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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida

Abstract With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C 5 ) and epi-isozizaene (C 15 ) for the first time in P. putida . We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing l -glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p -coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass. Graphical Abstract

09 BIOMASS FUELS↗
DOE OSTI2022

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13 C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum . 13 C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13 C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

13C-fingerprinting↗
DOE OSTI2021

Carbonate-hosted microbial communities are prolific and pervasive methane oxidizers at geologically diverse marine methane seep sites

At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated V max , and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample’s methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption.

58 GEOSCIENCES↗
DOE OSTI2022

Quantification of Metabolic Products from Microbial Hosts in Complex Media Using Optically Diffracting Hydrogels

In this work, we herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized N-isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log P) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of Zymomonas mobilis and Actinobacillus succinogenes, respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ~3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.

09 BIOMASS FUELS↗
DOE OSTI2025

Prenol production in a microbial host via the “Repass” Pathways

Prenol and isoprenol are promising advanced biofuels and serve as biosynthetic precursors for pharmaceuticals, fragrances, and other industrially relevant compounds. Despite engineering improvements that circumvent intermediate cytotoxicity and lower energy barriers, achieving high titer ‘mevalonate (MVA)-derived’ prenol has remained elusive. Difficulty in selective prenol production stems from the necessary isomerization of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) as well as the intrinsic toxicity of these diphosphate precursors. Here, the expression of specific isopentenyl monophosphate kinases with model-guided enzyme substitution of diphosphate isomerases and phosphatases enabled selective cycling of monophosphates and diphosphates, dramatically improving prenol titers and selectivity in Escherichia coli. Pairing this approach with the canonical MVA pathway resulted in 300 mg/L prenol at a 30:1 ratio with isoprenol. Further pairing with the “IPP-Bypass” pathway resulted in 526 mg/L prenol at a 72:1 ratio with isoprenol, the highest and purest MVA-derived prenol titer to date. Additionally, modifying this “IPP-Repass” for DMAPP production and coexpressing the prenyltransferase acPT1 yielded 48.3 mg/L of the potential therapeutic precursor drupanin from p-coumarate. These novel repass pathways establish a unique strategy for tuning diphosphate precursors to drive isoprenoid biosynthesis and prenylation reactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Prospective Study Reveals Host Microbial Determinants of Clinical Response to Fecal Microbiota Transplant Therapy in Type 2 Diabetes Patients

Increasing evidence shows that alterations in gut microbiome (GM) contribute to the development of type 2 diabetes mellitus (T2DM), and fecal microbiota transplantation (FMT) successfully treats various human diseases. However, the benefits of FMT therapy to T2DM patients remain unknown. We enrolled 17 patients with T2DM for nonblinded, one-armed intervention trial of FMT. A total of 20 healthy individuals were recruited as the baseline control. HbA1c% and metabolic parameter change were evaluated in 17 T2DM patients 12 weeks after they received FMT from healthy donors. The GM composition was characterized by 16S rRNA gene amplicon sequencing from fecal samples prior to and 12 weeks after FMT treatment. We found that the GM of T2DM patients was reconstituted by FMT. We observed a statistically significant decrease in HbA1c% (from 7.565 ± 0.148 to 7.190 ± 0.210, p<0.01), blood glucose (from 8.483 ± 0.497 to 7.286 ± 0.454 mmol/L, p<0.01), and uric acid (from 309.4 ± 21.5 to 259.1 ± 15.8 µmol/L, p<0.01) while a significant increase in postprandial C-peptide (from 4.503 ± 0.600 to 5.471 ± 0.728 ng/ml, p<0.01) at 12 weeks after FMT. Closely evaluating the changes in these assays, we found individual variability in response to FMT treatment. Out of 17 T2DM patients, 11 were found to significantly improve T2DM symptoms. The FMT responders have significantly higher levels of the family Rikenellaceae and the genus Anaerotruncus (family Ruminococcaceae) in their pretreated fecal in comparison to nonresponders, which could predict the clinical response with an area under the curve of 0.83. Our findings suggest that certain T2DM patients can potentially benefit from FMT, and the pretreated abundance of Rikenellaceae and Anaerotruncus in the fecal of patients may serve as potential biomarkers for selecting T2DM patients to receive FMT.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Host Onboarding Tool (HObT) v1.0.0

The Host OnBoarding Tool (Hobt) is a publicly accessible, web-based software designed to organize and share information about microbial hosts under development at the Agile BioFoundry (ABF). It streamlines the assessment, tracking, and sharing of information related to microbial host development and provides a centralized platform where users can rapidly evaluate hosts' readiness for various bio processes. HObT leverages the Tier System, a standardized host development framework that organizes and assesses microbial hosts based on their readiness for biomanufacturing. Each tier outlines key targets—including genetic tools, growth conditions, omics data, and predictive models—needed to transform new or emerging microbes into established production platforms. By applying clear criteria for advancement, the Tier System helps users quickly evaluate each organism's current development status, identify gaps in available knowledge or tools, and prioritize future strain improvement efforts. Through its user-friendly interface, HObT encourages contributions of new data and insights from researchers, fostering collaboration and accelerating host development. By providing structured guidance for microbial strain advancement, HObT and the Tier System support more systematic, rapid, and cost-effective development of non-traditional microbial hosts, ultimately enhancing the efficiency and impact of biomanufacturing research and applications.

Plahar, Hector [Lawrence Berkeley National Laborat↗
DOE OSTI2020

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides

Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH-specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next-generation host for the production of fatty acid-derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3-fold at bench scale to 352.6 mg/L. With further process optimization in a 2-L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The tier system: a host development framework for bioengineering

Development of microorganisms into mature bioproduction host strains has typically been a slow and circuitous process, wherein multiple groups apply disparate approaches with minimal coordination over decades. To help organize and streamline host development efforts, we introduce the Tier System for Host Development, a conceptual model and guide for developing microbial hosts that can ultimately lead to a systematic, standardized, less expensive, and more rapid workflow. The Tier System is made up of three Tiers, each consisting of a unique set of strain development Targets, including experimental tools, strain properties, experimental information, and process models. By introducing the Tier System, we hope to improve host development activities through standardization and systematization pertaining to nontraditional chassis organisms.

09 BIOMASS FUELS↗
DOE OSTI2022

Engineered hosts with exogenous ligninase and uses thereof

The present invention relates to methods and engineered microbial hosts useful for treating lignin or a derivative thereof. In some embodiments, the host has one or more exogenous nucleic acid sequences that encode a ligninase (e.g., a laccase and/or a peroxidase).

Singh, Seema↗
DOE OSTI2020

Ecological Assembly Processes Are Coordinated between Bacterial and Viral Communities in Fractured Shale Ecosystems

The ecological drivers that concurrently act upon both a virus and its host and that drive community assembly are poorly understood despite known interactions between viral populations and their microbial hosts. Hydraulically fractured shale environments provide access to a closed ecosystem in the deep subsurface where constrained microbial and viral community assembly processes can be examined. Here, we used metagenomic analyses of time-resolved-produced fluid samples from two wells in the Appalachian Basin to track viral and host dynamics and to investigate community assembly processes. Hypersaline conditions within these ecosystems should drive microbial community structure to a similar configuration through time in response to common osmotic stress. However, viral predation appears to counterbalance this potentially strong homogeneous selection and pushes the microbial community toward undominated assembly. In comparison, while the viral community was also influenced by substantial undominated processes, it assembled, in part, due to homogeneous selection. When the overall assembly processes acting upon both these communities were directly compared with each other, a significant relationship was revealed, suggesting an association between microbial and viral community development despite differing selective pressures. These results reveal a potentially important balance of ecological dynamics that must be in maintained within this deep subsurface ecosystem in order for the microbial community to persist over extended time periods. More broadly, this relationship begins to provide knowledge underlying metacommunity development across trophic levels.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

A Microbe Associated with Sleep Revealed by a Novel Systems Genetic Analysis of the Microbiome in Collaborative Cross Mice

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host–microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7 m +/+ Lepr db /J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Development of Enzyme Production Host (CRADA Final Report)

This CRADA aims to assess the effectiveness of various enzyme production hosts in efficiently expressing and producing proprietary enzymes for plastic recycling applications. The research includes screening different microbial hosts—notably Bacillus subtilis, Pichia pastoris, and E. coli—to determine their suitability for high-yield enzyme production. Additionally, the project focuses on optimizing scalable fermentation processes tailored to these hosts, with the goal of enabling efficient enzyme production at commercially viable scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Development of Enzyme Production Host (CRADA Final Report)

This CRADA aims to assess the effectiveness of various enzyme production hosts in efficiently expressing and producing proprietary enzymes for plastic recycling applications. The research includes screening different microbial hosts—notably Bacillus subtilis, Pichia pastoris, and E. coli—to determine their suitability for high-yield enzyme production. Additionally, the project focuses on optimizing scalable fermentation processes tailored to these hosts, with the goal of enabling efficient enzyme production at commercially viable scales.

60 APPLIED LIFE SCIENCES↗
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↗
DOE OSTI2023

Viral but not bacterial community successional patterns reflect extreme turnover shortly after rewetting dry soils

As central members of soil trophic networks, viruses have the potential to drive substantial microbial mortality and nutrient turnover. Pinpointing viral contributions to terrestrial ecosystem processes remains a challenge, as temporal dynamics are difficult to unravel in the spatially and physicochemically heterogeneous soil environment. In Mediterranean grasslands, the first rainfall after seasonal drought provides an ecosystem reset, triggering microbial activity during a tractable window for capturing short-term dynamics. In this report we simulated precipitation in microcosms from four distinct dry grassland soils and generated 144 viromes, 84 metagenomes and 84 16S ribosomal RNA gene amplicon datasets to characterize viral, prokaryotic and relic DNA dynamics over 10 days. Vastly different viral communities in each soil followed remarkably similar successional trajectories. Wet-up triggered a significant increase in viral richness, followed by extensive compositional turnover. Temporal succession in prokaryotic communities was much less pronounced, perhaps suggesting differences in the scales of activity captured by viromes (representing recently produced, ephemeral viral particles) and total DNA. Still, differences in the relative abundances of Actinobacteria (enriched in dry soils) and Proteobacteria (enriched in wetted soils) matched those of their predicted phages, indicating viral predation of dominant bacterial taxa. Rewetting also rapidly depleted relic DNA, which subsequently reaccumulated, indicating substantial new microbial mortality in the days after wet-up, particularly of the taxa putatively under phage predation. Production of abundant, diverse viral particles via microbial host cell lysis appears to be a conserved feature of the early response to soil rewetting, and results suggest the potential for ‘Cull-the-Winner’ dynamics, whereby viruses infect and cull but do not decimate dominant host populations.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

A Catabolic Powerhouse for Biorefineries: Characterization and Engineering Erwinia spp. Strain LJJL01 to Produce Bioproducts

Nonmodel microbial hosts can efficiently biotransform unconventional organic feedstocks into advanced bioproducts, leveraging their superior metabolic capabilities and resilience to process-relevant physicochemical conditions. In this study, we domesticated Erwinia spp. strain. LJJL01 (Er LJJL01) as a potent microbial chassis to advance the emerging biorefinery strategy for the valorization of lignin-rich biomass and plastic, thereby enabling the circular economy. The strain exhibits remarkable chemical tolerance and can biofunnel various substrates, including sugars, acids, polyols, and aromatics, in minimal salt media to produce native fine chemicals such as acetoin, 2,3-butanediol, and lactic acid. As a proof of concept, engineering this strain with advanced genetic tools enables the production of tailored high-value chemicals, such as cis,cis-muconate from plastic-derived terephthalate and polyhydroxybutyrate from lignocellulosic hydrolysate. We established Er LJJL01 as a potent microbial chassis for the green synthesis of bioproducts from unconventional organic feedstocks.

09 BIOMASS FUELS↗