Gilmour Andrew R, Wei Qiyao, Wang Chad, Hellinger Jessica, Pandarinath Vidya, d'Oelsnitz Simon, Powell Eli, Brodbelt Jennifer S, Ellington Andrew D, Thyer Ross
Systems, Synthetic, and Physical Biology, Rice University, Houston, TX.
Department of Bioengineering, Rice University, Houston, TX.
bioRxiv. 2025 Jun 26:2025.06.26.661824. doi: 10.1101/2025.06.26.661824.
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 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. 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.
3,4-二羟基苯丙氨酸（L-多巴）是一种源自L-酪氨酸的含儿茶酚氨基酸，主要作为翻译后修饰被引入蛋白质中。它也可以通过正交tRNA合成酶：tRNA对以位点特异性方式引入蛋白质中。然而，尽管人们对包含L-多巴的扩展遗传密码感兴趣，但在改善与其生产相关的代谢方面所做的努力却很有限。在此，我们报道并设计了一种LysR家族转录因子PP_2251，它可作为L-多巴的生物传感器，从而能够优化遗传回路，通过基于乳液PCR的定向进化方案——区室化配对复制（CPR）大幅提高L-多巴的产量。利用这种生物传感器，通过CPR对一种混杂的黄素依赖性单加氧酶（HpaB）进行了优化，以增强L-多巴的生物合成。工程改造后的HpaB变体能够支持L-多巴以80%的掺入效率高效翻译掺入蛋白质中，产量超过250 mg/L。L-多巴产量的提高不仅对蛋白质工程和遗传密码扩展具有重要意义，而且应该会影响多巴衍生天然产物的生产，包括儿茶酚胺神经递质和苄基异喹啉生物碱（BIA），这些都是具有重要药学意义的分子。
