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

Gilmour, A. R.; Wei, Q.; Wang, C.; Hellinger, J.; Pandarinath, V.; d'Oelsnitz, S.; Powell, E.; Brodbelt, J. S.; Ellington, A. D.; Thyer, R.

doi:10.1101/2025.06.26.661824

bioRxiv · 10.1101/2025.06.26.661824

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

Gilmour, A. R.·Wei, Q.·Wang, C.·Hellinger, J.·Pandarinath, V.·d'Oelsnitz, S.·Powell, E.·Brodbelt, J. S.·Ellington, A. D.·Thyer, R.

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Abstract

3,4-dihydroxyphenylalanine (L-DOPA) is a catechol-containing amino acid derived from L-tyrosine that can be introduced into proteins primarily as a post-translational modification. It can also be introduced in a site-specific manner into proteins via orthogonal tRNA synthetase:tRNA pairs. However, despite the interest in expanded genetic codes containing L-DOPA, efforts to improve metabolism relating to its production have been limited. Here, we report and engineer a LysR-family transcription factor, PP_2251, that can serve as a biosensor for L-DOPA, thereby allowing the optimization of genetic circuits for vastly improved L-DOPA production via compartmentalized partnered replication (CPR), an emulsion PCR-based directed evolution scheme. Using this biosensor, a promiscuous E. coli flavin-dependent monooxygenase (HpaB) was optimized via CPR for enhanced L-DOPA biosynthesis. Engineered HpaB variants could support efficient translational incorporation of L-DOPA into proteins with 80% incorporation efficiency and yields in excess of 250 mg.L-1. Improvements in L-DOPA production are not only of great interest for protein engineering and genetic code expansion but should impact the production of DOPA-derived natural products including catecholamine neurotransmitters and benzylisoquinoline alkaloids (BIAs), molecules of intense pharmaceutical interest.

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Gilmour, A. R., Wei, Q., Wang, C., Hellinger, J., Pandarinath, V., d'Oelsnitz, S., Powell, E., Brodbelt, J. S., Ellington, A. D., Thyer, R.. 2025-06-26. Directed evolution of L-DOPA sensing and production enables efficient incorporation of an expanded genetic code into proteins. https://doi.org/10.1101/2025.06.26.661824

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