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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 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Methane-cycling microbial communities from Amazon floodplains and upland forests respond differently to simulated climate change scenarios

Seasonal floodplains in the Amazon basin are important sources of methane (CH 4 ), while upland forests are known for their sink capacity. Climate change effects, including shifts in rainfall patterns and rising temperatures, may alter the functionality of soil microbial communities, leading to uncertain changes in CH 4 cycling dynamics. To investigate the microbial feedback under climate change scenarios, we performed a microcosm experiment using soils from two floodplains (i.e., Amazonas and Tapajós rivers) and one upland forest. We employed a two-factorial experimental design comprising flooding (with non-flooded control) and temperature (at 27 °C and 30 °C, representing a 3 °C increase) as variables. We assessed prokaryotic community dynamics over 30 days using 16S rRNA gene sequencing and qPCR. These data were integrated with chemical properties, CH 4 fluxes, and isotopic values and signatures. In the floodplains, temperature changes did not significantly affect the overall microbial composition and CH 4 fluxes. CH 4 emissions and uptake in response to flooding and non-flooding conditions, respectively, were observed in the floodplain soils. By contrast, in the upland forest, the higher temperature caused a sink-to-source shift under flooding conditions and reduced CH 4 sink capability under dry conditions. The upland soil microbial communities also changed in response to increased temperature, with a higher percentage of specialist microbes observed. Floodplains showed higher total and relative abundances of methanogenic and methanotrophic microbes compared to forest soils. Isotopic data from some flooded samples from the Amazonas river floodplain indicated CH 4 oxidation metabolism. This floodplain also showed a high relative abundance of aerobic and anaerobic CH 4 oxidizing Bacteria and Archaea. Taken together, our data indicate that CH 4 cycle dynamics and microbial communities in Amazonian floodplain and upland forest soils may respond differently to climate change effects. We also highlight the potential role of CH 4 oxidation pathways in mitigating CH 4 emissions in Amazonian floodplains.

16S rRNA sequencing↗
NASA NTRS2004

Microbial astronauts: assembling microbial communities for advanced life support systems

Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth. The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space. Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments "everything is everywhere" given enough time). This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch. Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time. Research at the John F. Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support. Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed. In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity. Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment. Copyright 2004 Springer-Verlag.

Review↗
NASA NTRS1986

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Source record↗
DOE OSTI2022

Syntrophic Co-Cultures of Clostridium Organisms to Produce Higher Alcohols & Other C6-C8 Metabolites (Final Report)

The goal of this project was to advance the systems biology understanding and predictive modeling of synthetic & syntrophic Clostridium microbial consortia, focusing on elucidation of metabolic networks and environmental signals in the consortia. The project has direct applicability to lignocellulosic-biomass based production of higher alcohols as advanced biofuels and C6-C8 metabolites, that can be used as chemicals or serve as biofuel precursors. This project studied and optimized three synthetic syntrophic systems. It examined the population dynamics using flow-cytometry, time-lapse microscopy and PCR analysis. 13C-based tracer analyses was used to examine the metabolite exchange between the syntrophic cell populations and the impact of those interactions on the transcriptome of the individual populations. To enhance our analytical and predictive capabilities, genome-scale models (GSMs) for these syntrophies was developed. RNAseq data for these syntrophic coculture systems were acquired to enable a molecular level understanding of the syntrophies aiming to identify the genetic networks of each organism in the co-culture and compare those against the networks of pure cultures.

09 BIOMASS FUELS↗
DOE OSTI2020

Growth, total lipid, and omega-3 fatty acid production by Nannochloropsis spp. cultivated with raw plant substrate

Improving productivity and lipid concentration in microalgae is important for the economic success of both biofuel and microalgae coproducts production. Nannochloropsis spp. are marine microalgae currently being grown at large-scale for the production of biofuel and lipid coproducts. In this work, we demonstrate improvements of growth and omega-3 production in Nannochloropsis gaditana CCMP526 and Nannochloropsis oceanica CCAP849/10 with plant substrate addition, a potentially economical option for increasing microalgal productivity. Furthermore, we examine growth in the presence of corn stover, switchgrass, sugarcane bagasse, and yard waste. By examining the microbial ecology of N. gaditana cultures with and without plant substrate and with and without antibiotics, we discovered a potential bacterial interaction in these cultures, but its presence is not necessary for algal growth improvements in the presence of plant substrate. Analysis of plant substrate morphology by scanning electron microscopy (SEM) after cultivation in media with and without N. gaditana shows a degradation of specific plant structural features and a colonization of the plant phloem by N. gaditana. An examination of N. gaditana in a Congo red plate assay indicates potential cellulolytic activity, but a preliminary examination of the potential cellulase transcripts does not reveal differential expression of a candidate in the presence of plant. This evidence demonstrates potential raw plant utilization by a marine microalga for increased productivity and provides a potentially economical option for increasing coproduct concentration at industrial scale without genetic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Self-Assembling Cell-Free Systems for Scalable Bioconversion

This project focused on developing cell-free systems to directly express multi-enzyme catalysts and perform CO2 bioconversions for industrial chemical production. The use of cell-free expression (CFE) systems derived from bacterial lysates is emerging as a promising approach for biomanufacturing. CFEs are genetically programmable, permit the expression of toxic enzymes, and allow for rapid prototyping of metabolic pathways. Research Contributions: 1. Understanding the Area Investigated: This research advances the understanding of cell-free systems by demonstrating their capability to perform complex multi-enzyme reactions. By directly expressing multi-gene systems, CFEs avoid the high costs and inefficiencies associated with producing and purifying enzymes for multi-step pathways. 2. Technical Effectiveness and Economic Feasibility: The project successfully engineered a CFE-based multienzyme biocatalyst for the de novo synthesis of serine and glycine from CO2 equivalents (formate and bicarbonate) and ammonia. This method achieved a 30% conversion rate of formate into these industrially important amino acids. Additionally, an 8-gene CFE biocatalyst was developed to produce malate, conserving 43% of carbon that would otherwise be lost as CO2. This approach has the potential to reach higher carbon efficiency than microbial production. 3. Public Benefit: The cell-free production of chemicals like serine, glycine, and malate using electrochemically generated formate could significantly reduce CO2 emissions. For example, satisfying the global malate market with this method could avoid approximately 400,000 tons of CO2 emissions annually. This work demonstrates the potential of CFE systems to produce platform chemicals, contributing to environmental sustainability and reducing reliance on petrochemicals. Future Prospects: The CFE-based biocatalyst process could be extended to produce a variety of chemicals, including other industrial di-acids, aromatics, terpenes, alcohols, and polymers. This project showcases the capabilities of cell-free expression systems for prototyping carbon-conserving pathways and sustainably bioproducing platform chemicals, marking a significant step towards economically-viable industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2017

The Transcriptional Response of Diverse Saccharomyces Cerevisiae Strains to Simulated Microgravity

Spaceflight imposes multiple stresses on biological systems resulting in genome-scale adaptations. Understanding these adaptations and their underlying molecular mechanisms is important to clarifying and reducing the risks associated with spaceflight. One such risk is infection by microbes present in spacecraft and their associated systems and inhabitants. This risk is compounded by results suggesting that some microbes may exhibit increased virulence after exposure to spaceflight conditions. The yeast, S. cerevisiae, is a powerful microbial model system, and its response to spaceflight has been studied for decades. However, to date, these studies have utilized common lab strains. Yet studies on trait variation in S. cerevisiae demonstrate that these lab strains are not representative of wild yeast and instead respond to environmental stimuli in an atypical manner. Thus, it is not clear how transferable these results are to the wild S. cerevisiae strains likely to be encountered during spaceflight. To determine if diverse S. cerevisiae strains exhibit a conserved response to simulated microgravity, we will utilize a collection of 100 S. cerevisiae strains isolated from clinical, environmental and industrial settings. We will place selected S. cerevisiae strains in simulated microgravity using a high-aspect rotating vessel (HARV) and document their transcriptional response by RNA-sequencing and quantify similarities and differences between strains. Our research will have a strong impact on the understanding of how genetic diversity of microorganisms effects their response to spaceflight, and will serve as a platform for further studies.

yeast↗
NASA NTRS2017

The Transcriptional Response of Diverse Saccharomyces Cerevisiae Strains to Simulated Microgravity

Spaceflight imposes multiple stresses on biological systems resulting in genome-scale adaptations. Understanding these adaptations and their underlying molecular mechanisms is important to clarifying and reducing the risks associated with spaceflight. One such risk is infection by microbes present in spacecraft and their associated systems and inhabitants. This risk is compounded by results suggesting that some microbes may exhibit increased virulence after exposure to spaceflight conditions. The yeast, S. cerevisiae, is a powerful microbial model system, and it's response to spaceflight has been studied for decades. However, to date, these studies have utilized common lab strains. Yet studies on trait variation in S. cerevisiae demonstrate that these lab strains are not representative of wild yeast and instead respond to environmental stimuli in an atypical manner. Thus, it is not clear how transferable these results are to the wild S. cerevisiae strains likely to be encountered during spaceflight. To determine if diverse S. cerevisiae strains exhibit a conserved response to simulated microgravity, we will utilize a collection of 100 S. cerevisiae strains isolated from clinical, environmental and industrial settings. We will place selected S. cerevisiae strains in simulated microgravity using a high-aspect rotating vessel (HARV) and document their transcriptional response by RNA-sequencing and quantify similarities and differences between strains. Our research will have a strong impact on the understanding of how genetic diversity of microorganisms effects their response to spaceflight, and will serve as a platform for further studies.

genetic diversity↗
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↗
NASA NTRS2018

The Transcriptional Response of Diverse Saccharomyces cerevisiae Strains to Simulated Microgravity

Spaceflight imposes multiple stresses on biological systems resulting in genome-scale adaptations. Understanding these adaptations and their underlying molecular mechanisms is important to clarifying and reducing the risks associated with spaceflight. One such risk is infection by microbes present in spacecraft and their associated systems and inhabitants. This risk is compounded by results suggesting that some microbes may exhibit increased virulence after exposure to spaceflight conditions. The yeast, S. cerevisiae, is a powerful microbial model system, and it's response to spaceflight has been studied for decades. However, to date, these studies have utilized common lab strains. Yet studies on trait variation in S. cerevisiae demonstrate that these lab strains are not representative of wild yeast and instead respond to environmental stimuli in an a typical manner. Thus, it is not clear how transferable these results are to the wild S. cerevisiae strains likely to be encountered during spaceflight. To determine if diverse S. cerevisiae strains exhibit a conserved response to simulated microgravity, we will utilize a collection of 100 S. cerevisiae strains isolated from clinical, environmental and industrial settings. We will place selected S. cerevisiae strains in simulated microgravity using a high-aspect rotating vessel (HARV) and document their transcriptional response by RNA-sequencing and quantify similarities and differences between strains. Our research will have a strong impact on the understanding of how genetic diversity of microorganisms effects their response to spaceflight, and will serve as a platform for further studies.

spaceflight↗
DOE OSTI2023

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Efficient utilization of agro-industrial waste, such as sugar beet pulp, is crucial for the bio-based economy. The fungus Aspergillus niger possesses a wide array of enzymes that degrade complex plant biomass substrates, and several regulators have been reported to play a role in their production. The role of the regulators GaaR, AraR, and RhaR in sugar beet pectin degradation has previously been reported. However, genetic regulation of the degradation of sugar beet pulp has not been assessed in detail. In this study, we generated a set of single and combinatorial deletion mutants targeting the pectinolytic regulators GaaR, AraR, RhaR, and GalX as well as the (hemi-)cellulolytic regulators XlnR and ClrB to address their relative contribution to the utilization of sugar beet pulp. We show that A. niger has a flexible regulatory network, adapting to the utilization of (hemi-)cellulose at early timepoints when pectin degradation is impaired.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An accurate and interpretable model for antimicrobial resistance in pathogenic Escherichia coli from livestock and companion animal species

Understanding the microbial genomic contributors to antimicrobial resistance (AMR) is essential for early detection of emerging AMR infections, a pressing global health threat in human and veterinary medicine. Here we used whole genome sequencing and antibiotic susceptibility test data from 980 disease causing Escherichia coli isolated from companion and farm animals to model AMR genotypes and phenotypes for 24 antibiotics. We determined the strength of genotype-to-phenotype relationships for 197 AMR genes with elastic net logistic regression. Model predictors were designed to evaluate different potential modes of AMR genotype translation into resistance phenotypes. Our results show a model that considers the presence of individual AMR genes and total number of AMR genes present from a set of genes known to confer resistance was able to accurately predict isolate resistance on average (mean F 1 score = 98.0%, SD = 2.3%, mean accuracy = 98.2%, SD = 2.7%). However, fitted models sometimes varied for antibiotics in the same class and for the same antibiotic across animal hosts, suggesting heterogeneity in the genetic determinants of AMR resistance. We conclude that an interpretable AMR prediction model can be used to accurately predict resistance phenotypes across multiple host species and reveal testable hypotheses about how the mechanism of resistance may vary across antibiotics within the same class and across animal hosts for the same antibiotic.

Chung, Henri C.↗
DOE OSTI2021

Understanding Multilevel Selection May Facilitate Management of Arbuscular Mycorrhizae in Sustainable Agroecosystems

Studies in natural ecosystems show that adaptation of arbuscular mycorrhizal (AM) fungi and other microbial plant symbionts to local environmental conditions can help ameliorate stress and optimize plant fitness. This local adaptation arises from the process of multilevel selection, which is the simultaneous selection of a hierarchy of groups. Studies of multilevel selection in natural ecosystems may inform the creation of sustainable agroecosystems through developing strategies to effectively manage crop microbiomes including AM symbioses. Field experiments show that the species composition of AM fungal communities varies across environmental gradients, and that the biomass of AM fungi and their benefits for plants generally diminish when fertilization and irrigation eliminate nutrient and water limitations. Furthermore, pathogen protection by mycorrhizas is only important in environments prone to plant damage due to pathogens. Consequently, certain agricultural practices may inadvertently select for less beneficial root symbioses because the conventional agricultural practices of fertilization, irrigation, and use of pesticides can make these symbioses superfluous for optimizing crop performance. The purpose of this paper is to examine how multilevel selection influences the flow of matter, energy, and genetic information through mycorrhizal microbiomes in natural and agricultural ecosystems, and propose testable hypotheses about how mycorrhizae may be actively managed to increase agricultural sustainability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The ancestral environment of teosinte populations shapes their root microbiome

Summary Background The composition of the root microbiome affects the host’s growth, with variation in the host genome associated with microbiome variation. However, it is not known whether this intra-specific variation of root microbiomes is a consequence of plants performing targeted manipulations of them to adapt to their local environment or varying passively with other traits. To explore the relationship between the genome, environment and microbiome, we sampled seeds from teosinte populations across its native range in Mexico. We then grew teosinte accessions alongside two modern maize lines in a common garden experiment. Metabarcoding was performed using universal bacterial and fungal primers to profile their root microbiomes. Results The root microbiome varied between the two modern maize lines and the teosinte accessions. We further found that variation of the teosinte genome, the ancestral environment (temperature/elevation) and root microbiome were all correlated. Multiple microbial groups significantly varied in relative abundance with temperature/elevation, with an increased abundance of bacteria associated with cold tolerance found in teosinte accessions taken from high elevations. Conclusions Our results suggest that variation in the root microbiome is pre-conditioned by the genome for the local environment (i.e. non-random). Ultimately, these claims would be strengthened by confirming that these differences in the root microbiome impact host phenotype, for example, by confirming that the root microbiomes of high-elevation teosinte populations enhance cold tolerance.

Genetics & Heredity↗
DOE OSTI2025

Sulfoquinovose is exclusively metabolized by the gut microbiota and degraded differently in mice and humans

Abstract Background Sulfoquinovose (SQ) is a green-diet-derived sulfonated glucose and a selective substrate for a limited number of human gut bacteria. Complete anaerobic SQ degradation via interspecies metabolite transfer to sulfonate-respiring bacteria produces hydrogen sulfide, which has dose- and context-dependent health effects. Here, we studied potential SQ degradation by the mammalian host and the impact of SQ supplementation on human and murine gut microbiota diversity and metabolism. Results 13 CO 2 breath tests with germ-free C57BL/6 mice gavaged with 13 C-SQ were negative. Also, SQ was not degraded by human intestinal cells in vitro, indicating that SQ is not directly metabolized by mice and humans. Addition of increasing SQ concentrations to human fecal microcosms revealed dose-dependent responses of the microbiota and corroborated the relevance ofAgathobacter rectalisandBilophila wadsworthiain cooperative degradation of SQ to hydrogen sulfide via interspecies transfer of 2,3-dihydroxy-1-propanesulfonate (DHPS). Similar to the human gut microbiome, the genetic capacity for SQ or DHPS degradation is sparsely distributed among bacterial species in the gut of conventional laboratory mice.Escherichia coliandEnterocloster clostridioformiswere identified as primary SQ degraders in the mouse gut. SQ and DHPS supplementation experiments with conventional laboratory mice and their intestinal contents showed that SQ was incompletely catabolized to DHPS. Although someE. clostridioformisgenomes encode an extended sulfoglycolytic pathway for both SQ and DHPS fermentation, SQ was only degraded to DHPS by a mouse-derivedE. clostridioformisstrain. Conclusions Our findings suggest that SQ is solely a nutrient for the gut microbiota and not for mice and humans, emphasizing its potential as a prebiotic. SQ degradation by the microbiota of conventional laboratory mice differs from the human gut microbiota by absence of DHPS degradation activity. Hence, the microbiota of conventional laboratory mice does not fully represent the SQ metabolism in humans, indicating the need for alternative model systems to assess the impact of SQ on human health. This study advances our understanding of how individual dietary compounds shape the microbial community structure and metabolism in the gut and thereby potentially influence host health.

Microbiology↗
DOE OSTI2022

In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Optimization of Heterologous Glucoraphanin Production In Planta

Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target for production in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphane before and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin in heterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there may be missing auxiliary genes that play a role in improving production in planta. Here, in an effort to identify auxiliary genes required for high glucoraphanin production, we leveraged transient expression in Nicotiana benthamiana to screen a combination of previously uncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategy alleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosynthetic pathway provides a pathway amenable for high glucoraphanin production.

59 BASIC BIOLOGICAL SCIENCES↗