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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗
DOE OSTI2025

Visolis Microbial Chemical Intermediate Library Screening (CRADA Final Report)

Visolis is developing a commercial process for the bioproduction of a chemical intermediate, towards the derivative production of a portfolio of bio-based chemicals with large application potential, from drop-in commodity chemicals, to innovative specialty chemical offerings, to materials for multiple end markets and industries, which will ultimately benefit consumers and the public. Visolis already has several variants of a microbe that produce the chemical intermediate at distinct levels (i.e. high, medium, and low). They are currently pursuing transcriptomics analysis for some of these strains, towards a better understanding of the biology behind what makes certain strains perform better than others. There exist genome-scale library approaches for the microbe that could be used to screen gene disruption, overexpression, or repression candidates for perturbations to the production of the chemical intermediate. These approaches often reveal opportunities for further production improvement that are not otherwise accessible using hypothesis-driven metabolic engineering approaches. However, Visolis, while it could generate or procure such genome-scale libraries, does not have the automated strain engineering workflows required to screen thousands of variants, obtaining a production phenotype and a genotype for each. The purpose of this collaboration is to use LBNL and SNL capabilities to enable Visolis to effectively screen thousands of genome-scale library strain variants for phenotype/genotype relationships that will complement Visolis’ transcriptomic investigations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Data for "Metabolic Engineering Strategies to Produce Medium-Chain Oleochemicals via Acyl-ACP:CoA Transacylase Activity"

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation – an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

Bioproducts↗
DOE OSTI2024

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, in this study, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2026

Demonstrating a butylamine-based deconstruction method for poplar biomass and conversion by diverse microbial strains

Low-boiling alkylamines such as butylamine offer promise as effective biomass pretreatment solvents that can be readily recovered and recycled; however, their capability to support microbial conversion of nutrients present in hydrolysates represents an important area for investigation. Here we employed butylamine to pretreat poplar biomass and characterize its effects on the release of fermentable sugars after solvent removal and enzymatic hydrolysis, as well as the biocompatibility of the produced hydrolysates with three organisms commonly used as bioconversion hosts. We observed that residual butylamine and the derivative butylacetamide were present in high enough concentrations to exert toxicity to strains of Aspergillus niger, Pseudomonas putida, and Rhodosporidium toruloides that produce malic acid, isoprenol and bisabolene, respectively. Removal of the toxic compounds by charcoal filtration and nutrient supplementation resulted in a hydrolysate containing >100 g L −1 of sugars that enabled strong growth, substrate consumption and bioproduct accumulation, outperforming defined cultivation media. This is the first demonstration of a butylamine-based deconstruction process for poplar biomass at a pilot-scale to achieve conversion of high sugar concentrations to valuable bioproducts with engineered microbes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Hybrid biological-chemical strategy for converting polyethylene into a recyclable plastic monomer using engineered Corynebacterium glutamicum

Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular weight necessitates harsh decomposition conditions, resulting in diverse decomposition outcomes that present significant challenges for downstream applications, especially for bioconversion. In this study, we demonstrate a hybrid biological-chemical conversion process for PE, converting its decomposition products, namely short-chain diacids, into a monomer, β-keto-δ-lactone (BKDL), for highly recyclable polydiketoenimine plastics using engineered Corynebacterium glutamicum. Since BKDL synthesis requires a substantial supply of malonyl-CoA, we employed an alternative biosynthesis pathway that leverages C. glutamicum's natural proficiency in amino acid production. We optimized this pathway in vivo by minimizing carbon loss to CO2 and byproducts, improving the transporter system, and maximizing co-factor regeneration. Furthermore, we co-optimized the PE deconstruction process to produce predominantly C4 to C6 diacids and integrated three catabolic pathways into the engineered strain to enhance diacid utilization, maximizing the carbon conversion from PE. Finally, an engineered polyketide synthase was introduced into C. glutamicum to enable BKDL synthesis. This work demonstrates the potential of a chemo-biological hybrid strategy for recycling plastic waste, highlighting its promise in addressing environmental challenges and promoting sustainable materials.

Zhan, Chunjun↗
DOE OSTI2024

ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction

Adenosine-5’-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics’ impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Degradable Biocomposite Thermoplastic Polyurethanes

In this project, the team developed tough and degradable biocomposite thermoplastic polyurethanes (TPUs) by incorporating bacterial spores into TPUs as a biofunctional living filler. The team screened various bacteria and selected the Bacillus subtilis ATCC 6633 strain as the final candidate, primarily due to its genomic availability, sporulation ability and TPU assimilation activity. The heat-shock tolerance of ATCC 6633 spores was further improved through evolutionary engineering via Adaptive Laboratory Evolution (ALE), demonstrating a 17.7-fold enhanced germination efficiency post heat-shock treatment compared to the wild-type strain (WT). The team fabricated biocomposite TPUs by incorporating lyophilized powder of heat-shock tolerized (HST) spores during the hot melt extrusion (HME) of TPU at 135 °C. The baseline TPU used in this project is a commercially available soft-grade TPU (BCF45) manufactured by BASF. Colony forming unit (CFU) assays quantified that WT and HST spores in the TPU matrix retained approximately 20% and 100% survivability, respectively, after HME. Tensile testing demonstrated that the spores behaved as a polymer-reinforcing filler, positively affecting the overall tensile properties of the biocomposite TPU. For example, biocomposite TPU with WT and HST spores (BC TPU WT and BC TPU HST , respectively) exhibited up to 25% and 37% improved toughness, respectively, compared to TPU without spores. BC TPU HST showed remarkably improved disintegration in autoclaved compost (92% mass loss in 5 months), which simulated a microbially poor environment for TPU degradation. When compared to TPU without spores (44% mass loss in 5 months) the acceleration of degradation is marked. Respirometry confirmed that 72% of BC TPU HST was biomineralized into CO2 within 6 months, indicating that spores in the biocomposite TPU were germinated by utilizing nutrients in the autoclaved compost, facilitating TPU degradation at the end of the material's life. The team demonstrated the scale-up of biocomposite TPU fabrication using continuous extrusion and injection molding techniques. Processing conditions optimized in a lab-scale microcompounder were successfully transferred to a continuous extruder with a 30-fold increased throughput. Biocomposite TPUs prepared using these industry-relevant processes showed comparable toughness improvements to samples prepared in the lab-scale extruder. Excitingly, following compounding in the pilot-extruder the composite material could be injection molded, while retaining high spore viability and similar toughness improvements. The team also found that spores in biocomposite TPU served as antioxidants, preventing toughness decay during the recycled extrusion of BC TPU HST . Long-term storage tests over one year showed that the addition of spores had no negative effect on the longevity of the TPU. Furthermore, the team demonstrated the fabrication of spore-bearing biocomposite polymers with other polyesters such as PBAT, PLA, and PCL. We obtained promising preliminary data that showed overall toughness improvements for all polymers with spore addition. Finally, life cycle assessment (LCA) and techno-economic analysis (TEA) were carried out, which indicated minimal additional cost of fabrication. Overall, a tough and degradable biocomposite thermoplastic was successfully developed through this project, with all tasks completed successfully, achieving >100% of the objectives.

36 MATERIALS SCIENCE↗
DOE OSTI2024

IMAGINE BioSecurity: Mesocosm-Based Methods to Evaluate Biocontainment Strategies and Impact of Industrial Microbes Upon Native Ecosystems

Project Goals: The Integrative Modeling and Genome-scale Engineering for Biosystems Security (IMAGINE BioSecurity) SFA project seeks to establish an understanding of the behavior of engineered microbes in controlled versus environmental conditions to predictively devise new strategies for responding to biological escape. To this end, the IMAGINE Team has established a plant-soil mesocosm platform to track and quantify the fate of industrial microbes in environmental systems and assess the efficacy of biocontainment constraints upon genetically engineered microbe escape frequency and the impact of industrial microbes upon native ecological microbiomes. Abstract Text: Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees, the effect of associated bio-products, and the impact on native ecologies. To this end, we have developed an approach that utilizes soil mesocosms and integrated systems analyses to evaluate the efficacy of novel biocontainment strategies and to assess the impact of production systems upon terrestrial microbiome dynamics. We demonstrate the utility of this approach by modeling a contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from both strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, and stains of Escherichia coli that are contained via genomic recoding. The resultant data demonstrate that this system has broad utility across diverse microbial chassis and biocontainment strategies, enables us to track the fate of our contaminating microbe with high sensitivity in the soil, as well as monitor broader impacts of the perturbation on the underlying soil system. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

BASIC BIOLOGICAL SCIENCES,INORGANIC, ORGANIC, PHYS↗
DOE OSTI2025

A Rhodopseudomonas strain with a substantially smaller genome retains the core metabolic versatility of its genus

ABSTRACT Rhodopseudomonas are a group of phototrophic microbes with a marked metabolic versatility and flexibility that underpins their potential use in the production of value-added products, bioremediation, and plant growth promotion. Members of this group have an average genome size of about 5.5 Mb, but two closely related strains have genome sizes of about 4.0 Mb. To identify the types of genes missing in a reduced genome strain, we compared strain DSM127 with other Rhodopseudomonas isolates at the genomic and phenotypic levels. We found that DSM127 can grow as well as other members of the Rhodopseudomonas genus and retains most of their metabolic versatility, but it has many fewer genes associated with high-affinity transport of nutrients, iron uptake, nitrogen metabolism, and biodegradation of aromatic compounds. This analysis indicates genes that can be deleted in genome reduction campaigns and suggests that DSM127 could be a favorable choice for biotechnology applications using Rhodopseudomonas or as a strain that can be engineered further to reside in a specialized natural environment. IMPORTANCE Rhodopseudomonas are a cohort of phototrophic bacteria with broad metabolic versatility. Members of this group are present in diverse soil and water environments, and some strains are found associated with plants and have plant growth-promoting activity. Motivated by the idea that it may be possible to design bacteria with reduced genomes that can survive well only in a specific environment or that may be more metabolically efficient, we compared Rhodopseudomonas strains with typical genome sizes of about 5.5 Mb to a strain with a reduced genome size of 4.0 Mb. From this, we concluded that metabolic versatility is part of the identity of the Rhodopseudomonas group, but high-affinity transport genes and genes of apparent redundant function can be dispensed with.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Genome-resolved insights into microbial diversity and elemental cycling in Winogradsky columns

We retained 18 MAGs with ≥50% completion and <10% contamination (i.e., at least medium quality). Of these, 10 had >90% completion and <5% contamination; however, only one (Paceibacteria Bin.003_MG) can be described as high-quality, as the others lacked a full suite of 5S, 16S, and 23S rRNA genes. To maximize the diversity of our recovered MAGs, we also retained one MAG (Chromatiaceae Bin.008_AM) with >40% (but less than 50%) completion and <5% contamination, as well as one (Rhodopseudomonas Bin.015_MK) with >90% completion and <20% (but>10%) contamination. Interestingly, significant chimerism was not detected in this MAG (40) , suggesting that the elevated contamination (20%) may instead reflect two closely related strains collapsing into a single bin. Consistent with this, contig coverage was bimodal, with roughly 17% of the assembly at ~115x and the remaining 83% at ~282x, while GC content remained uniform across both groups (~64%), arguing against contamination from a taxonomically distinct source.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Digital Droplet PCR and Mesocosm-Based Methods to Evaluate Biocontainment Strategies in a Native Soil Ecosystem

Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect the complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees within a complex soil microbiome and differentiation between closely related strains. To this end, we have developed an approach that utilizes soil mesocosms and integrated digital droplet PCR (ddPCR) system to evaluate the efficacy of novel biocontainment strategies. We demonstrate the utility of this approach by modeling contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, strains of Synechocystis sp. PCC 6803 contained via gene knockout or toxin anti-toxin system, and strains of Escherichia coli that are contained via genomic recoding. We also show that ddPCR can be used to detect gene copies from E. coli equal to those counted by traditional spot plating assays. The resultant data demonstrates that this system has broad utility across diverse microbial chassis and biocontainment strategies and enables researchers to track the fate of our contaminating microbe with high sensitivity in the soil. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Extraction of high-purity medium-chain-length polyhydroxyalkanoates via combined mechanical treatment and mild oxidation

Polyhydroxyalkanoates (PHAs) are a class of polyester polymers of microbial origin and considered biodegradable alternatives to conventional plastics. To make PHAs cost-competitive with synthetic plastics, their production cost, including extraction cost, should be significantly reduced. Herein, this study aimed to develop an effective extraction method based on combined mechanical disruption and chemical treatment for the recovery of high-purity medium-chain-length PHAs (mcl-PHAs) at a lower cost. mcl-PHAs-accumulating Pseudomonas strains were used for PHA extraction via high-pressure homogenization (HPH) coupled with surfactant treatment and mild alkaline hydrogen peroxide oxidation. The mechanical-chemical method was found to have strong synergy for PHA extraction while minimizing PHA depolymerization. The optimized condition resulted in nearly 90 % mcl-PHA recovery with high purity (>91 %). The extracted high-purity PHAs exhibited thermal and mechanical properties suitable for downstream applications entailing flexible and elastomeric materials.

42 ENGINEERING↗
DOE OSTI2025

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

ABSTRACT Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a Pseudomonas putida strain pre-tolerized (“PT”) background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, whereas a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled the development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. IMPORTANCE Acetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential to be used together with sugars, especially in microbes with versatile catabolism such as P. putida . However, the use of mixed carbon streams necessitates additional optimization. Furthermore, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study, we generate acetate-tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, although independent phenotypes, can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing, we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

de Siqueira, Guilherme M. V. (ORCID:00000002364563↗
DOE OSTI2024

Biosensors for the detection of chorismate and cis,cis -muconic acid in Corynebacterium glutamicum

Abstract Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. One-Sentence Summary High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

ABSTRACT Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here, we show that the MEP pathway is essential in Mm, while the MEV pathway is dispensable in culture, with the ΔMEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo acute infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2 , responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions. IMPORTANCE Organisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, maintaining membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways. Importantly, some bacteria encode both pathways, including M. marinum . We found that only one pathway is essential in M. marinum , while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

Qabar, Christine M. [Department of Plant and Micro↗
DOE OSTI2026

Cas3-Mediated Genome Reduction: Demonstration in Cupriavidus Necator H16 Improves Growth on Heterotrophic and Autotrophic Carbon Sources

Genome reduction is widely used to improve microbial bioprocessing hosts by reducing the burden of inessential physiology. Rationally identifying genomic regions that are dispensable or even detrimental to bioprocessing is challenged by our inability to map genome sequence to function across complex regulation and physiology. Thus, there is a need for tools that rapidly generate reduced genome strains with improved performance in process-relevant conditions. Here, we report a Cascade-Cas3-enabled method called TRIM3 that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library. Mutants with improved performance were isolated following growth-coupled selection and analyzed by long-read DNA sequencing to identify deletions in their genomes. We deploy this system iteratively in the industrial host Cupriavidus necator H16 on fructose and on formate. After two rounds of TRIM3, we isolate a strain containing a total reduction of 1.4 Mb (18.4% of the genome) that grows 25% faster in a bioreactor on fructose and a strain with a total reduction of 0.5 Mb (7.3% of the genome) that grows 14% faster on formate. This work demonstrates a method for random, iterative, growth-selectable genome reduction that represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.

09 BIOMASS FUELS↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗