Fig. 1: Pyl biosynthetic pathway evolution via Alt-PANCE. | Nature Communications

Fig. 1: Pyl biosynthetic pathway evolution via Alt-PANCE.

From: Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway

Fig. 1

A Pyl biosynthesis involves condensation of two lysine molecules by three enzymes—the radical SAM enzyme PylB, ATP-dependent PylC, and PylD. B Biosynthetic pylBCD operon was cloned into a selection phage (SP), while the constitutively expressed pylST and phage shock promoter-controlled gIII were cloned into accessory plasmid (AP) vectors. Numbered portions indicate selection circuit elements that were altered throughout evolution to control selection stringency, including (1) supplementation with BocK, (2) expression level of PylRS, (3) mutant variants of PylRS used, and (4) the number of amber codons present in gIII. C Each Alt-PANCE round entailed two phage passages: selective passage (left) entails operon activity-dependent expression of PIII (pink rods with circular tips); mutagenic passage (right) entails mutagenesis plasmid (MP)-dependent mutagenesis of the SP.

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