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DOE OSTI2025

Integrated Biorefinery of Brewer’s Spent Grain for Second-Generation Ethanol, Mycoprotein, and Bioactive Vinasse Production

Brewer’s spent grain (BSG), the main lignocellulosic by-product of the beer industry, represents an abundant yet underutilized resource with high potential for valorization. This study presents an integrated biorefinery approach to convert BSG into second-generation (2G) ethanol, bioactive vinasse for plant growth promotion, and fungal biomass as a potential mycoprotein source. The biomass was first subjected to biological delignification using the white-rot fungus Ganoderma lucidum, after which two valorization routes were explored: (i) evaluation of the fungal biomass as a mycoprotein candidate and (ii) alcoholic fermentation for ethanol production. For the latter, three pretreatment strategies were assessed (diluted sulfuric acid and two deep eutectic solvents (DESs) based on choline chloride combined with either glycerol or lactic acid) followed by a one-pot enzymatic saccharification and fermentation using Kluyveromyces marxianus SLP1. The highest ethanol yield on substrate (YP/S) was achieved with [Ch]Cl:lactic acid pretreatment (0.46 g/g, 89.32% of theoretical). Vinasse, recovered after distillation, was characterized for organic acid content and tested on Solanum lycopersicum seed germination, showing promising biostimulant activity. Overall, this work highlights the potential of BSG as a sustainable feedstock within circular economy models, enabling the production of multiple bio-based products from a single residue.

Ganoderma lucidum↗

DOE OSTI2024

Cellulose Nanofibrils Dewatered with Poly(Lactic Acid) for Improved Bio-Polymer Nanocomposite Processing

Cellulose nanofibrils (CNFs) have theoretically ideal properties for bio-based composite applications; however, the incorporation of these materials into polymers is made challenging by the strong binding of water to CNFs and the irreversible agglomeration of CNFs during drying. Previous methods used “contact dewatering”, wherein the addition of wood flour (WF) to CNFs facilitated the mechanical removal of water from the system via cold pressing, which showed potential in producing dried CNF fibrils attached to wood fibers for biocomposite applications. In this work, the viability of contact dewatering with poly(lactic) acid (PLA) powder for PLA/CNF composites was evaluated. The energy efficiency of dewatering, preservation of nanoscale CNF morphology, and mechanical properties were examined by mixing wet CNFs with pulverized PLA at various loading levels, pressing water out of the system, and compression molding and shear mixing composites for testing. The most impressive results from this dewatering method were the preservation of micron-to-nanoscale fibers with high aspect ratios in PLA-CNF composites; increased strength and modulus of 1.7% and 4.2%, respectively, compared to neat PLA; equivalent or better properties than spray-dried nanocellulose at similar loading levels; and an 11-194x reduction in drying energy compared to spray-drying CNFs.

Chemistry↗

DOE OSTI2025

Plant Defense Proteins: Recent Discoveries and Applications

Proteins play pivotal roles in safeguarding plants against numerous biotic and abiotic stresses. Understanding their biological functions and mechanisms of action is essential for advancing plant biology, agriculture, and biotechnology. This review considers the diversity and potential applications of plant defense proteins including pathogenesis-related (PR) proteins, chitinases, glucanases, protease inhibitors, lectins, and antimicrobial peptides. Recent advances, such as the omics technologies, have enabled the discovery of new plant defense proteins and regulatory networks that govern plant defense responses and unveiled numerous roles of plant defense proteins in stress perception, signal transduction, and immune priming. The molecular affinities and enzymatic activities of plant defense proteins are essential for their defense functions. Applications of plant defense proteins span agriculture, biotechnology, and medicine, including the development of resistant crop varieties, bio-based products, biopharmaceuticals, and functional foods. Future research directions include elucidating the structural bases of defense protein functions, exploring protein interactions with ligands and other proteins, and engineering defense proteins for enhanced efficacy. Overall, this review illuminates the significance of plant defense proteins against biotic stresses in plant biology and biotechnology, emphasizing their potential for sustainable agriculture and environmental management.

G-proteins↗

DOE OSTI2025

Enabling Industrial Re-Use of Large-Format Additive Manufacturing Molding and Tooling

Large-format additive manufacturing (LFAM) is an enabling manufacturing technology capable of producing large parts with highly complex geometries for a wide variety of applications, including automotive, infrastructure/construction, and aerospace mold and tooling. In the past decade, the LFAM industry has seen widespread use of bio-based, glass, and/or carbon fiber reinforced thermoplastic composites which, when printed, serve as a lower-cost alternative to metallic parts. One of the highest-volume materials utilized by the industry is carbon fiber (CF)-filled polycarbonate (PC), which in out-of-autoclave applications can achieve comparable mechanical performance to metal at a significantly lower cost. Previous work has shown that if this material is recovered at various points throughout the manufacturing process for both the lab and pilot scale, it can be mechanically recycled with minimal impacts on the functional performance and printability of the material while significantly reducing the feedstock costs. End-of-life (EOL) CF-PC components were processed through industrial shredding, melt compounding, and LFAM equipment, followed by evaluation of the second-life material properties. Experimental assessments included quantitative analysis of fiber length attrition, polymer molecular weight degradation using gel permeation chromatography (GPC), density changes via pycnometry, thermal performance using dynamic mechanical analysis (DMA), and mechanical performance (tensile properties) in both the X- and Z-directions. Results demonstrated a 24.6% reduction in average fiber length compared to virgin prints, accompanied by a 21% decrease in X-direction tensile strength and a 39% reduction in tensile modulus. Despite these reductions, Z-direction tensile modulus improved by 4%, density increased by 6.8%, and heat deflection temperature (HDT) under high stress retained over 97% of its original value. These findings underscore the potential for integrating mechanically recycled CF-PC into industrial LFAM applications while highlighting the need for technological innovations to mitigate fiber degradation and enhance material performance for broader adoption. This critical step toward circular material practices in LFAM offers a pathway to reducing feedstock costs and environmental impact while maintaining functional performance in industrial applications.

additive manufacturing↗

DOE OSTI2024

Feasibility of Recovering and Recycling Polymer Composites from End-of-Life Marine Renewable Energy Structures: A Review

Over the last few decades, several marine renewable energy (MRE) technologies, such as wave energy converters (WECs) and current energy converters (CECs), have been developed. As opposed to traditional materials such as metal alloys, the structure of these technologies is made up of polymer and polymer composite materials. Most structures have been made using thermoset polymer composites; however, since thermoset polymer composites are not recyclable and lack sustainability, and with recent innovations in recyclable resins, bio-based resins, and the development of additive manufacturing technologies, thermoplastic polymers are increasingly being used. Nevertheless, the methodologies for identifying end-of-life options and recovering these polymer composites, as well as the recycling and reuse processes for MRE structures, are not well-studied. Specifically, since these MRE structures are subjected to salinity, moisture, varying temperature, biofouling, and corrosion effects depending on their usage, the recyclability after seawater aging and degradation needs to be explored. Hence, this review provides an in-depth review of polymer composites used in marine applications, the hygrothermal aging studies conducted so far to understand the degradation of these materials, and the reuse and recycling methodologies for end-of-life MRE structures, with a particular emphasis on sustainability.

16 TIDAL AND WAVE POWER↗

DOE OSTI2026

CAZyme domain architectures suggest fine-scale functional differentiation among anaerobic fungi and bacteria during lignocellulose conversion to volatile fatty acids

Anaerobic fermentation with microbial communities (microbiomes) is an emerging platform for conversion of lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), which can be achieved by arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow faeces, goat faeces, and anaerobic digester sludge, selecting for fungal or bacterial dominated communities that convert sorghum biomass into VFAs. Subsequently we reconstructed metabolic networks across these enrichments based on recovered bacterial metagenome-assembled genomes (MAGs) and fungal isolate genomes and profiled their metabolic activity using metatranscriptomics to identify potential functional niches. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix-dominated fungal enrichments converted lignocellulose to lactate, acetate and formate. Analysis of carbohydrate-active enzymes (CAZymes) revealed fine-scale differences between microbes that expressed unique multi-functional enzymes linking two or more CAZymes together with distinct carbohydrate binding motifs, implicating lignocellulose structure as a key driver of selection and niche differentiation. Most of these multi-functional enzymes localized complementary degradation functions together, likely conferring synergistic degradation effects within and between microbiome members. We anticipate that these findings will help inform efforts to develop synthetic microbiomes with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals.

Lawson, Christopher E [University of Toronto;]↗

DOE OSTI2026

Recycling of PET by Dissolution-Purification-Recovery (DPR) Process Using Bioderived Solvent

Polyethylene terephthalate (PET) waste continues to accumulate at staggering rates, with the majority still routed to landfills due to limitations in current recycling methods. While mechanical recycling is widely implemented, it struggles to effectively remove colorants, additives, and harmful contaminants - resulting in discolored, degraded materials with limited reuse potential. In this presentation, we demonstrate a scalable dissolution-purification-recovery (DPR) process designed to selectively reclaim high-purity PET from post-consumer bottle flake. The method utilizes a food grade bio-based solvent for targeted PET dissolution, followed by activated carbon treatment for impurity removal and antisolvent-induced precipitation for polymer recovery. The resulting recycled PET exhibits complete decolorization, significantly reduced metal contamination, and minimal loss in molecular weight. These performance metrics indicate strong suitability for remanufacturing into new bottles, offering a circular alternative to traditional mechanical recycling pathways.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

Application of Machine Learning to Improve Biobased Glucaric Acid Production: Cooperative Research and Development Final Report, CRADA Number CRD-20-17260

The Agile Biofoundry (ABF) is a multi-national lab consortium funded by the DOE Bioenergy Technologies Office that has developed a biofoundry that enables the rapid deployment of bioproducts into the market. Working with four ABF laboratory members (National Laboratory of the Rockies (NLR), Pacific Northwest National Laboratory (PNNL), Lawrence Berkeley National Laboratory (LBNL), and Argonne National Laboratory (ANL)), Kalion will use advanced high throughput techniques, advanced analytics, and machine learning to optimize the production environment for glucaric acid. Low-cost, high-purity glucaric acid enables products in a wide range of fields to improve the chemical properties of products and applications ranging from water treatment, polymers & textiles, coatings, detergents, and pharmaceuticals. With its broad range of applications, glucaric acid has the potential to demonstrate how such bio-based materials offer great benefit to the US economy.

09 BIOMASS FUELS↗

NASA NTRS2018

Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk

Dragline spider silk is among the strongest and toughest bio-based materials, capable of outperforming most synthetic polymers and even some metal alloys.1,2,3,4 These properties have gained spider silk a growing list of potential applications that, coupled with the impracticalities of spider farming, have driven a decades-long effort to produce recombinant spider silk proteins (spidroins) in engineered heterologous hosts.2 However, these efforts have so far been unable to yield synthetic silk fibers with mechanical properties equivalent to natural spider silk, largely due to an inability to stably produce highly repetitive, high molecular weight (MW) spidroins in heterologous hosts.1,5 Here we address these issues by combining synthetic biology techniques with split intein (SI)- mediated ligation for the bioproduction of spidroins with unprecedented MW (556 kDa), containing 192 repeat motifs of the Nephila clavipes MaSp1 dragline spidroin. Fibers spun from these synthetic spidroins display ultimate tensile strength (σ), modulus (E), extensibility (ε), and toughness (UT) of 1.03 +/- 0.11 GPa, 13.7 +/- 3.0 GPa, 18 +/- 6%, and 114 +/- 51 MJ/m3, respectively-equivalent to the performance of natural N. clavipes dragline silk.6 This work demonstrates for the first time that microbially produced synthetic silk fibers can match the performance of natural silk fibers by all common metrics (σ, E, ε, UT), providing a more dependable source of high-strength fibers to replace natural spider silks for mechanically demanding applications. Furthermore, our biosynthetic platform can be potentially expanded for the assembly and production of other protein-based materials with high MW and repetitive sequences that have so far been impossible to synthesize by genetic means alone.

spider silk↗

NASA NTRS2020

TPSAS-NF1676L-35856-DND

Southeast Virginia sits under one of the major flight corridors of the eastern U.S., and it’s not unusual to look up and see streaks of line-shaped clouds crisscrossing the sky. These aircraft-made condensation trails, or contrails for short, form when the hot water vapor in the jet engine exhaust cools and condenses onto soot and other atmospheric particles. What’s remarkable about these clouds is that they can have a significant impact on our regional and global climate. In fact, the climate effects of contrails are thought to be more important than the impact from all of the carbon dioxide emitted from aviation ever! Researchers at NASA’s Langley Research Center are exploring how new, bio-based jet fuels can reduce the amount of soot emitted from the aircraft engines and decrease the climate impact of contrail cirrus clouds. Join Dr. Rich Moore as he highlights the exciting results from recent formation flight experiments conducted by NASA and international collaborators.

Richard Moore↗

NASA NTRSDate not supplied

Evaluating Crystallinity in Thermoplastic composites

Polymer matrix composites (PMCs) offer many benefits for the aerospace industry due to their potential for weight reduction when compared to metal or ceramic based materials. Most PMCs currently in flight use thermoset matrices, however, thermoplastic resins are being explored as alternatives due to their ability to be remelted, which is of particular interest due to the potential for in-situ repair and manufacturing required in space. Most thermoplastic resins are semicrystalline polymers. The properties of semicrystalline thermoplastics are largely influenced by their crystallinity, which can vary due to many factors including thermal treatments, environmental conditions, and mechanical deformation. Monitoring the crystallinity of thermoplastic composites is key to ensuring these materials reliably meet the high demands required by space exploration. This talk discusses the use of multiple techniques such as Polarized Light Optical Microscopy and Fourier-Transform Infrared Spectroscopy to characterize the crystallinity in various thermoplastic composites, including carbon fiber reinforced PMCs and novel bio-based Martian and Lunar regolith composites designed for in-situ manufacturing. This work aims to provide the fundamental data necessary to understand the effects of crystallinity on thermoplastic PMCs, which is key to advancing their use in space applications.

Thermoplastics↗

NASA NTRS2026

Micromechanical Design of Carbon Nanotube Ribbon Reinforced Polymer Composite Materials

Lightweight materials are an important component of the design of aerospace structures. Carbon nanotube materials have been considered for this purpose due to the strength and stiffness of individual nanotubes, and the commercial availability of bulk formats such as fibers. These fibers can have a ribbon cross section which results in a different design space for their composites relative to traditional reinforcements which have a round cross section. This work applies brick-and-mortar micromechanical models and classical lamination theory with an inverse approach to investigate the design space of these composites. Using this approach, the influences of fiber geometry and axial and transverse mechanical properties are mapped. Finally, a sensitivity study is performed and the relative impacts of ±10% variations in the constituent material and geometric properties are ranked. Lamina axial moduli were found to range from a maximum of 3x to 1x minimum relative to a target quasi-isotropic laminate modulus depending on the anisotropy and shear modulus of the lamina. The fiber targets depended strongly on the fiber volume fraction in the lamina and the fiber axial modulus target was found to range from 4.8x to 3.2x the quasi-isotropic laminate target. The sensitivity analysis found that the largest driver of performance was the volume fraction, followed by the fiber axial modulus. While bio-based brick-and-mortar composites, such as nacre, can benefit from reinforcement aspect ratios above 10, for carbon nanotube ribbon (or carbon fiber)/polymer composites the sensitivity study indicated that the optimal cross-sectional aspect ratio was relatively smaller, potentially less than three.

Micromechanics↗

DOE OSTI2024

Use of a Lignin-Based Admixture for Tailoring the Rheological Properties of Mortars for 3D Printing: Preprint

Efforts toward decarbonizing construction materials and industrial processes related to cement and concrete can be aided via multifaceted approaches that target alternative admixtures as well as precision control of fabrication. Chemical admixtures for water reduction have played a crucial role in the development of advanced concrete mixtures. Newer biomass processing techniques developed for aviation fuel production from corn stover biomass produce a more reactive lignin byproduct that is suitable for chemical modifications to mimic the properties of polycarboxylate ether admixtures with a smaller carbon footprint. The present study examines the use of lignin-based water-reducing admixture in cement pastes and mortar mixtures for 3D printing. The experimental program explores the use of different dosages of lignin-based admixture to produce 3D-printed samples with appropriate extrudability and buildability. The rheological characterization was performed to determine the flow curve of various mixtures. Finally, the heat of hydration of cement pastes was monitored via isothermal calorimetry to assess the impact of lignin-based admixtures on the hydration process of cement. The results of this study indicate that the use of biomass by-products, such as lignin-based admixtures have great potential to effectively control the fresh-state properties of cement-based materials.

bio-based admixtures↗

DOE OSTI2025

Structure-performance relationships in lignin-based transesterification vitrimers: The role of lignin structural features

Lignin has been hailed as an ideal renewable alternative for petrochemical-based prepolymers in material synthesis for a sustainable and circular economy, due to its abundant aromatic network and high carbon content. However, the properties and performance of lignin-derived macromolecules are strongly influenced by the lignin itself. While numerous studies have explored the impact of lignin content on the thermomechanical performance of lignin-based vitrimers, literature on how the inherent structural features of lignin affect these properties is scanty. In this study, hardwood organosolv lignin was fractionated in ethyl acetate, ethanol, and acetone to obtain lignin fractions with varying structural characteristics. These fractions were then modified through carboxylation and crosslinked with epoxidized soybean oil (ESO) at a hydroxyl to epoxy group ratio of 1:1 to produce lignin-based transesterification vitrimers (LVs). The thermal properties (i.e. glass transition temperature and thermal stability), tensile strength, storage modulus, and stress relaxation behavior of the LVs were studied and carefully related to the structural features of lignin. The results revealed a positive relationship between strong hydroxyl content in modified lignin and the tensile strength (5.10–9.71 MPa), storage modulus (1099.4 – 1372.8 MPa), crosslinking density, and stress relaxation of the LVs. Additionally, both the storage modulus and tensile strength exhibited a positive relationship with the ratio of rigid linkages in modified lignin, while lignin molecular weight was found to significantly impact the thermal properties of LVs (i.e Tg and thermal stability). This study not only highlights the valorization of lignin in vitrimer synthesis but also provide insights for designing lignin-based materials with tailored properties for specific applications.

Bio-based polymer↗

DOE OSTI2026

Counterintuitive Compatibilization of Poly(-Valerolactone) and Poly(L-Lactic Acid) by Statistical Copolymers toward Compostable and Recyclable Packaging

Poly(δ-valerolactone) (PVL) and poly(l-lactic acid) (PLLA) are bioderivable, compostable, and chemically recyclable plastics with synergistic properties for addressing plastic waste accumulation in receiving environments. Though recyclable-by-design polymers often fall short of competing with incumbent materials, blending affords a means to leverage individual component strengths toward ideal tunable properties. Polymer blends are often immiscible, but a range of methodologies are available to promote mixing. Here, we report on the compatibilization of 9 immiscible PVL and PLLA blends with three different compatibilization agents: thermoplastic starch, synthesized PVL-co-PLLA statistical copolymers (SCPs), and synthesized PVL-b-PLLA block-type copolymers. Resulting degrees of compatibilization are observed through scanning electron microscopy, corroborated by thermal and mechanical analyses monitoring performance as a function of microdomain size. Small-angle and wide-angle X-ray scattering experiments are conducted to observe the influence of compatibilizers on individual crystalline phases to further elucidate material behavior. Molecular dynamics simulations provide key insights into the interfacial interactions between homopolymers and compatibilizers. Finally, a suite of end-of-life avenues is established by biodegradation in industrial composting conditions, chemical recycling by deconstruction to hydroxymethyl esters, and direct chemical depolymerization to lactone precursors in mixed feed. Overall, we highlight several promising blends and the counterintuitive SCP compatibilization phenomenon toward high-performance, sustainable materials.

Compatibilization, copolymer compatibilizer, bio-b↗

DOE OSTI2026

Simultaneous Overexpression of FERULOYL‐CoA 6′‐HYDROXYLASE 1 and COUMARIN SYNTHASE Leads to Coumarin‐Enriched Lignin and Improved Saccharification in Greenhouse‐ and Field‐Grown Poplar

ABSTRACT The urgent need for renewable resources has increased the interest in woody biomass to manufacture bio‐based products. However, lignin recalcitrance limits the enzymatic conversion of wood into fermentable sugars, posing a major challenge for biomass deconstruction. To address this problem, we aimed at incorporating the coumarin scopoletin into the lignin polymer of poplar ( Populus tremula × P . alba ) by expressing FERULOYL‐CoA 6′‐HYDROXYLASE 1 ( F6′H1 ) and COUMARIN SYNTHASE ( COSY ) in lignifying cells. Three constructs were evaluated: two bicistronic constructs, SCOP1 ( COSY followed by F6′H1 ) and SCOP2 ( F6′H1 followed by COSY ), and one monocistronic, SCOP3 (only F6′H1 ). SCOP1 poplars produced most free scopoletin without altering overall lignin, cellulose or hemicellulose content. SCOP2 poplars were overall less efficient in scopoletin production and most of these lines showed a severe biomass yield penalty, whereas SCOP3 caused plant lethality. NMR and metabolic analyses confirmed that scopoletin cross‐coupled with G and S monomers during lignification in SCOP1 lines. In addition to scopoletin, the detection of benzodioxane structures revealed the incorporation of dihydroxycoumarins. Overall coumarin incorporation in lignin amounted up to 2.3%. After alkaline pretreatment, wood from greenhouse‐grown SCOP1 poplars released up to 29% more glucose compared to the wild type upon limited saccharification. Field‐testing of three SCOP1 lines showed a 6 to 11% increase in saccharification efficiency, with the line containing the lowest scopoletin levels maintaining normal growth. These results demonstrate that engineering lignin composition in poplar can improve saccharification, and emphasize the importance of construct design, translational research and field validation.

alternative lignin monomers↗

DOE OSTI2025

In-Silico Design of Next Generation Cellulose-Derived Packaging Materials (CRADA Final Report)

Developing sustainable solutions for single-use packaging is an important objective to combat the environmental crisis of plastics pollution. Most embodiments of cellulose-based packaging materials, including CellophaneTM, are completely biodegradable in both terrestrial and marine environments. However, petroleum-derived alternatives offer some performance advantages for metrics such as moisture barriers and mechanical properties. This project leverages molecular dynamics simulation to investigate how molecular modifications to cellulose-based polymer assemblies impact their material properties. An important performance criterion for the modified materials was to retain biodegradability; thus, modifications by naturally occurring, biodegradable additives were the focus of this study. Specifically, we developed models with xylan and lignin of varying monomeric compositions into the cellulose matrix. The mechanical properties were investigated by performing stress-strain simulations, and the water barrier and hydrophobicity were investigated by simulating the water contact angle. Our findings indicate that the incorporation of xylan into the cellulose matrix tends to increase the mechanical properties with an optimal loading of ~27 wt%. We also predict that orienting the nanoscale directionality of the xylan chains such that they are perpendicular to the cellulose fibrils will dramatically increase mechanical strength. In contrast, the incorporation of lignin tends to weaken the composite at all loadings investigated. Simulations of water contact angle predicted that coating polymers on the surface of the cellulose assembly creates a more hydrophobic surface than incorporating them throughout the matrix. Of the coatings investigated, lignin resulted in the most hydrophobic surface, followed by pectin and keratin, which both imparted modest increases in hydrophobicity. Future experimental work done by Futamura will focus on designing material prototypes to capitalize on the predictions of performance enhancement obtained from molecular modeling. While substantial progress was made by the simulations performed in this project, there still exists a vast parameter space that we were unable to investigate, including branching, functional group decoration, and degree of polymerization of polymer additives. However, the methods developed in this initial investigation will facilitate more rapid evaluation of the impact of molecular characteristics on the performance of biopolymer composite materials and thereby accelerate future materials discovery efforts in this area.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Developing multi-gene CRISPRa/i programs to accelerate DBTL cycles in ABF hosts engineered for chemical production

This project developed and implemented a modular CRISPR activation and interference (CRISPRa/i) platform to accelerate strain optimization and pathway development for industrially relevant microbial hosts. By integrating multiplexed transcriptional perturbation tools with data-driven Design–Build–Test–Learn (DBTL) workflows, the team achieved reductions in cycle time and enhanced production of industrial aromatics, particularly 4-aminocinnamic acid (4-ACA), in Pseudomonas putida. Key accomplishments included: ● Development of a robust, tunable CRISPRa/i system in P. putida that enabled efficient multi-target gene regulation via guide RNA (gRNA) programs ● Completion of two full DBTL cycles, guided by machine learning (ML) models trained on transcriptomic and performance data, reducing engineering time by over 30% ● Optimization of multi-gene regulatory programs to balance expression of host and pathway modules, improve 4-ACA titers, and resolve metabolic bottlenecks ● Demonstration of system portability through a limited proof-of-concept extension in Acinetobacter baylyi, underscoring the generalizability of the approach ● Evaluation of strain performance on lignocellulosic biomass-derived substrates, demonstrating the feasibility of converting renewable carbon into aromatic building blocks These results illustrate the feasibility of applying ML-guided CRISPRa/i perturbation strategies to accelerate strain development in complex microbial systems. The resulting tools and datasets contribute to DOE objectives by improving platform predictability, reducing development costs, and enabling broader access to sustainable, economically viable bioproduction technologies.

09 BIOMASS FUELS↗