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DOE OSTI2021

Science–based maize stover removal can be sustainable

Maize (Zea mays L.) stover can be harvested for multiple uses or left in the field to sustain soil organic carbon (SOC), cycle essential plant nutrients, and protect soil health. This 13-yr field study quantified effects of no (0 Mg ha –1 yr –1 ), low (1.0–1.4 Mg ha –1 yr –1 ), moderate (3.5–4.0 Mg ha –1 yr –1 ), or high rates (4.7–5.4 Mg ha –1 yr –1 ) of stover harvest from either continuous maize or maize–soybean [Glycine max (L.) Merr.] rotation on grain yield, plant nutrient concentrations, and multiple soil properties at two sites in Iowa. Stover harvest increased plant macro- and micro-nutrient removal, but did not affect average grain yields of either crops. Soil inorganic carbon (IC), SOC, bulk density, pH, and cation exchange capacity (CEC) showed no significant differences due to stover harvest. Plant tissue and soil-test nutrient concentration effects were also minor and site-specific. Stover harvest significantly (p < .05) decreased exchangeable K and Ca concentrations by 8.3–23.8% and 0.3–22.5% but overall soil health indicator effects were minimal. Overall, based on crop yields, plant nutrient and soil-test concentrations, soil health indicators, and carbon sequestration estimates, maize stover harvest can be sustainable provided: (a) grain yields consistently exceed 11 Mg ha –1 , (b) stover removal does not exceed 40% of the aboveground biomass (i.e., 3.5–4.0 Mg ha –1 yr –1 ), and (c) plant nutrients (especially K) are closely monitored.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Effect of microstructure on fatigue crack propagation in additive manufactured nickel-based superalloy Haynes 282: an experiment and crystal plasticity study

Haynes 282 is a γ' precipitation-strengthened nickel-based superalloy known for its exceptional high-temperature creep resistance and excellent fabricability. Recent advancements in powder-bed fusion-based additive manufacturing (PBF-AM) have enabled the fabrication of Haynes 282 with accurate, site-specific control of the grain orientation and morphology at the microscale. This ability opens up new avenues for microstructure design, to improve the material’s fatigue crack resistance and service life. This paper investigates the fatigue crack growth behavior of hybrid microstructure Haynes 282 fabricated via PBF-AM. Previous experiments revealed a higher crack propagation rate in the coarse columnar-grained microstructural regions when compared against fine-grained areas. Here, a strain gradient crystal plasticity model was adapted to study the fracture-related mechanical fields at the crack tip in the two microstructures. The simulation results showed a consistent influence of grain structure and texture on crack propagation, as was seen in the experiment. The model analysis revealed higher crack propagation driving force along crack direction in the coarse-grained sharply textured microstructure and higher driving force for crack kinking in fine-grained more diffusely textured microstructure, which is ascribed to the combined effect of yield stress, hardening rate, texture and grain morphology. The presented modeling approach will facilitate the development of the AM-based accurate microstructure design by deepening the fundamental study in AM-specific microstructure-properties relations.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Efficient marine environmental characterisation to support monitoring of geological CO 2 storage

Carbon capture and storage is key for mitigating greenhouse gas emissions, and offshore geological formations provide vast CO 2 storage potential. Monitoring of sub-seabed CO 2 storage sites requires that anomalies signifying a loss of containment be detected, and if attributed to storage, quantified and their impact assessed. However, monitoring at or above the seabed is only useful if one can reliably differentiate abnormal signals from natural variability. Baseline acquisition is the default option for describing the natural state, however we argue that a comprehensive baseline assessment is likely expensive and time-bound, given the multi-decadal nature of CCS operations and the dynamic heterogeneity of the marine environment. We present an outline of the elements comprising an efficient marine environmental baseline to support offshore monitoring. We demonstrate that many of these elements can be derived from pre-existing and ongoing sources, not necessarily related to CCS project development. We argue that a sufficient baseline can be achieved by identifying key emergent properties of the system rather than assembling an extensive description of the physical, chemical and biological states. Further, that contemporary comparisons between impacted and non-impacted sites are likely to be as valuable as before and after comparisons. Furthermore, as these emergent properties may be nuanced between sites and seasons and comparative studies need to be validated by the careful choice of reference site, a site-specific understanding of the scales of heterogeneity will be an invaluable component of a baseline.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Elastic-wave sensitivity-guided adaptive seismic survey design for cost-effective monitoring of geological carbon storage

Effective seismic monitoring is essential for verifying CO₂ containment, detecting potential leakage, and optimizing operational decisions in geologic carbon storage. Here, this study presents a time-adaptive, elastic-wave sensitivity-guided framework for designing cost-effective seismic monitoring layouts for tracking CO₂ plume migration. The method is based on elastic-wave sensitivity analysis, which quantifies how variations in subsurface properties impact seismic wavefields. Two complementary design strategies are developed: one based on selecting a fixed number of seismic sources (Method A), and the other based on selecting source–receiver pairs contributing to a fixed fraction of cumulative elastic-wave sensitivity energy (Method B). The optimization workflow to identify source–receiver configurations with the highest detection potential is demonstrated using a hypothetical GCS scenario at the Kimberlina site in California using simulations of elastic-wave sensitivity data at multiple post-injection timesteps. Results show that both strategies adapt to evolving plume geometries and wavefield sensitivities, with Method B offering broader spatial coverage and Method A ensuring simpler deployment. This framework enables site-specific, cost-effective, and risk-informed seismic survey designs, enhancing the ability to monitor CO₂ migration over time in evolving geological environments

58 GEOSCIENCES↗

DOE OSTI2025

Photon counting with intensified charge coupled device (ICCD) – I. In-depth detector characterization

While the adsorption properties of transition metal catalysts have been widely studied, leading to the discovery of various scaling relations, descriptors of catalytic activity, and well-established computational models, a similar understanding of semiconductor catalysts has not yet been achieved. In this work, we present a high-throughput density functional theory investigation into the adsorption properties of 5 oxides of interest to the photocatalytic CO 2 reduction reaction: TiO 2 (rutile and anatase), SrTiO 3 , NaTaO 3 , and CeO 2 . Using a systematic approach, we exhaustively identify unique surfaces and construct adsorption structures to undergo geometry optimizations. We then perform a data-driven analysis, which reveals the presence of weak adsorption energy scaling relations, the propensity of adsorbates of interest to interact with oxygen surface sites, and the importance of slab deformation upon adsorption. Our findings are presented in the context of experimental observations and in comparison to previously studied classes of catalysts, such as pure metals and tellurium-containing semiconductors, and reinforce the need for a comprehensive approach to the study of site-specific surface phenomena on semiconductors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Systematic computational study of oxide adsorption properties for applications in photocatalytic CO 2 reduction

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Probing Interactions between Adsorbed Polymers and γ-Al 2 O 3 Using Solid-State NMR

The interactions between polymers and metal oxide surfaces affect the physical properties of polymer composites and the selectivity of catalysts used for plastic conversion. Gauging the strengths of these interactions and how they depend on adsorption conformations and polymer structure is nevertheless quite challenging. Herein, we evaluate whether a recently introduced dynamic nuclear polarization-based surface-to-atom distance measurement method can be used to gauge the specific interactions between five vinyl polymers and an oxide support (alumina). The adsorption conformations of polymer monolayers were studied using site-specific 13 C{ 27 Al} rotational-echo saturation-pulse double-resonance (RESPDOR) experiments. The method is effective in revealing the average distance between the adsorbed polymer and the surface, as well as the fraction of the monomers that are adsorbed. Polymers that possess larger side chains are more weakly adsorbed, as evidenced by their formation of more numerous loop and tail structures. In conclusion, the measured RESPDOR dephasing data further reveals that polymer adsorption conformations are largely disordered and involve few hydrogen bonding interactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Recent Developments in Revealing the Impact of Complex Metal Oxide Reconstruction on Catalysis

Surface reconstruction of complex metal oxides refers to the alterations of the topmost catalyst surface, relative to the bulk structure, following exposure to pretreatment or reaction conditions during heterogeneous catalysis. (1) This structural and electronic transformation may involve (1) changes of the exposed crystallographic facets, (2) changes in chemical composition, such as the enrichment of specific elements, and, (3) morphological variations, including surface roughness and defects (Scheme 1). Beyond relaxation, surface reconstruction here also encompasses disruptions to the periodicity of the sublayers and changes to its stoichiometry. This change significantly influences the chemical properties of the catalyst surface, such as redox sites, (2) acid/base pairs, hydroxyl groups, (3) and surface defects. Surface reconstruction occurs due to the thermodynamic drive to lower the surface energy of the catalyst under the surrounding environment (e.g., temperature, chemical potential of species present). In addition to the treatment or reaction conditions, the extent of reconstruction also depends on the elements in the metal oxide structure. For instance, annealing ABO 3 perovskites (SrTiO 3 , BaTiO 3 , and BaZrO 3 ) at high temperatures (500 °C) in O 2 has a very different impact on the A/B ratio at the surface (ranging from 0.9 to 2.5), depending on the identity of A and B. (4) Metal oxide surface reconstruction under an electric field and exposure to acidic and alkaline media has recently gained increased attention in electrocatalysis, such as in the oxygen evolution reaction (OER); (5,6) however, in this Viewpoint we focus on the impact of surface reconstruction on thermal catalysis, a critical enduring research theme with ongoing challenges. Here, we start showcasing approaches to characterize surface reconstruction at the topmost surface layer, such as low-energy ion scattering (LEIS), discussing advantages and limitations. Next, we connect characterization of surface reconstruction to site-specific kinetic analysis, enabled via steady-state isotopic transient kinetic analysis (SSITKA), and we express our viewpoint on building structure–performance relationships by deeply understanding reconstructed surfaces. Later, we comment on the potential of tuning surface reconstruction to enhance catalytic performance. Finally, we explore the interplay between surface reconstruction and the “intelligent behavior”.

alcohol conversion↗

DOE OSTI2024

Dynamic control and quantification of active sites on ceria for CO activation and hydrogenation

Ceria (CeO 2 ) is a widely used oxide catalyst, yet the nature of its active sites remains elusive. This study combines model and powder catalyst studies to elucidate the structure-activity relationships in ceria-catalyzed CO activation and hydrogenation. Well-defined ceria clusters are synthesized on planar CeO 2 (111) and exhibit dynamic and tunable ranges of Ce coordination numbers, which enhance their interaction with CO. Reduced ceria clusters (e.g., Ce 3 O 3 ) bind CO strongly and facilitate its dissociation, while near-stoichiometric clusters (e.g., Ce 3 O 7 ) adsorb CO weakly and promote oxidation via carbonate formation. Unlike planar ceria surfaces, supported ceria clusters exhibit dynamic properties and enhanced catalytic activity, that mimic those of powder ceria catalysts. Insight from model studies provide a method to quantify active sites on powder ceria and guide further optimization of ceria catalysts for syngas conversion. This work marks a leap toward model-guided catalyst design and highlights the importance of site-specific catalysis.

03 NATURAL GAS↗

DOE OSTI2025

Site-specific photo-crosslinking in a double crossover DNA tile facilitated by squaraine dye aggregates: advancing thermally stable and uniform DNA nanostructures

We investigated the role of dichloro-squaraine (SQ) dye aggregates in facilitating thymine–thymine interstrand photo-crosslinking within double crossover (DX) tiles, to develop thermally stable and structurally uniform two-dimensional (2D) DNA-based nanostructures. By strategically incorporating SQ modified thymine pairs, we enabled site-selective [2 + 2] photocycloaddition under 310 nm UV light. Strong dye–dye interactions, particularly through the formation of aggregates, facilitated covalent bond formation between proximal thymines. To evaluate the impact of dye aggregation on crosslinking efficiency, ten DX tile variants with varying SQ-modified thymine positions were tested. Our results demonstrated that SQ dye aggregates significantly enhanced crosslinking, driven by precise SQ-modified thymine dimer placement within the DNA tiles. Analytical techniques, including denaturing PAGE and UV-visible spectroscopy, validated successful crosslinking in DNA tiles with multiple SQ-modified thymine pairs. This non-phototoxic method offers a potential route for creating thermally stable, homogeneous higher-order DNA–dye assemblies with potential applications in photoactive and exciton-based fields such as optoelectronics, nanoscale computing, and quantum computing. Furthermore, the insights from this study establish a foundation for further exploration of advanced DNA–dye systems, enabling the design of next-generation DNA nanostructures with enhanced functional properties.

2D DNA template↗

DOE OSTI2024

Engineered mRNA–ribosome fusions for facile biosynthesis of selenoproteins

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA–ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins’ active sites. Overall, our work demonstrates the feasibility of creating functional mRNA–rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Artificial Replication of Field Soiling Losses on PV Modules

In this paper, we experimentally demonstrate an improved replication of field soiling losses using an indoor artificial soiling chamber and tests on anti-soiling coated PV modules and coupons. The primary focus is to use site-specific soil collected from module surface and replicate the natural soiling processes including dust concentration in the air, slow and gradual dust accumulation and sedimentation on the module surface during the dominant soiling season of the site of interest. The experiments were conducted on two sample sets having different anti-soiling properties. The first set contains commercial modules with two different surface properties retrieved after three years of exposure from a single PV plant in a mid-Atlantic location; the other set contain glass coupons with three different coating materials that were installed and exposed over 4 months, at Lemoore, California. Major field-representative factors considered here for the close replication in the chamber include: the use of dust collected from modules surfaces at the outdoor sites to give the same dust chemistry; dust particle size distribution and concentration; the charge size of dust (< 0.15 g per injection); field humidity, and module temperature. The effectiveness of antisoiling coatings (or surface properties) for both sample sets were ranked in the artificial testing and were found to be closely matching with the field rank orders of the respective sites and sample sets. This paper provides the rank ordering results to objectively demonstrate the replication of field soiling losses in the artificial soiling chamber.

artificial soiling↗

DOE OSTI2024

Ground-motions site and event specificity: Insights from assessing a suite of simulated ground motions in the San Francisco Bay Area

This article presents the results of a research that is part of a larger collaborative effort between the Lawrence Berkeley National Laboratory and the Pacific Earthquake Engineering Research Center, funded by the US Department of Energy Office of Cybersecurity, Energy Security and Emergency Response. The main objective of this study is to assess a suite of near and far-field simulated ground motions obtained from 20 realizations of an M7 Hayward Fault earthquake in the San Francisco Bay Area, California USA, and inform the selection of rupture simulation parameters leading to strong motions. To this aim, comparisons are conducted with NGA-W2 and directivity ground-motion models and a selected population of records. An archetypal steel moment-resisting frame is utilized to assess infrastructure response distributions. The analyses carried out for each simulated event and subdomain with consistent properties in terms of shallow shear-wave velocity proved to be instrumental for better interpreting the differences between simulated motions and empirical models. The main reasons identified for variances between simulations and empirical relationships included (1) directivity effects fully captured by the simulations across the full breadth of rupture models; (2) site vicinity to ruptures that incorporate large-slip patches, particularly if these are in the forward-directivity direction; and (3) presence of geologic structures that can “trap” seismic waves and produce ground motions with large amplitude and long signal duration. The analyses carried out in this work provide a path for interpreting ground-motion site and event specificity obtained from a suite of physics-based simulations, differing only in the rupture model characterization, to inform the selection of simulation scenarios for site-specific engineering analyses under strong excitations. Evidence from this work points to the possibility that current hazard models may underestimate ground-motion intensities in areas where the combined effect of directivity and site conditions results in large ground-motion amplitudes.

58 GEOSCIENCES↗

DOE OSTI2023

Utah FORGE: Optimization of a Plug-and-Perf Stimulation (Fervo Energy)

Information around the plug-and-perf treatment design at Utah FORGE by Fervo Energy. Objective and Purpose: - Develop a multistage hydraulic stimulation approach designed specifically to target the top three factors that control the technical and commercial viability of an EGS system: i) Achieving sufficient injectivity to support high cross-well flow rates ii) Distributing flow evenly across the wellbore and reservoir to maximize heat mining efficiency, ensure sustained heat transfer, and mitigate thermal breakthrough iii) Overcoming the effects of stress heterogeneity, stress shadowing, and variations in natural fracture properties during the stimulation treatment, leading to a more predictable stimulated reservoir volume and offset well placement - The following activities will be performed: i) Design, plan, and execute a multistage plug-and-perf stimulation treatment at a Fervo site with data acquisition and well testing activities aimed at addressing key technical aspects of the issues above ii) Perform data processing and interpretation of field results to translate the results form the Fervo site to a site-specific design at the Utah FORGE site iii) Design, plan, and execute a multistage plug-and-perf stimulation treatment design at the Utah FORGE site Methods and Approach: - Design a detailed data acquisition plan to maximize learning around: i) DFIT testing ii) Petrophysical logging, image logging iii) Permanent DAS/DTS fiber optic monitoring iv) Deep borehole microseismic monitoring v) Shallow borehole induced seismicity monitoring vi) Injection/production testing (RTA analysis, tracer testing) vii) Integrated numerical modeling and production forecasting

15 GEOTHERMAL ENERGY↗

DOE OSTI2021

Storm Water Pollution Prevention Plan (SWPPP) for the Limited Area, Multi-Purpose (LAMP) High Bay Laboratory

Kier + Wright, as Qualified SWPPP Developer (QSD), puts forth this Storm Water Pollution Prevention Plan (SWPPP) for the Limited Area, Multi-Purpose (LAMP) High Bay Laboratory facility (Project) located at Sandia National Laboratories, 7011 East Avenue, CA. The property is owned by the U.S. Department of Energy, and managed and operated by National Technology & Engineering Solutions of Sandia, LLC. The project proposes converting an asphalt parking lot into a new high bay machine shop building and a low bay office building. Per the California State Water Resources Control Board’s (California State Water Board) Construction General Permit (CGP), a SWPPP is required when 1 acre or more of land is disturbed. The project site area of 1.6 acres exceeds the minimum acreage threshold of 1 acre and therefore requires SWPPP implementation. QSD has determined the sediment risk for this project, based on soil type at the site and starting and ending dates of construction, to be low (Section 3.4.1 and Appendix B). Receiving water for this project is the Arroyo Seco. QSD has determined the Arroyo Seco to be a high-risk receiving water because it has the three beneficial uses of “spawn”, “cold”, and “migratory” (Sections 3.3 and 3.4.2 and Appendix B). QSD has determined the overall risk level for the site to be Risk Level 2, based on a combination of low sediment risk and high receiving water risk (Appendix B). As such, QSD has delineated a variety of Best Management Practices (BMPs) to be employed during project construction to reduce or eliminate pollutants in stormwater runoff or any other discharges from the Project site. In addition to site-specific BMPs, this SWPPP report provides instruction for on site monitoring. Electronic copies of required documentation such as inspection reports, REAPs, annual report documentation, etc. shall be submitted to NTESS Sandia Delegated Representative via Newforma.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

IEA Wind TCP Task 49: Reference Site Conditions for Floating Wind Arrays

This report, prepared within Work package 1 of IEA Wind Task 49, presents reference site conditions for floating wind arrays to serve as a design basis for the techno-economic design of reference floating wind arrays. Data of the reference sites presented here are publicly available in an open database and thus support fast development and comparable design of floating wind arrays for various relevant conditions. The development of these reference sites drew on existing open access datasets and ongoing research projects of task participants. Six classes were identified that describe relevant key conditions for the design and development of floating wind arrays: met-ocean conditions, seabed conditions, coastal infrastructure, environmental impact, socio-economic impact, as well as regulations and permissions. The reference sites for the techno-economic design of floating wind arrays are based on a concept with building blocks to synthesize purpose-built site representations. In each of the identified classes with influencing design factors, building blocks are used to describe the characteristic properties and their spread. However, the latter three classes (i.e., environmental impact, socio-economic impact, regulations and permissions) are not included in the reference site conditions due to limited knowledge and lack of reliable criteria to quantify their impact on the techno-economic design in numeric parameters. Building blocks with key parameters for the techno-economic design of floating wind arrays are provided for met-ocean conditions, seabed conditions, and coastal infrastructure. For met-ocean conditions, multiple sites were selected for detailed analysis that represent a range of conditions across the pipeline of floating wind projects. Wind conditions and sea states are separated, and each location considers both the severity of wind and waves e.g. one site may have a moderate wave condition but severe wind condition. From this pipeline, eleven representative sites were selected where both site-specific analysis was available within the consortium, and where they represent different parts of the global pipeline. The eleven sites are: Hannibal (Italy), Humboldt (US), Ulsan (South Korea), MoneyPoint One (Ireland), Havbredey (UK), Fukushima (Japan), Utsira Nord (Norway), Gulf of Maine (US), Sud de la Bretagne II (France), Sorlige Nordsjo II (Norway). Each of these sites is summarized in the main report while more details about the studies and analyses behind the datasets are provided in the appendix. For seabed conditions, general information about the geotechnical parameters is provided and a baseline is established for the geotechnical parameters and stratigraphy that may be encountered on the sites. A set of six 'synthetic cases' is defined as building blocks providing the different parameters required for design under each case/soil condition. For the coastal infrastructure, general information about the main requirements is provided that a port should comply with to provide a satisfactory service during the construction of floating offshore wind arrays. Minimum port infrastructural requirements are provided for three types of ports.

17 WIND ENERGY↗

DOE OSTI2023

Stormwater Pollution Prevention Plan (SWPPP) for Site Wide Landscaping Project

Kier + Wright, as Qualified SWPPP Developer (QSD), puts forth this Storm Water Pollution Prevention Plan (SWPPP) for the SNL/CA Site Landscaping Project at Sandia National Laboratories/California (SNL/CA), 7011 East Avenue, Livermore, California (SNL/CA). The property is owned by the U.S. Department of Energy, and managed and operated by National Technology & Engineering Solutions of Sandia (NTESS), LLC. The project proposes landscape improvements throughout SNL/CA. Per the California State Water Resources Control Board’s (California State Water Board) Construction General Permit (CGP), a SWPPP is required when 1 acre or more of land is disturbed. The project site area exceeds the minimum acreage threshold of 1 acre and therefore requires SWPPP implementation. QSD has determined the sediment risk for this project, based on soil type at the site and starting and ending dates of construction, to be low (Section 3.4.1 and Appendix B). Receiving water for this project is the Arroyo Seco. QSD has determined the Arroyo Seco to be a high-risk receiving water because it has the three beneficial uses of “spawn”, “cold”, and “migratory” (Sections 3.3 and 3.4.2 and Appendix B). QSD has determined the overall risk level for the site to be Risk Level 2, based on a combination of low sediment risk and high receiving water risk (Appendix B). As such, QSD has delineated a variety of Best Management Practices (BMPs) to be employed during project construction to reduce or eliminate pollutants in stormwater runoff or any other discharges from the Project site. In addition to site-specific BMPs, this SWPPP report provides instruction for on site monitoring. Electronic copies of required documentation such as inspection reports, REAPs, annual report documentation, etc. shall be submitted to NTESS Sandia Delegated Representative via Newforma.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2022

Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant

Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS ( Ma PylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of Ma PylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve Ma PylRS to efficiently incorporate N ε -Boc- l -lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRS opt ) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRS opt can be used to enhance previously engineered PylRS constructs such as Ma PylRS N166S , and PylRS opt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.

59 BASIC BIOLOGICAL SCIENCES↗