Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Microplastic exposure inhibits nitrate uptake and assimilation in wheat plants.

Microplastic (MP) contamination in soil severely impairs plant growth. However, mechanisms underlying the effects of MPs on plant nutrient uptake remain largely unknown. In this study, we revealed that NO3- content was significantly decreased in shoots and roots of wheat plants exposed to high concentrations (50-100 mg L-1) of MPs (1 µm and 0.1 µm; type: polystyrene) in the hydroponic solution. Isotope labeling experiments demonstrated that MP exposure led to a significant inhibition of NO3- uptake in wheat roots. Further analysis indicated that the presence of MPs markedly inhibited root growth and caused oxidative damage to the roots. Additionally, superoxide dismutase and peroxidase activities in wheat roots decreased under all MP treatments, whereas catalase and ascorbate peroxidase activities significantly increased under the 100 mg L-1 MP treatment. The transcription levels of most nitrate transporters (NRTs) in roots were significantly downregulated by MP exposure. Furthermore, exposure to MPs distinctly suppressed the activity of nitrate reductase (NR) and nitrite reductase (NiR), as well as the expression levels of their coding genes in wheat shoots. These findings indicate that a decline in root uptake area and root vitality, as well as in the expression of NRTs, NR, and NiR genes caused by MP exposure may have adverse effects on NO3- uptake and assimilation, consequently impairing normal growth of plants.

Energy deprivation affects nitrogen assimilation and fatty acid biosynthesis leading to leaf chlorosis under waterlogging stress in the endangered Abies koreana.

Energy deprivation triggers various physiological, biochemical and molecular changes in plants under abiotic stress. We investigated the oxidative damages in the high altitude grown conifer Korean fir (Abies koreana) exposed to waterlogging stress. Our experimental results showed that waterlogging stress led to leaf chlorosis, 35 days after treatment. A significant decrease in leaf fresh weight, chlorophyll and sugar content supported this phenotypic change. Biochemical analysis showed a significant increase in leaf proline, lipid peroxidase and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical content of waterlogged plants. To elucidate the molecular mechanisms, we conducted RNA-sequencing (RNA-seq) and de novo assembly. Using RNA-seq analysis approach and filtering (P < 0.05 and false discovery rate <0.001), we obtained 134 unigenes upregulated and 574 unigenes downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis placed the obtained differentially expressed unigenes in α-linoleic pathway, fatty acid degradation, glycosis, glycolipid metabolism and oligosaccharide biosynthesis process. Mapping of unigenes with Arabidopsis using basic local alignment search tool for nucleotides showed several critical genes in photosynthesis and carbon metabolism downregulated. Following this, we found the repression of multiple nitrogen (N) assimilation and nucleotide biosynthesis genes including purine metabolism. In addition, waterlogging stress reduced the levels of polyunsaturated fatty acids with a concomitant increase only in myristic acid. Together, our results indicate that the prolonged snowmelt may cause inability of A. koreana seedlings to lead the photosynthesis normally due to the lack of root intercellular oxygen and emphasizes a detrimental effect on the N metabolic pathway, compromising this endangered tree's ability to be fully functional under waterlogging stress.

The Role of Nitrate Supply in Bioactive Compound Synthesis and Antioxidant Activity in the Cultivation of Porphyra linearis (Rhodophyta, Bangiales) for Future Cosmeceutical and Bioremediation Applications.

Porphyra sensu lato has economic importance for food and pharmaceutical industries due to its significant physiological activities resulting from its bioactive compounds (BACs). This study aimed to determine the optimal nitrate dosage required in short-term cultivation to achieve substantial BAC production. A nitrate experiment using varied concentrations (0 to 6.5 mM) revealed optimal nitrate uptake at 0.5 mM in the first two days and at 3 and 5 mM in the last five days. Polyphenols and carbohydrates showed no differences between treatments, while soluble proteins peaked at 1.5 and 3 mM. Total mycosporine-like amino acids (MAAs) were highest in algae incubated at 5 and 6.5 mM, and the highest antioxidant activity was observed in the 5 mM, potentially related to the MAAs amount. Total carbon and sulfur did not differ between treatments, while nitrogen decreased at higher nitrate. This discovery highlights the nuanced role of nitrate in algal physiology, suggesting that biological and chemical responses to nitrate supplementation can optimize an organism's health and its commercially significant bioactive potential. Furthermore, given its ability to absorb high doses of nitrate, this alga can be cultivated in eutrophic zones or even in out-/indoor tanks, becoming an excellent option for integrated multi-trophic aquaculture (IMTA) and bioremediation.

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis.

Freezing stress can seriously affect plant growth and development, but the mechanisms of these effects and plant responses to freezing stress require further exploration. Here, we identified a NAM, ATAF1/2, and CUC2 (NAC)-family transcription factor (TF), NAC056, that can promote freezing tolerance in Arabidopsis. NAC056 mRNA levels are strongly induced by freezing stress in roots, and the nac056 mutant exhibits compromised freezing tolerance. NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor (CBF) pathway genes. Interestingly, we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants; therefore, NAC056-CBF1-NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress. In addition, 35S::NAC056 transgenic plants show enhanced freezing tolerance, which is partially reversed in the cbfs triple mutant. Thus, NAC056 confers freezing tolerance through the CBF pathway, mediating plant responses to balance growth and freezing stress tolerance.

Transcriptomic analysis of nitrogen metabolism pathways in Klebsiella aerogenes under nitrogen-rich conditions.

The acceleration of the nitrogen cycle and the nitrogen excess observed in some coastal waters has increased interest into understanding the biochemical and molecular basis of nitrogen metabolism in various microorganisms. To investigate nitrogen metabolism of a novel heterotrophic nitrification and aerobic denitrification bacterium Klebsiella aerogenes strain (B23) under nitrogen-rich conditions, we conducted physiological and transcriptomic high-throughput sequencing analyses on strain B23 cultured on potassium nitrate-free or potassium nitrate-rich media. Overall, K. aerogenes B23 assimilated 82.47% of the nitrate present into cellular nitrogen. Further, 1,195 differentially expressed genes were observed between K. aerogenes B23 cultured on potassium nitrate-free media and those cultured on potassium nitrate-rich media. Gene annotation and metabolic pathway analysis of the transcriptome were performed using a series of bioinformatics tools, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Non-Redundant Protein Database annotation. Accordingly, the nitrogen metabolism pathway of K. aerogenes B23 was analyzed; overall, 39 genes were determined to be involved in this pathway. Differential expression analysis of the genes involved in the nitrogen metabolism pathway demonstrated that, compared to the control, FNR, NarK/14945, fdx, gshA, proB, proA, gapA, argH, artQ, artJ, artM, ArgR, GAT1, prmB, pyrG, glnS, and Ca1 were significantly upregulated in the nitrogen-treated K. aerogenes B23; these genes have been established to be involved in the regulation of nitrate, arginine, glutamate, and ammonia assimilation. Further, norV, norR, and narI were also upregulated in nitrogen-treated K. aerogenes B23; these genes are involved in the regulation of NO metabolism. These differential expression results are important for understanding the regulation process of key nitrogen metabolism enzyme genes in K. aerogenes B23. Therefore, this study establishes a solid foundation for further research into the expression regulation patterns of nitrogen metabolism-associated genes in K. aerogenes B23 under nitrogen-rich conditions; moreover, this research provides essential insight into how K. aerogenes B23 utilizes nutritional elements.

Biochar leachate reduces primary nitrogen assimilation by inhibiting nitrogen fixation and microbial nitrate assimilation.

Biochar contains biotoxic aromatic compounds, and their influence on nitrogen-fixing cyanobacteria, the critical nitrogen fixer in paddy soil, has never been tested. Here, the physiological, metabolomic, and transcriptomic analyses of Nostoc sp. PCC7120 in response to biochar leachate were performed. The results suggested that biochar leachate inhibited the efficiency of photosynthesis, nitrogen fixation, and nitrate assimilation activities of nitrogen-fixing cyanobacteria. Biochar leachate containing aromatic compounds and odd- and long-chain saturated fatty acids impaired the membrane structure and antenna pigments, damaged the D1 protein of the oxygen evolution complex, and eventually decreased the electron transfer chain activity of photosystem II. Moreover, the nitrogen fixation and nitrate assimilation abilities of nitrogen-fixing cyanobacteria were inhibited by a decrease in photosynthetic productivity. A decrease in iron absorption was another factor limiting nitrogen fixation efficiency. Our study highlights that biochar with relatively high contents of dissolved organic matter poses a risk to primary nitrogen assimilation reduction and ecosystem nitrogen loss. Further evidence of the potential negative effects of biochar leachates on the fixation and assimilation capacity of nitrogen by soil microbes is needed to evaluate the impact of biochar on soil multifunctionality prior to large-scale application.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

[Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor].

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.

Salt Stress-Related Mechanisms in Leaves of the Wild Barley Hordeum spontaneum Generated from RNA-Seq Datasets.

This study aims to detect salt stress-related genes and mechanisms of the wild barley Hordeum spontaneum. Among the generated RNA-Seq datasets, several regulated transcripts are influenced by levels of cellular carbon, nitrogen and oxygen. Some of the regulated genes act on photorespiration and ubiquitination processes, as well as promoting plant growth and development under salt stress. One of the genes, encoding alanine:glyoxylate aminotransferase (AGT), participates in signaling transduction and proline biosynthesis, while the gene encoding asparagine synthetase (ASN) influences nitrogen storage and transport in plants under stress. Meanwhile, the gene encoding glutamate dehydrogenase (GDH) promotes shoot and root biomass production as well as nitrate assimilation. The upregulated genes encoding alpha-aminoadipic semialdehyde synthase (AASAS) and small auxin-up RNA 40 (SAUR40) participate in the production of proline and signaling compounds, respectively, while the gene encoding E3 ubiquitin-protein ligase regulates the carbon/nitrogen-nutrient response and pathogen resistance, in addition to some physiological processes under biotic and abiotic stresses via signal transduction. The gene encoding the tetratricopeptide repeat (TPR)-domain suppressor of STIMPY (TSS) negatively regulates the carbon level in the cell. In conclusion, this study sheds light on possible molecular mechanisms underlying salt stress tolerance in wild barley that can be utilized further in genomics-based breeding programs of cultivated species.

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Nitrogen fertilization and CO2 concentration synergistically affect the growth and protein content of Agropyron mongolicum.

Background: The nitrogen (N) and protein concentrations in plant tissues exposed to elevated CO2 (eCO2) generally decline , such declines in forage grass composition are expected to have negative implications for the nutritional and economic value of grass. Plants require N for the production of a photosynthetically active canopy and storage proteins in the tissues, whose functionality will strongly influence productivity and quality. The objective of this study was to investigate whether eCO2 plus N-fertilization increases growth and N nutrition of Agropyron mongolicum, and the dependence of this improvement on the coordination between root and leaf development. Methods: We analyzed A. mongolicum from field-grown within the open-top chambers (OTCs) facility under two atmospheric CO2 (ambient, 400 ± 20 µmol mol-1, aCO2, and elevated, 800 ± 20 µmol mol-1, eCO2) and three N-fertigation treatments (control, low N-fertigation , and high N-fertigation) for two months. Results: Elevated CO2 plus N-fertigation strongly increased shoot and root biomass, and the nitrogen and protein concentrations of A. mongolicum compared to those plants at aCO2 levels. Increased N content in leaves and reduced specific leaf area (SLA) at a high N supply could alleviate photosynthetic acclimation to eCO2 and drive the production of greater shoot biomass with the potential for higher photosynthesis, productivity, and nutritional quality. The increased root length (RL), the ratio of total aboveground N taken up per RL (TN/RL), stomatal conductance (Gs), and transpiration rate (Tr) contribute to the transpiration-driven mass flow of N, consequently increasing N uptake by roots. In addition, a smaller percentage of N remained as unassimilated nitrate ( NO 3 - ) under eCO2, indicating that assimilation of NO 3 - into proteins was not inhibited by eCO2. These findings imply that grass productivity and quality will enhance under anticipated elevated CO2 concentration when effective management measures of N-fertilization are employed.

The C2H2 Zinc Finger Protein MaNCP1 Contributes to Conidiation through Governing the Nitrate Assimilation Pathway in the Entomopathogenic Fungus Metarhizium acridum.

Zinc finger proteins are an important class of multifunctional regulators. Here, the roles of a C2H2 zinc finger protein MaNCP1 (Metarhizium acridum nitrate-related conidiation pattern shift regulatory factor 1) in nitrogen utilization and conidiation were explored in the entomopathogenic fungus M. acridum. The results showed that MaNCP1-disruption mutant (ΔMaNCP1) impaired the ability to utilize nitrate, ammonium and glutamine and reduced the expression of nitrate assimilation-related genes, suggesting that MaNCP1 was involved in governing nitrogen utilization. In addition, the conidial yield of the ΔMaNCP1 strain, cultured on the microcycle conidiation medium (SYA), was significantly decreased, which could be restored or even enhanced than that of the WT strain through increasing the nitrate content in SYA medium. Further study showed that MaAreA, a core regulator in the nitrogen catabolism repression (NCR) pathway, was a downstream target gene of MaNCP1. Screening the differential expression genes between WT and ΔMaNCP1 strains revealed that the conidial yield of M. acridum regulated by nitrate might be related to NCR pathway on SYA medium. It could be concluded that MaNCP1 contributes to the nitrate assimilation and conidiation, which will provide further insights into the relationship between the nitrogen utilization and conidiation in fungi.

Light-emitting diode combined with humic acid improve the nutritional quality and enzyme activities of nitrate assimilation in rocket (Eruca sativa (Mill.) Thell.).

Belonging to the Brassicaceae family, rocket (Eruca sativa (Mill.) Thell.), is considered to be a nitrate-accumulating leafy vegetable. Many studies show that light-emitting diode (LED) lights can be a suitable tool to decrease anti-nutritional compounds (e.g., nitrate (and enhance antioxidant and nutritional quality for phytochemical-rich vegetable production. The positive influence of humic acid on health-promoting compounds in different crops is also well documented. This study aimed to investigate the effects of supplemental LED lights of various spectral compositions, namely 25-100% red, 25-100% blue, and 100% white, as well as their combination with humic acid on the physiological and biochemical responses of rocket plants. ANOVA results showed that almost all the measured traits were significantly affected by LED and humic acid treatments. Generally, LED combined with humic acid improved the accumulation of nutritional compounds (e.g., polyphenols, flavonoid, ascorbic acid, carbohydrate, tannin), increased the activity of key enzymes involved in nitrogen metabolism (e.g., nitrate reductase, nitrite reductase, and glutamine synthetase), and lowered nitrate and ammonium concentrations. The results of principal component analysis indicated that the combination of LED lights, regardless of the spectra, with humic acid was the most effective treatment to enhance the nutritional value and activity of enzymes involved in nitrate assimilation. In sum, these findings may be used as a reference in rocket production for supplemental LED light optimization and its combination with humic acid.

Interaction of Nitrate Assimilation and Photorespiration at Elevated CO2.

It has been shown repeatedly that exposure to elevated atmospheric CO2 causes an increased C/N ratio of plant biomass that could result from either increased carbon or - in relation to C acquisition - reduced nitrogen assimilation. Possible reasons for diminished nitrogen assimilation are controversial, but an impact of reduced photorespiration at elevated CO2 has frequently been implied. Using a mutant defective in peroxisomal hydroxy-pyruvate reductase (hpr1-1) that is hampered in photorespiratory turnover, we show that indeed, photorespiration stimulates the glutamine-synthetase 2 (GS) / glutamine-oxoglutarate-aminotransferase (GOGAT) cycle, which channels ammonia into amino acid synthesis. However, mathematical flux simulations demonstrated that nitrate assimilation was not reduced at elevated CO2, pointing to a dilution of nitrogen containing compounds by assimilated carbon at elevated CO2. The massive growth reduction in the hpr1-1 mutant does not appear to result from nitrogen starvation. Model simulations yield evidence for a loss of cellular energy that is consumed in supporting high flux through the GS/GOGAT cycle that results from inefficient removal of photorespiratory intermediates. This causes a futile cycling of glycolate and hydroxy-pyruvate. In addition to that, accumulation of serine and glycine as well as carboxylates in the mutant creates a metabolic imbalance that could contribute to growth reduction.

Metabolic and biotechnological insights on the analysis of the Pdh bypass and acetate production in the yeast Dekkera bruxellensis.

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast.

CmNDB1 and a Specific Domain of CmMYB1 Negatively Regulate CmMYB1-Dependent Transcription of Nitrate Assimilation Genes Under Nitrogen-Repleted Condition in a Unicellular Red Alga.

Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (-N)-activated TF, in a unicellular red alga Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.

Direct Air Capture of CO2 through Carbonate Alkalinity Generated by Phytoplankton Nitrate Assimilation.

Despite the consensus that keeping global temperature rise within 1.5 °C above pre-industrial level by 2100 reduces the chance for climate change to reach the point of no return, the newest Intergovernmental Panel on Climate Change (IPCC) report warns that the existing commitment of greenhouse gas emission reduction is only enough to contain the warming to 3-4 °C by 2100. The harsh reality not only calls for speedier deployment of existing CO2 reduction technologies but demands development of more cost-efficient carbon removal strategies. Here we report an ocean alkalinity-based CO2 sequestration scheme, taking advantage of proton consumption during nitrate assimilation by marine photosynthetic microbes, and the ensuing enhancement of seawater CO2 absorption. Benchtop experiments using a native marine phytoplankton community confirmed pH elevation from ~8.2 to ~10.2 in seawater, within 3-5 days of microbial culture in nitrate-containing media. The alkaline condition was able to sustain at continued nutrient supply but reverted to normalcy (pH ~8.2-8.4) once the biomass was removed. Measurements of δ13C in the dissolved inorganic carbon revealed a significant atmospheric CO2 contribution to the carbonate alkalinity in the experimental seawater, confirming the occurrence of direct carbon dioxide capture from the air. Thermodynamic calculation shows a theoretical carbon removal rate of ~0.13 mol CO2/L seawater, if the seawater pH is allowed to decrease from 10.2 to 8.2. A cost analysis (using a standard bioreactor wastewater treatment plant as a template for CO2 trapping, and a modified moving-bed biofilm reactor for nitrate recycling) indicated that a 1 Mt CO2/year operation is able to perform at a cost of ~$40/tCO2, 2.5-5.5 times cheaper than that offered by any of the currently available direct air capture technologies, and more in line with the price of $25-30/tCO2 suggested for rapid deployment of large-scale CCS systems.

Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol.

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.

Barley transcription factor HvNLP2 mediates nitrate signaling and affects nitrogen use efficiency.

Plants have evolved complex mechanisms to adapt to the changing nitrogen levels in the environment. In Arabidopsis, more than a dozen nitrate signaling regulatory genes have been characterized, including the NODULE INCEPTION-LIKE PROTEIN (AtNLP) genes, which play essential roles in nitrate signaling. However, whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) remains unknown. Here, we isolated a barley (Hordeum vulgare L.) mutant, hvnlp2-1, from a TILLING (Targeting Local Lesions IN Genomes) population and constructed two RNAi lines, hvnlp2-2 and hvnlp2-3, to study the function of HvNLP2. The expression of the nitrate-responsive genes was substantially inhibited after nitrate treatment in the hvnlp2 mutants, indicating that HvNLP2 controls nitrate signaling. Nitrate content was significantly higher in the hvnlp2 mutants, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes. HvNLP2 is localized to the nucleus in the presence of nitrate. Further investigation showed that HvNLP2 binds to and activates the nitrate-responsive cis-elements. Moreover, hvnlp2 exhibited reduced biomass, seed yield, and NUE. Therefore, HvNLP2 controls nitrate signaling and plays an important role in NUE.