Directed evolution of L-DOPA sensing and production enables efficient incorporation of an expanded genetic code into proteins

Abstract

L-3,4-dihydroxyphenylalanine (L-DOPA), is a versatile catechol-containing amino acid derived from L-tyrosine that is both a key metabolite for the production of many valuable natural products and a naturally occurring post-translational modification in proteins. While advances in microbial natural product biosynthesis and site-specific incorporation of L-DOPA have both paved the way for relevant applications of L-DOPA, progress has been stifled by a lack of robust in vivo detection tools and high-efficiency biosynthetic enzymes. Here, we report and engineer a LysR-family transcription factor, PP_2251, that can serve as a transcriptional regulator for L-DOPA with a dynamic range of >100-fold when coupled with a fluorescent reporter. Leveraging this improved regulator in conjunction with compartmentalized partnered replication (CPR), we engineered a promiscuous E. coli flavin-dependent monooxygenase (HpaB) for enhanced L-DOPA biosynthesis. This represents the first use of high-throughput emulsion PCR to improve a metabolic enzyme and yielded HpaB variants that showed >40-fold activity within our genetic circuit and a 6-fold increase in L-DOPA titer compared to wild-type HpaB. We further demonstrated that the highest performing HpaB variant was capable of supporting efficient translational incorporation of L-DOPA into protein, achieving 97% incorporation efficiency and yields approaching 250 mg.L-1. Collectively, these advancements enable the in vivo detection, biosynthesis, and site-specific incorporation of L-DOPA for synthetic and chemical biology applications.