吡咯赖氨酸（PYL，O）存在于自然界作为22次蛋白原氨基酸。尽管 Pyl 是蛋白质的基本组成部分，但其化学和生物合成的困难和低效率阻碍了 Pyl 的研究。在这里，我们通过合理工程和整个生物合成途径的定向进化来改善 Pyl 生物合成。适应大肠杆菌中Pyl 生物合成基因的毒性，我们还开发了交替噬菌体辅助非连续进化 (Alt-PANCE)，可交替突变和选择性噬菌体生长。与合理设计的祖先相比，进化的途径使含 Pyl 的报告蛋白的产量提高了 32 倍。进化的 PylB 突变体在细胞内以高达 4.5 倍的升高水平存在，并且显示出高达 2.2 倍的蛋白酶抗性增加。该研究表明，Alt-PANCE 提供了一种用于进化表现出毒副作用的蛋白质的通用方法，并进一步提供了一种能够在大肠杆菌中产生大量 Pyl 蛋白质的改进途径。
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Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway
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 improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. 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.