TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses.

Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana, Nicotiana tabacum, and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

Modelling metabolic fluxes of tomato stems reveals that nitrogen shapes central metabolism for defence against Botrytis cinerea.

Among plant pathogens, the necrotrophic fungus Botrytis cinerea is one of the most prevalent, leading to severe crop damage. Studies related to its colonization of different plant species have reported variable host metabolic responses to infection. In tomato, high N availability leads to decreased susceptibility. Metabolic flux analysis can be used as an integrated method to better understand which metabolic adaptations lead to effective host defence and resistance. Here, we investigated the metabolic response of tomato infected by B. cinerea in symptomless stem tissues proximal to the lesions for 7 d post-inoculation, using a reconstructed metabolic model constrained by a large and consistent metabolic dataset acquired under four different N supplies. An overall comparison of 48 flux solution vectors of Botrytis- and mock-inoculated plants showed that fluxes were higher in Botrytis-inoculated plants, and the difference increased with a reduction in available N, accompanying an unexpected increase in radial growth. Despite higher fluxes, such as those involved in cell wall synthesis and other pathways, fluxes related to glycolysis, the tricarboxylic acid cycle, and amino acid and protein synthesis were limited under very low N, which might explain the enhanced susceptibility. Limiting starch synthesis and enhancing fluxes towards redox and specialized metabolism also contributed to defence independent of N supply.

Genomic Analysis of Novel Sulfitobacter Bacterial Strains Isolated from Marine Biofilms.

Bacteria from the genus Sulfitobacter are distributed across various marine habitats and play a significant role in sulfur cycling. However, the metabolic features of Sulfitobacter inhabiting marine biofilms are still not well understood. Here, complete genomes and paired metatranscriptomes of eight Sulfitobacter strains, isolated from biofilms on subtidal stones, have been analyzed to explore their central energy metabolism and potential of secondary metabolite biosynthesis. Based on average nucleotide identity and phylogenetic analysis, the eight strains were classified into six novel species and two novel strains. The reconstruction of the metabolic pathways indicated that all strains had a complete Entner-Doudoroff pathway, pentose phosphate pathway, and diverse pathways for amino acid metabolism, suggesting the presence of an optimized central carbon metabolism. Pangenome analysis further revealed the differences between the gene cluster distribution patterns among the eight strains, suggesting significant functional variation. Moreover, a total of 47 biosynthetic gene clusters were discovered, which were further classified into 37 gene cluster families that showed low similarity with previously documented clusters. Furthermore, metatranscriptomic analysis revealed the expressions of key functional genes involved in the biosynthesis of ribosomal peptides in in situ marine biofilms. Overall, this study sheds new light on the metabolic features, adaptive strategies, and value of genome mining in this group of biofilm-associated Sulfitobacter bacteria.

Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex.

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

Primary Human M2 Macrophage Subtypes Are Distinguishable by Aqueous Metabolite Profiles.

The complexity of macrophage (MΦ) plasticity and polarization states, which include classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ phenotypes, is becoming increasingly appreciated. Within the M2 MΦ polarization state, M2a, M2b, M2c, and M2d MΦ subcategories have been defined based on their expression of specific cell surface receptors, secreted cytokines, and specialized immune effector functions. The importance of immunometabolic networks in mediating the function and regulation of MΦ immune responses is also being increasingly recognized, although the exact mechanisms and extent of metabolic modulation of MΦ subtype phenotypes and functions remain incompletely understood. In this study, proton (1H) nuclear magnetic resonance (NMR) metabolomics was employed to determine the polar metabolomes of M2 MΦ subtypes and to investigate the relationship between aqueous metabolite profiles and M2 MΦ functional phenotypes. Results from this study demonstrate that M2a MΦs are most distinct from M2b, M2c, and M2d MΦ subtypes, and that M2b MΦs display several metabolic traits associated with an M1-like MΦ phenotype. The significance of metabolome differences for metabolites implicated in glycolysis, the tricarboxylic acid (TCA) cycle, phospholipid metabolism, and creatine-phosphocreatine cycling is discussed. Altogether, this study provides biochemical insights into the role of metabolism in mediating the specialized effector functions of distinct M2 MΦ subtypes and supports the concept of a continuum of macrophage activation states rather than two well-separated and functionally distinct M1/M2 MΦ classes, as originally proposed within a classical M1/M2 MΦ framework.

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.).

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.

Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering.

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism.

Transcriptome Metabolic Characterization of Tuber borchii SP1-A New Spanish Strain for In Vitro Studies of the Bianchetto Truffle.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

Deciphering the physiological response of Escherichia coli under high ATP demand.

One long-standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild-type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate-controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.

Management of plant central metabolism by SnRK1 protein kinases.

SUCROSE NON-FERMENTING1 (SNF1)-RELATED KINASE 1 (SnRK1) is an evolutionarily conserved protein kinase with key roles in plant stress responses. SnRK1 is activated when energy levels decline during stress, reconfiguring metabolism and gene expression to favour catabolism over anabolism, and ultimately to restore energy balance and homeostasis. The capacity to efficiently redistribute resources is crucial to cope with adverse environmental conditions and, accordingly, genetic manipulations that increase SnRK1 activity are generally associated with enhanced tolerance to stress. In addition to its well-established function in stress responses, an increasing number of studies implicate SnRK1 in the homeostatic control of metabolism during the regular day-night cycle and in different organs and developmental stages. Here, we review how the genetic manipulation of SnRK1 alters central metabolism in several plant species and tissue types. We complement this with studies that provide mechanistic insight into how SnRK1 modulates metabolism, identifying changes in transcripts of metabolic components, altered enzyme activities, or direct regulation of enzymes or transcription factors by SnRK1 via phosphorylation. We identify patterns of response that centre on the maintenance of sucrose levels, in an analogous manner to the role described for its mammalian orthologue in the control of blood glucose homeostasis. Finally, we highlight several knowledge gaps and technical limitations that will have to be addressed in future research aiming to fully understand how SnRK1 modulates metabolism at the cellular and whole-plant levels.

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids.

Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.

Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by Crypthecodinium cohnii from Glycerol, Glucose and Ethanol.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.

Reprogramming of Plant Central Metabolism in Response to Abiotic Stresses: A Metabolomics View.

Abiotic stresses rewire plant central metabolism to maintain metabolic and energy homeostasis. Metabolites involved in the plant central metabolic network serve as a hub for regulating carbon and energy metabolism under various stress conditions. In this review, we introduce recent metabolomics techniques used to investigate the dynamics of metabolic responses to abiotic stresses and analyze the trend of publications in this field. We provide an updated overview of the changing patterns in central metabolic pathways related to the metabolic responses to common stresses, including flooding, drought, cold, heat, and salinity. We extensively review the common and unique metabolic changes in central metabolism in response to major abiotic stresses. Finally, we discuss the challenges and some emerging insights in the future application of metabolomics to study plant responses to abiotic stresses.

Grape ASR Regulates Glucose Transport, Metabolism and Signaling.

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

Morphological and metabolic profiling of a tropical-adapted potato association panel subjected to water recovery treatment reveals new insights into plant vigor.

Potato (Solanum tuberosum L.) is one of the world's most important crops, but it is facing major challenges due to climatic changes. To investigate the effects of intermittent drought on the natural variability of plant morphology and tuber metabolism in a novel potato association panel comprising 258 varieties we performed an augmented block design field study under normal irrigation and under water-deficit and recovery conditions in Ica, Peru. All potato genotypes were profiled for 45 morphological traits and 42 central metabolites via nuclear magnetic resonance. Statistical tests and norm of reaction analysis revealed that the observed variations were trait specific, that is, genotypic versus environmental. Principal component analysis showed a separation of samples as a result of conditional changes. To explore the relational ties between morphological traits and metabolites, correlation-based network analysis was employed, constructing one network for normal irrigation and one network for water-recovery samples. Community detection and difference network analysis highlighted the differences between the two networks, revealing a significant correlational link between fumarate and plant vigor. A genome-wide association study was performed for each metabolic trait. Eleven single nucleotide polymorphism (SNP) markers were associated with fumarate. Gene Ontology analysis of quantitative trait loci regions associated with fumarate revealed an enrichment of genes regulating metabolic processes. Three of the 11 SNPs were located within genes, coding for a protein of unknown function, a RING domain protein and a zinc finger protein ZAT2. Our findings have important implications for future potato breeding regimes, especially in countries suffering from climate change.

Role of metAB in Methionine Metabolism and Optimal Chicken Colonization in Campylobacter jejuni.

Campylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

Systematic mapping of protein-metabolite interactions in central metabolism of Escherichia coli.

Metabolite binding to proteins regulates nearly all cellular processes, but our knowledge of these interactions originates primarily from empirical in vitro studies. Here, we report the first systematic study of interactions between water-soluble proteins and polar metabolites in an entire biological subnetwork. To test the depth of our current knowledge, we chose to investigate the well-characterized Escherichia coli central metabolism. Using ligand-detected NMR, we assayed 29 enzymes towards binding events with 55 intracellular metabolites. Focusing on high-confidence interactions at a false-positive rate of 5%, we detected 98 interactions, among which purine nucleotides accounted for one-third, while 50% of all metabolites did not interact with any enzyme. In contrast, only five enzymes did not exhibit any metabolite binding and some interacted with up to 11 metabolites. About 40% of the interacting metabolites were predicted to be allosteric effectors based on low chemical similarity to their target's reactants. For five of the eight tested interactions, in vitro assays confirmed novel regulatory functions, including ATP and GTP inhibition of the first pentose phosphate pathway enzyme. With 76 new candidate regulatory interactions that have not been reported previously, we essentially doubled the number of known interactions, indicating that the presently available information about protein-metabolite interactions may only be the tip of the iceberg.

Modification of the glycolytic pathway in Pyrococcus furiosus and the implications for metabolic engineering.

The key difference in the modified Embden-Meyerhof glycolytic pathway in hyperthermophilic Archaea, such as Pyrococcus furiosus, occurs at the conversion from glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) where the typical intermediate 1,3-bisphosphoglycerate (1,3-BPG) is not present. The absence of the ATP-yielding step catalyzed by phosphoglycerate kinase (PGK) alters energy yield, redox energetics, and kinetics of carbohydrate metabolism. Either of the two enzymes, ferredoxin-dependent glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) or NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), responsible for this "bypass" reaction, could be deleted individually without impacting viability, albeit with differences in native fermentation product profiles. Furthermore, P. furiosus was viable in the gluconeogenic direction (growth on pyruvate or peptides plus elemental sulfur) in a ΔgapnΔgapor strain. Ethanol was utilized as a proxy for potential heterologous products (e.g., isopropanol, butanol, fatty acids) that require reducing equivalents (e.g., NAD(P)H, reduced ferredoxin) generated from glycolysis. Insertion of a single gene encoding the thermostable NADPH-dependent primary alcohol dehydrogenase (adhA) (Tte_0696) from Caldanaerobacter subterraneus, resulted in a strain producing ethanol via the previously established aldehyde oxidoreductase (AOR) pathway. This strain demonstrated a high ratio of ethanol over acetate (> 8:1) at 80 °C and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date.

Promoted performance of microbial fuel cells using Escherichia coli cells with multiple-knockout of central metabolism genes.

The effect of central metabolic activity of Escherichia coli cells acting as biocatalysts on the performance of microbial fuel cells (MFCs) was studied with glucose used as the energy source. Milliliter-scale two-chambered MFCs were used with 2-hydroxy-1,4-naphthoquinone (HNQ) as an electron mediator. Among the single-gene deletions examined, frdA, pdhR, ldhA, and adhE increased the average power output of the constructed MFC. Next, multiple-gene knockout mutants were constructed using P1 transduction. The Δ5 (ΔfrdAΔpdhRΔldhAΔadhEΔpta) strain showed the highest ave. power output (1.82 mW) and coulombic efficiency (21.3%). Our results show that the combination of multiple-gene knockout in E. coli cells leads to the development of an excellent catalyst for MFCs. Finally, preventing a decrease in the pH of the anodic solution was a key factor for improving the power output of the Δ5 strain, and a maximum ave. power output of 2.21 mW was achieved with 5% NaHCO3 in the buffer. The ave. power density of the constructed MFC was 0.27 mW/cm3, which is comparable to an enzymatic fuel cell of a Milliliter-scale using glucose dehydrogenase.