Using structurally fungible biosensors to evolve improved alkaloid biosyntheses

Abstract

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids.

Evolving a Generalist Biosensor for Bicyclic Monoterpenes

“…While we wanted to make one, broad-ranging sensor for the variety of monoterpene pathways and applications that are even now being developed, we were surprised to find that only generalists emerged from our selections, despite the application of both counterselection and additional rounds that attempted to “push” a lineage toward fenchol specificity. These results stand in contrast to previous results with benzylisoquinoline alkaloids (BIAs) despite using a similar mutagenesis strategy, where we were able to find highly specific sensors for five different compounds starting from a similar TetR family member, RamR . The seeming intransigence of CamR to change, rather than broaden, effector specificity could be due to several factors.…”

Divergent Directed Evolution of a TetR-type Repressor Towards Aromatic Molecules

“…Directed evolution campaigns to evolve generalist aTFs to be more speci c towards a single target molecule show a high rate of success 50,56,73 . Evolving narrow or ligand-speci c aTFs for induction by new molecules was also recently reported using one or a combination of random mutagenesis, computational protein design, and site saturation mutagenesis 52,53,74−76 .…”

“…This demonstrates the bene t of structure-guided site-saturation mutagenesis of the ligand-binding pocket coupled with random mutagenesis of the entire ligand-binding domain to simultaneously evolve a single ligandspeci c aTF to respond to multiple molecules. Similar methods were used for example to improve response and eliminate crosstalk of the promiscuous aTFs RamR 73 and LysG 56 , or to expand substrate speci city of AraC 74 ,PcaV 75 ,and VanR 76 and over a single generation of mutagenesis.…”

Divergent Directed Evolution of a TetR-type Repressor Towards Aromatic Molecules

“…This demonstrates the benefit of structure-guided site-saturation mutagenesis of the ligand-binding pocket coupled with random mutagenesis of the entire ligand-binding domain to simultaneously evolve a single ligand-specific aTF to respond to multiple molecules. Similar methods were used for example to improve response and eliminate crosstalk of the promiscuous aTFs RamR 73 and LysG 56 , or to expand substrate specificity of AraC 74 ,PcaV 75 ,and VanR 76 and over a single generation of mutagenesis.…”

“…Directed evolution campaigns to evolve generalist aTFs to be more specific towards a single target molecule show a high rate of success 50,56,73 . Evolving narrow or ligand-specific aTFs for induction by new molecules was also recently reported using one or a combination of random mutagenesis, computational protein design, and site saturation mutagenesis 52,53,[74][75][76] .…”