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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Here, our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Drivers of stability and transience in composition-functioning links during serial propagation of litter-decomposing microbial communities

ABSTRACT Biotic factors that influence the temporal stability of microbial community functioning are an emerging research focus for the control of natural and engineered systems. The discovery of common features within community ensembles that differ in functional stability over time is a starting point to explore biotic factors. We serially propagated a suite of soil microbial communities through five generations of 28-day microcosm incubations to examine microbial community compositional and functional stability during plant litter decomposition. Using dissolved organic carbon (DOC) abundance as a target function, we hypothesized that microbial diversity, compositional stability, and associated changes in interactions would explain the relative stability of the ecosystem function between generations. Communities with initially high DOC abundance tended to converge towards a “low DOC” phenotype within two generations, but across all microcosms, functional stability between generations was highly variable. By splitting communities into two cohorts based on their relative DOC functional stability, we found that compositional shifts, diversity, and interaction network complexity were associated with the stability of DOC abundance between generations. Further, our results showed that legacy effects were important in determining compositional and functional outcomes, and we identified taxa associated with high DOC abundance. In the context of litter decomposition, achieving functionally stable communities is required to utilize soil microbiomes to increase DOC abundance and long-term terrestrial DOC sequestration as one solution to reduce atmospheric carbon dioxide concentrations. Identifying factors that stabilize function for a community of interest may improve the success of microbiome engineering applications. IMPORTANCE Microbial community functioning can be highly dynamic over time. Identifying and understanding biotic factors that control functional stability is of significant interest for natural and engineered communities alike. Using plant litter–decomposing communities as a model system, this study examined the stability of ecosystem function over time following repeated community transfers. By identifying microbial community features that are associated with stable ecosystem functions, microbial communities can be manipulated in ways that promote the consistency and reliability of the desired function, improving outcomes and increasing the utility of microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Editorial: Bioconversion and Biorefinery of C1 Compounds

The past decade has seen significant progress in the field of metabolic engineering and synthetic biology. The exponentially growing multi-omics data and technological advances in the development of efficient genetic manipulation tools and techniques have allowed scientists to explore and expand their understanding of microbial metabolisms and further develop sophisticated engineering strategies to realize the use of industrial "workhorses" and non-conventional microorganisms for sustainable bioconversion and biorefinery. There is of great interest for the research community in using C1 compounds (i.e., CO 2 , CO/syngas, methane, methanol) as the next generation feedstocks for microbial cell factories and biocatalysts to promote the sustainable development of a green economy (Figure 1). Considering lowering input costs is also a main consideration for successful business ventures, the use of inexpensive, abundant, and widely accessible C1 compounds is envisioned as a promising route for the sustainable production of fine chemicals, fuels, and other high-value products. Many C1 compounds are waste gases from industrial activities and may have detrimental effects on climate change upon emission into the atmosphere. Therefore, promoting the use of C1 compounds as renewable carbon feedstocks can greatly contribute to the reduction of anthropogenic emission of air pollutants.

09 BIOMASS FUELS↗
DOE OSTI2021

Biofuels for a sustainable future

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

09 BIOMASS FUELS↗
DOE OSTI2024

A synthetic co-culture for bioproduction of ammonia from methane and air

Abstract Fixed nitrogen fertilizers feed 50% of the global population, but most fixed nitrogen production occurs using energy-intensive Haber–Bosch-based chemistry combining nitrogen (N2) from air with gaseous hydrogen (H2) from methane (CH4) at high temperatures and pressures in large-scale facilities sensitive to supply chain disruptions. This work demonstrates the biological transformation of atmospheric N2 into ammonia (NH3) using CH4 as the sole carbon and energy source in a single vessel at ambient pressure and temperature, representing a biological “room-pressure and room-temperature” route to NH3 that could ultimately be developed to support compact, remote, NH3 production facilities amenable to distributed biomanufacturing. The synthetic microbial co-culture of engineered methanotroph Methylomicrobium buryatense (now Methylotuvimicrobium buryatense) and diazotroph Azotobacter vinelandii converted three CH4 molecules to l-lactate (C3H6O3) and powered gaseous N2 conversion to NH3. The design used division of labor and mutualistic metabolism strategies to address the oxygen sensitivity of nitrogenase and maximize CH4 oxidation efficiency. Media pH and salinity were central variables supporting co-cultivation. Carbon concentration heavily influenced NH3 production. Smaller-scale NH3 production near dispersed, abundant, and renewable CH4 sources could reduce disruption risks and capitalize on untapped energy resources. One-Sentence Summary Co-culture of engineered microorganisms Methylomicrobium buryatense and Azotobacter vinelandii facilitated the use of methane gas as a sole carbon feedstock to produce ammonia in an ambient temperature, atmospheric pressure, single-vessel system.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Leverage Microbial Innovations to Address Methane Emission Challenges: Input for FY24 Annual LDRD Report

Sandia researchers are addressing the urgent challenge of minimizing dilute and distributed methane emissions. The team is focused on generating stable methane-consuming microbial consortia for deployment in engineered environmental systems. This innovative work aims to produce stable inocula of these consortia and implement viral controls for microbes that generate methane, significantly reducing emissions.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

The Sugar Metabolic Model of Aspergillus niger Can Only Be Reliably Transferred to Fungi of Its Phylum

Fungi play a critical role in the global carbon cycle by degrading plant polysaccharides to small sugars and metabolizing them as carbon and energy sources. We mapped the well-established sugar metabolic network of Aspergillus niger to five taxonomically distant species (Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium and Dichomitus squalens) using an orthology-based approach. The diversity of sugar metabolism correlates well with the taxonomic distance of the fungi. The pathways are highly conserved between the three studied Eurotiomycetes (A. niger, A. nidulans, P. subrubescens). A higher level of diversity was observed between the T. reesei and A. niger, and even more so for the two Basidiomycetes. These results were confirmed by integrative analysis of transcriptome, proteome and metabolome, as well as growth profiles of the fungi growing on the corresponding sugars. In conclusion, the establishment of sugar pathway models in different fungi revealed the diversity of fungal sugar conversion and provided a valuable resource for the community, which would facilitate rational metabolic engineering of these fungi as microbial cell factories.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Devices and methods for generating electrical current from methane

Methods, microbial fuel cells and microbial consortia for generating electrical current are provided according to the present invention which include providing a microbial consortium to an anode chamber of a microbial fuel cell, wherein the microbial consortium includes: 1) an engineered methanogen that contains a heterologous nucleic acid sequence encoding methyl-coenzyme M reductase derived from an anaerobic methane oxidizer, 2) an exoelectrogen microbe that produces electrically-conductive appendages and/or one or more types of electron carrier, and 3) a sludge, methane-acclimated sludge, a sludge isolate component, a methane-acclimated sludge isolate component chosen from Paracoccus spp., Geotoga spp., Geobacter spp., Methanosarcina spp., Garciella spp., humic acids; or a combination of any two or more thereof.

Wood, Thomas K.↗
DOE OSTI2026

Engineered plants for the production of the antioxidants arbutin and gallate

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.

Antioxidant↗
DOE OSTI2025

Transportability of exogenous microbial community correlates with interwell connectivity in deep aquifers

Subsurface resource engineering operations often utilize continuous injection of externally-sourced water into geological reservoirs for formation pressure maintenance, resource recovery or energy/waste storage. Such injected water generally contains naturally occurring microbes. Little is known, however, about how the injectate microbes transport through geological media as a community, how such transportability is affected by injector-producer connectivity, and whether such knowledge can be utilized for flowpath characterization. In this study, we analyzed daily-to-weekly timeseries microbial community data from the injected- and produced-fluids of a ten-month flow test at a deep, well-characterized engineered aquifer. We found that the injectate microbial community was distinct from the indigenous community at the amplicon sequence variant (ASV) level, and that the transportability of injectate community towards a given producer, quantified by an “nASV-Overlap” metric we propose, had strong and significant positive correlation with known injector-producer connectivities at our site. This suggests that the better the connectivity, the higher the probability for more injectate species to flow through the interwell region and arrive at a producer. Because interwell connectivity is an important yet usually unknown parameter in subsurface resource engineering, such correlation in turn points to nASV-Overlap as a useful indicator of interwell connectivity for aquifer characterization and long-term monitoring. Based on our findings, an nASV-Overlap-based microbial tracing approach was developed for characterizing and monitoring the relative connectivities across multiple producers with a given injector. A side-by-side comparison between the new nASV-Overlap approach and traditional artificial tracer methods is presented, and their respective strengths and limitations are discussed.

Deep biosphere↗
DOE OSTI2025

Production of Vanillin From Ferulic Acid by Pseudomonas putida KT2440 Using Metabolic Engineering and In Situ Product Recovery

Vanillin is the most in-demand flavouring compound in the world and because vanillin extracted from vanilla pods cannot meet the global demand, most vanillin on the market today is chemically synthesised. Increasing demands by consumers for natural ingredients have inspired efforts to develop vanillin derived from microbial sources. These efforts have been challenged by low titers, likely caused by the toxicity of vanillin to most microbial biocatalysts. In this study, we engineered a Pseudomonas putida KT2440-derived strain that accumulated vanillin from ferulic acid to 0.64 g/L. To increase the overall titre, we applied a hydrophobic polystyrene-based resin to vanillin-accumulating cultures, which enabled an increase in total vanillin recovery to an apparent titre of 3.35 g/L. This study demonstrates that P. putida can accumulate vanillin from ferulic acid to higher titers when vanillin is removed from the cultivation medium, mitigating its toxicity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Biosensor and optogenetics for systems biology of yeast branched-chain alcohol production and tolerance

In this project we combined synthetic biology, systems biology, protein engineering and metabolic engineering to study, control, and improve the production of branched chain alcohols (BCAs), a class of advanced biofuels preferred by the DOE, in the yeast Saccharomyces cerevisiae. This involved the development and application of optogenetic systems as a new modality of dynamic control of native and engineered metabolic pathways, using light as inducible or repressible agent. The optogenetic systems include gene circuits for light control of gene expression, as well as light-assembled synthetic organelles and photo-switchable protein binders to control metabolic and protein function with light at the protein level. In addition, we developed the first genetically encoded biosensor for BCA production in yeast, which we used to design high throughput assays to identify highly productive strains, pathways, and enzymes. We also showed that this biosensor can be functionally co-expressed with optogenetic circuits in the same strain, raising the possibility of establishing, for the first time, computer-interfaced closed-loop controls of engineered metabolic pathways. The yeast gene deletion library was also utilized to conduct the first systems-level study on BCA toxicity in yeast, which uncovered key fundamental principles of yeast sensitivity and tolerance to these alcohols, allowing us to design highly tolerant strains with increased BCA production. This project, thus comprises the development of several new technologies, which we integrated to make new discoveries on the dynamics of BCA production and their mechanisms of cellular toxicity and tolerance, as well as to establish new paradigms to engineer and control metabolic pathways and microbial fermentations with light, for the production of BCAs and other products of interest to the DOE.

2-metyl-1-butanol↗
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↗
DOE OSTI2021

Solutions in microbiome engineering: prioritizing barriers to organism establishment

Abstract Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite, increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2) environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Environmental microbiome engineering for the mitigation of climate change

Abstract Environmental microbiome engineering is emerging as a potential avenue for climate change mitigation. In this process, microbial inocula are introduced to natural microbial communities to tune activities that regulate the long‐term stabilization of carbon in ecosystems. In this review, we outline the process of environmental engineering and synthesize key considerations about ecosystem functions to target, means of sourcing microorganisms, strategies for designing microbial inocula, methods to deliver inocula, and the factors that enable inocula to establish within a resident community and modify an ecosystem function target. Recent work, enabled by high‐throughput technologies and modeling approaches, indicate that microbial inocula designed from the top‐down, particularly through directed evolution, may generally have a higher chance of establishing within existing microbial communities than other historical approaches to microbiome engineering. We address outstanding questions about the determinants of inocula establishment and provide suggestions for further research about the possibilities and challenges of environmental microbiome engineering as a tool to combat climate change.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Transgenic Mixed‐Linkage‐Glucan Enhancement Affects Root Characteristics and Decomposition in Soils of Contrasting Vegetation History

ABSTRACT Development of transgenic bioenergy sorghum [ Sorghum bicolor (L.) Moench] with increased contents of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is an important step towards enhancing quality of bioenergy feedstocks. Since MLG‐enhancement leads to greater biomass digestibility, our overarching hypothesis is that root residues of MLG‐enhanced plants may be more readily decomposed in the soil, potentially creating new opportunities for optimizing soil carbon (C) sequestration, nutrient cycling, and overall agricultural sustainability. The study examined morphological, chemical, and enzymatic characteristics of fine and coarse roots of four bioenergy sorghum genotypes. Then, we incubated the roots within soils with contrasting vegetation histories while measuring C mineralization, microbial biomass C (MBC), and activity of hydrolytic enzymes and calculating vector length and vector angle enzymatic stoichiometry parameters. The results indicated that MLG‐enhancing transformations increased root total nitrogen (N) contents, decreased C/N ratios, and were associated with higher MLG concentrations in fine than in coarse roots. Incubations with transgenic roots led to 16%–38% higher MBC and 19%–41% lower microbial metabolic quotient (qCO 2 ). While enzyme activity differed markedly among the studied genotypes, it did not directly respond to MLG levels in root tissues. The increase in MBC without concurrent increases in C mineralization or hydrolytic enzyme activities in transgenic genotypes suggests that MLG enhancement promoted microbial anabolic retention of root‐derived C rather than stimulating catabolic decomposition. Enzymatic vector results indicated that these parameters reflect a variety of drivers behind microbial enzyme production, including availability of specific substrates, such as MLG here, and/or deficiency in specific nutrients, such as phosphorus (P). The study confirms the positive impacts from the roots of engineered MLG‐enhanced bioenergy plants on soil microbial activity and highlights the interactive influences on the MLG‐enhancement effects from root size and inherent soil properties.

Mahmoodabadi, Majid [Department of Plant, Soil, an↗
DOE OSTI2022

Life and death in the soil microbiome: how ecological processes influence biogeochemistry

We report soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter — Earth’s largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗