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

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 x 10(6) 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.

Current and Emerging Approaches for Natural Product Biosynthesis in Microbial Cells

“…Cells can be sorted into distinct pools based upon novel product formation, using an intrinsic feature of the molecule, such as fluorescence. This technique has already been demonstrated with E. coli producing novel metal porphyrins using fluorescence, [84] and may be applicable in the future for other natural products. Metagenomics (reviewed recently elsewhere [85,86] ) has attempted to solve the cultivation problem by removing the culturing step and simply cloning the gene or genes responsible for the natural product biosynthesis by isolation of genomic DNA in situ.…”

Iron Transport in Escherichia Coli: All has not been said and Done

“…Besides Zn(II), Fe(II), Co(II) and probably Mn(II) or Cd(II) are transported (Kwon et al 2004;Grass et al 2005a, b). In this, ZupT resembles its eukaryotic orthologues that also have broad substrate specificity (Gaither & Eide 2000;Guerinot 2000).…”

Ultrahigh‐Throughput FACS‐Based Screening for Directed Enzyme Evolution

“…Kwon et al have developed a method to screen ferrochelatase activity in vivo in E. coli cells, [36] based upon the reduction in fluorescence intensity at 650 nm accompanying reaction. By screening a random library of 2.4 10 6 cells for reduced fluorescence, they obtained several mutants with enhanced catalytic activity.…”

“…1.4-2.4-fold increased catalytic turnover. [36] ferrochelatase Activity of Product entrapment inside the cell because of its lipoModel screening. [38] b-galactosidase philic tail.…”