPPIX) and CoPPIX-substituted hemoproteins in living cells. This method enables the use of CoPPIX-substituted enzymes as biocatalysts and widens the scope of proteins that may be studied by metal substitution. 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.