Improved pyrrolysine biosynthesis through continuous directed evolution of the complete pathway

Abstract Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improved Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we devised an approach termed Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway exhibited a 32-fold improved yield of Pyl-containing super- folder green fluorescent protein (sfGFP) compared to the rationally engineered ancestor, whereas the WT pathway produced no detectable quantities of Pyl-containing sfGFP. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl- proteins in E. coli.