Search NASA⌕ Search
Home/Archive/DOE OSTI

DOE OSTI · 3014231

Sequence-based generative AI design of versatile tryptophan synthases

Lambert, Théophile [California Institute of Technology (CalTech), Pasadena, CA (United States); Univ. Paris-Saclay, Orsay (France)] (ORCID:0009000584858729)·Tavakoli, Amin [California Institute of Technology (CalTech), Pasadena, CA (United States)]·Dharuman, Gautham [Argonne National Laboratory (ANL), Argonne, IL (United States)]·Yang, Jason [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000331841550)·Bhethanabotla, Vignesh [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000194016967)·Kaur, Sukhvinder [Elegen Corp, San Carlos, CA (United States)]·Hill, Matthew [Elegen Corp, San Carlos, CA (United States)]·Ramanathan, Arvind [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000216225488)·Anandkumar, Anima [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000269746797)·Arnold, Frances H. [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:000000024027364X)
View original source ↗Read source document ↗Export metadata ↓

Abstract

Enzymes are powerful and sustainable catalysts, but their widespread application is limited by the difficulty of identifying functional starting points for optimization, creating a major bottleneck in early- stage biocatalyst discovery. Designing libraries of such starting enzymes remains particularly challenging. Here, we use the GenSLM protein language model to generate novel β-subunit of tryptophan synthase (TrpB) enzymes that express in Escherichia coli and are both stable and catalytically active. Many generated TrpBs also display significant substrate promiscuity, outperforming their natural counterparts on non-native substrates. Some even surpass laboratory-evolved TrpBs. Comparison of the most-active and most-promiscuous generated TrpB to its closest natural homolog confirms that the enhanced versatility is absent from the natural enzyme, highlighting the creative potential of generative models. These results demonstrate that the generated TrpBs not only preserve natural structure and function but also acquire non-natural properties, establishing generative models as powerful tools for biocatalyst discovery and engineering.

Explore related subjects

biocatalysisenzymesmachine learning

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Lambert, Théophile [California Institute of Technology (CalTech), Pasadena, CA (United States); Univ. Paris-Saclay, Orsay (France)] (ORCID:0009000584858729), Tavakoli, Amin [California Institute of Technology (CalTech), Pasadena, CA (United States)], Dharuman, Gautham [Argonne National Laboratory (ANL), Argonne, IL (United States)], Yang, Jason [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000331841550), Bhethanabotla, Vignesh [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000194016967), Kaur, Sukhvinder [Elegen Corp, San Carlos, CA (United States)], Hill, Matthew [Elegen Corp, San Carlos, CA (United States)], Ramanathan, Arvind [Argonne National Laboratory (ANL), Argonne, IL (United States)] (ORCID:0000000216225488), Anandkumar, Anima [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:0000000269746797), Arnold, Frances H. [California Institute of Technology (CalTech), Pasadena, CA (United States)] (ORCID:000000024027364X). 2026-01-14. Sequence-based generative AI design of versatile tryptophan synthases. https://doi.org/10.1038/s41467-026-68384-6

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related reports

DOE OSTI2026

Photoenzymatic Csp 3 –Csp 3 bond formation via enzyme-templated radical–radical coupling

Cross-couplings are essential reactions in modern chemical synthesis, enabling the rapid construction of complex molecules from simple precursors. Transition metal catalysts are prized for these transformations because their reactivity and selectivity can be tuned via judicious selection of the metal and ligand. Although enzymes offer analogous opportunities for tuning via protein engineering, their application to cross-coupling remains limited, as nature relies on alternative paradigms for building molecular complexity. Here, we report the cross-coupling of alkyl halides and benzylic carboxylic acids using an engineered flavin-dependent lactate monooxygenase—a photoenzyme. The enzyme achieves this feat by exploiting the redox versatility of the flavin cofactor. Stoichiometric experiments, ultrafast spectroscopy, and computational studies support a mechanism in which photoexcited flavin quinone initiates the reaction via oxidative decarboxylation to generate a benzylic radical. The resulting flavin semiquinone can reduce the alkyl halide to form a second organic radical within the protein active site, which rapidly engages in C(sp 3 )–C(sp 3 ) bond formation. A variant was engineered to control the stereochemical outcome of this radical–radical coupling event, highlighting the ability of the protein to alter the energetic barrier for a mechanistic step that is traditionally understood to be near barrierless. This work demonstrates that the scope for nonnative reaction mechanisms in biocatalysis far exceeds previously established bounds and has potential to solve a variety of reactivity challenges in cross-coupling chemistry.

biocatalysis↗
DOE OSTI2025

A high throughput assay to detect enzymatic polyethylene oxidation

Biological plastics deconstruction and upcycling have emerged as sustainable alternatives to traditional recycling technologies for plastics waste. The discovery and engineering of efficient thermostable poly(ethylene terephthalate) (PET) hydrolases have made biological PET recycling possible at scale; however, enzymes for non-PET plastics, which account for approximately 70% of all plastics produced, remain largely undiscovered. To accelerate the discovery of such enzymes, we develop a high-throughput screen to detect initial polymer oxidation, specifically that of the C-H bond to an aldehyde. We test 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H), which reacts with generated aldehydes to form a fluorescent hydrazone on plasma oxidized low-density polyethylene (LDPE) films. Hydrazone generation correlated well with the area of aldehyde peaks as measured by Fourier Transform Infrared Spectroscopy (FTIR) (R2 = 0.92). Moreover, we demonstrate that the probe reliably identifies LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films (1.7 – 3.0 fold change relative to background), serving as an effective screen as demonstrated by receiver operating characteristic area under the curve of 0.95. Furthermore, this assay offers an LDPE oxidation screening platform that can be readily parallelized and automated for accelerated discovery of enzymes involved in polyolefin deconstruction.

biocatalysis↗
DOE OSTI2025

Copper-dependent halogenase catalyses unactivated C−H bond functionalization

Carbon–hydrogen (C–H) bonds are the foundation of essentially every organic molecule, making them an ideal place to do chemical synthesis. The key challenge is achieving selectivity for one particular C(sp 3 )−H bond. In recent years, metalloenzymes have been found to perform C(sp 3 )−H bond functionalization. Despite substantial progresses in the past two decades, enzymatic halogenation and pseudohalogenation of unactivated C(sp 3 )−H—providing a functional handle for further modification—have been achieved with only non-haem iron/α-ketoglutarate-dependent halogenases, and are therefore limited by the chemistry possible with these enzymes. Here, in this work, we report the discovery and characterization of a previously unknown halogenase ApnU, part of a protein family containing domain of unknown function 3328 (DUF3328). ApnU uses copper in its active site to catalyse iterative chlorinations on multiple unactivated C(sp 3 )−H bonds. By taking advantage of the softer copper centre, we demonstrate that ApnU can catalyse unprecedented enzymatic C(sp 3 )−H bond functionalization such as iodination and thiocyanation. Using biochemical characterization and proteomics analysis, we identified the functional oligomeric state of ApnU as a covalently linked homodimer, which contains three essential pairs—one interchain and two intrachain—of disulfide bonds. The metal-coordination active site in ApnU consists of binuclear type II copper centres, as revealed by electron paramagnetic resonance spectroscopy. This discovery expands the enzymatic capability of C(sp 3 )−H halogenases and provides a foundational understanding of this family of binuclear copper-dependent oxidative enzymes.

biocatalysis↗