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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

ABSTRACT Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α−1,2), nigerose (α−1,3), and isomaltose (α−1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of threeCellvibrio japonicusstrains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in theamy13Egene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis ofamy13Efound that this gene was essential for kojibiose and isomaltose metabolism inC. japonicus. Interestingly, a Δamy13Emutant was not deficient for cyclodextrin or pullulan utilization inC. japonicus; however, heterologous expression of the gene inE. coliwas sufficient for cyclodextrin-dependent growth. Biochemical analyses found thatCjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of theCjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. IMPORTANCE Understanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Engineering Novosphingobium aromaticivorans to produce cis,cis -muconic acid from biomass aromatics

ABSTRACT The platform chemical cis,cis- muconic acid ( cc MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of cc MA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to cc MA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in cc MA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of cc MA from PCA and produced greater than 100% yield of cc MA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of cc MA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce cc MA from deconstructed biomass. IMPORTANCE The production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis -muconic acid from renewable and abundant biomass aromatics.

09 BIOMASS FUELS↗
DOE OSTI2020

Improving Arsenic Tolerance of Pyrococcus furiosus by Heterologous Expression of a Respiratory Arsenate Reductase

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea. Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. Furthermore, x-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired

ABSTRACT Denitrification is a form of anaerobic respiration wherein nitrate (NO 3 − ) is sequentially reduced via nitrite (NO 2 − ), nitric oxide, and nitrous oxide (N 2 O) to dinitrogen gas (N 2 ) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria,Rhodopseudomonas palustrisCGA0092 andRhodobacter capsulatusSB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO 2 − to N 2 , and SB1003 reduced N 2 O to N 2 . For each bacterium, N 2 O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N 2 O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO 3 − served as a stable, non-catalyzable molecule that was sufficient to activate N 2 O reduction. Using a β-galactosidase reporter, we found that NO 3 − acted, at least in part, by stimulating N 2 O reductase gene expression. In SB1003, NO 2 − but not NO 3 − activated N 2 O reduction, but NO 2 − was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. IMPORTANCE Denitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Deletion of Re -citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of Heliomicrobium modesticaldum

ABSTRACT Heliomicrobium modesticaldum,a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typicalSi-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodesRe-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO 2 as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle. IMPORTANCE Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

Arbuscular Mycorrhizal Tree Communities Have Greater Soil Fungal Diversity and Relative Abundances of Saprotrophs and Pathogens than Ectomycorrhizal Tree Communities

Trees associating with different mycorrhizas often differ in their effects on litter decomposition, nutrient cycling, soil organic matter (SOM) dynamics, and plant-soil interactions. For example, due to differences between arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) tree leaf and root traits, ECM-associated soil has lower rates of C and N cycling and lower N availability than AM-associated soil. These observations suggest that many groups of nonmycorrhizal fungi should be affected by the mycorrhizal associations of dominant trees through controls on nutrient availability. To test this overarching hypothesis, we explored the influence of predominant forest mycorrhizal type and mineral N availability on soil fungal communities using next-generation amplicon sequencing. Soils from four temperate hardwood forests in southern Indiana, United States, were studied; three forests formed a natural gradient of mycorrhizal dominance (100% AM tree basal area to 100% ECM basal area), while the fourth forest contained a factorial experiment testing long-term N addition in both dominant mycorrhizal types. We found that overall fungal diversity, as well as the diversity and relative abundance of plant pathogenic and saprotrophic fungi, increased with greater AM tree dominance. Additionally, tree community mycorrhizal associations explained more variation in fungal community composition than abiotic variables, including soil depth, SOM content, nitrification rate, and mineral N availability. Our findings suggest that tree mycorrhizal associations may be good predictors of the diversity, composition, and functional potential of soil fungal communities in temperate hardwood forests. These observations help explain differing biogeochemistry and community dynamics found in forest stands dominated by differing mycorrhizal types.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Microfluidic droplets with amended culture media cultivate a greater diversity of soil microorganisms

ABSTRACT Uncultivated but abundant soil microorganisms have untapped potential for producing broad ranges of natural products, as well as for bioremediation. However, cultivating soil microorganisms while maintaining a broad microorganism diversity to enable phenotyping and functional analysis of as diverse individual isolates as possible remains challenging. In this study, we developed and tested the ability of several culture media formulations that contain defined soil metabolites or soil extracts to maintain microorganism diversity during culture. We also assessed their performance in microfluidic droplet cultivation where single-soil microorganism isolates were encapsulated and cultivated in picoliter-volume water-in-oil emulsion droplets to enable clonal growth needed for downstream functional analyses. Our results show that droplet cultivation with media supplemented by soil extract or soil metabolites enables the recovery of soil microorganisms with higher diversity (up to 1.5-fold higher richness) compared to bulk cultivation methods. Importantly, 1.7-fold more of less abundant (<1%) phyla and 11-fold more of unique genera were recovered, demonstrating the utility of this method for interrogating highly diverse soil microorganisms for broad ranges of applications. IMPORTANCE Although soil microorganisms hold a significant value in bioproduction and bioremediation, only a small fraction—less than 1%—can be cultured under specific media and cultivation conditions. This indicates that there are ample opportunities in harvesting the diverse environmental microorganisms if isolating and recovering these uncultured microorganisms are possible. This paper presents a new cultivation technique composed of isolating single-soil microorganism cell from anin situsoil microorganism community in microfluidic droplets and conducting in-droplet cultivation in media supplemented by soil extract or soil metabolites. This method enables the recovery of a broader diversity of the original microorganism community, laying the groundwork for a high-throughput phenotyping of these diverse microorganisms from their natural habitats.

Biotechnology & Applied Microbiology↗
DOE OSTI2023

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks

The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers

ABSTRACT The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway inCaldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C 5 and C 6 sugars. In addition, C. besciigrows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO.C. besciinatively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway,C. besciiwas engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present inC. bescii,the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model ofC. bescii. IMPORTANCE Lignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacteriumCaldicellulosiruptor besciiwas successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling ofC. besciiwas applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Enhanced transport of bacteria along root systems by protists can impact plant health

ABSTRACT Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpodasp.), a flagellate (Cercomonassp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixingSinorhizobium melilotito infectible root tips. Co-inoculation of protists plusS. melilotiresulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated withS. melilotialone. Co-inoculation of the ciliate, Colpodasp., withS. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix.Colpodasp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum ofS. melilotiresulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling. IMPORTANCE Protists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria,Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S.melilotialone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

ABSTRACT Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified inPseudomonasandBurkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacteriumPseudomonas protegensPf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation ofpgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation ofgacAdecreased the translation of PgnC, which is consistent with the result thatpgnCleader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria. IMPORTANCE Antimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE inPseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

Differential Response of Bacterial Microdiversity to Simulated Global Change

Microbial communities play an integral role in global biogeochemical cycling, but our understanding of how global change will affect microbial community structure and functioning remains limited. Microbiome analyses typically aggregate large amounts of genetic diversity which may obscure finer variation in traits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Multi-technique characterization of iron reduction by an Antarctic Shewanella : an analog system for putative Martian biosignature identification

ABSTRACT Microbes from terrestrial extreme environments enable testing of biosignature production in conditions relevant to astrobiological targets. Mars, which was likely more conducive to life during early warmer and wetter epochs, has inspired missions that search for signs of early life in the surficial rock record, including mineral or organic biosignatures. Microbial iron reduction is a common and ancient metabolism that may have also operated on other rocky celestial bodies. To investigate biosignature production during iron reduction, aShewanellasp. (strain BF02_Schw) isolated from a subglacial discharge known as Blood Falls, Antarctica, was incubated with the electron acceptor ferrihydrite (Fh). Biosignatures associated with Fh reduction were identified using a suite of techniques currently utilized or proposed for Mars missions, including X-ray diffraction and infrared, Mössbauer, and Raman spectroscopy. The biotic origin of features was validated by transcriptional changes observed between treatments with and without Fh and comparison to killed controls. In live treatments, Fh was reduced to magnetite and goethite, both detected in Martian lacustrine basins. Several soluble and volatile metabolites were also detected, including riboflavin and dimethyl sulfide (DMS), which could be astrobiological indicators of active microbial processes. While none of the identified biosignatures individually would serve as definitive proof of life (past or present), detecting concomitant features associated with known terrestrial biotic processes would provide compelling rationale for more targeted life detection missions. Terrestrial extremophiles can support the exploration of astrobiologically relevant microbial processes, validation of life detection instrumentation, and potentially the discovery of new biomarkers. IMPORTANCE Culture-based experiments with terrestrial extremophiles can elucidate biosignatures that may be analogous to those produced under extraterrestrial conditions, and thus inform sampling and technology strategies for future missions. Here, we demonstrate the production of several biosignatures under iron-reducing conditions byShewanellasp. BF02_Schw, originally isolated from an Antarctic analog feature. These biosignatures could be detectable using flight-ready instrumentation. Growth experiments with terrestrial extremophiles can identify biosignatures measurable by current methodologies and inform the development and optimization of techniques for detecting extant or extinct life on other worlds.

Biotechnology & Applied Microbiology↗
DOE OSTI2021

Redundancy in Aromatic O -Demethylation and Ring-Opening Reactions in Novosphingobium aromaticivorans and Their Impact in the Metabolism of Plant-Derived Phenolics

ABSTRACT Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds, followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM 12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O -demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolic compounds by N. aromaticivorans . The results showed the involvement of DesA in O -demethylation of syringic and vanillic acids, LigM in O- demethylation of vanillic acid and 3-MGA, and a new O- demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring-opening dioxygenase involved in 3-MGA, PCA, and GA metabolism and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O -demethylation of 3-MGA to GA. We predict that this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

Biotechnology & Applied Microbiology↗