A High‐Throughput Screen for Porphyrin Metal Chelatases: Application to the Directed Evolution of Ferrochelatases for Metalloporphyrin Biosynthesis

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Abstract
Porphyrins are of particular interest in a variety of applications ranging from biocatalysis and chemical synthesis to biosensor and electronic technologies as well as cancer treatment. Recently, we have developed a versatile system for the high-level production of porphyrins in engineered E. coli cells with the aim of diversifying substitution patterns and accessing porphyrin systems not readily available through chemical synthesis. However, this approach failed to produce significant amounts of the metalloporphyrin in vivo from overproduced protoporphyrin due to insufficient metal insertion. Therefore, we systematically assessed the activity of the B. subtilis ferrochelatase in vivo and in vitro. A true high-throughput-screening approach based on catalytic in vivo ferrochelatase activity was developed by using fluorescence-activated cell sorting (FACS). This assay was used to screen a library of 2.4×106 ferrochelatase mutants expressed in protoporphyrin-overproducing recombinant E. coli cells. Several selected protein variants were purified, and their improved catalytic activity was confirmed in vitro. In addition to ferrochelatase activity, metal transport into E. coli was identified as another limitation for in vivo heme overproduction. Overexpression of the metal transporter zupT as part of the assembled pathway increased the overall metalloporphyrin production twofold. This report represents the most exhaustive in vitro evolution study of a ferrochelatase and demonstrates the effectiveness of our novel high-throughput-screening system for directed evolution of ferrochelatases based on their catalytic activity.