Nitrogen-limiting Conditions Research Articles

The GCN4 Transcription Factor: A Review of Its Functional Progress in Fungi

Nitrogen serves as a pivotal nutrient for the proliferation, maturation, and pathogenicity of fungi. Despite its importance, nitrogen starvation is a common challenge encountered during fungal development and host invasion. A key regulatory transcription factor, known as general control non-derepressible 4 (GCN4), has been characterized in various fungal groups, including model fungal, pathogens, and basidiomycetes. This factor is triggered by nitrogen limitation and subsequently stimulates the expression of a multitude of genes involved in amino acid synthesis, thereby countering the effects of nitrogen deficiency. This paper provides a comprehensive review on the activation mechanisms, the structural characteristics and stability of GCN4, and how GCN4 activates its downstream target genes to regulate the physiological processes of fungi. This study lays the theoretical groundwork for future research endeavors that seek to enhance nitrogen utilization, preserve the delicate balance of carbon–nitrogen metabolism, and stimulate growth, development, and secondary metabolism in fungi, especially under nitrogen-limited conditions.

Peptide Signals Regulate Nitrogen Deficiency Adaptation of Dicotyledonous Model Plants.

Nitrogen is a crucial macroelement essential for plant growth and development. In Arabidopsis Thaliana, classical phytohormones such as auxin and cytokinin orchestrate local and systemic signalling networks coordinate plant growth and development in response to nitrogen deficiency. Nowadays, emerging signalling pathways involving small peptides like CLAVATA3/EMBRYO SURROUNDINGR REGION (CLE) and C-TERMINALLY ENCODED PEPTIDE (CEP) and their corresponding kinase receptors, also regulate Arabidopsis' adaptation to nitrogen scarcity. Unlike Arabidopsis, which adapts to nitrogen deficiency by changing root development, legumes have the unique ability to form nitrogen-fixing root nodules through symbiotic interactions with soil rhizobia. During the symbiotic nodulation in Medicago, CLE and CEP peptides and their receptors consist of an autoregulatory network governing the number of nodules in accordance with the soil nitrogen level. Additionally, other plant peptides, such as phytosulfokine (PSK) and root meristem growth factors (RGF), have been identified as new regulators of leguminous root nodule development under nitrogen-limited condition. However, the precise mechanism by which these peptides coordinate nitrogen deficiency response and the development of nitrogen-fixing organs remains to be fully elucidated. This review summarises the adaptive strategies of dicotyledons to nitrogen deficiency, with a particular focus on the regulation of Medicago nitrogen-fixing nodule development by the peptides.

The ammonium transporter AmtB is dispensable for the uptake of ammonium in the phototrophic diazotroph Rhodopseudomonas palustris

Ammonium (NH4+) transport across cell membranes plays an important role in assimilation or removal of the environmental nitrogen. The membrane protein AmtB has been considered to be the NH4+ transporter that is responsible for the NH4+ uptake. The phototrophic diazotroph Rhodopseudomonas palustris harboring two amtB genes has been widely used in wastewater treatment and bioremediation. However, the role of AmtB in NH4+ uptake remains unclear in R. palustris. Here, we employed an innovative approach combining stable isotope probing (SIP) with Raman spectroscopy to determine the physiological functions of AmtB1 and AmtB2 in R. palustris. This powerful technique allowed us to investigate NH4+ uptake at the single-cell level. The generated R. palustris ΔamtB1 ΔamtB2 mutant lacking AmtB1 and AmtB2 proteins was still capable of utilizing 15NH4+ even the 15NH4+ concentration was as low as 5 μM. These data demonstrate that both of the AmtB proteins are not essential for R. palustris to take up NH4+ regardless of environmental NH4+ levels. However, both AmtB1 and AmtB2 can contribute to NH4+ uptake under nitrogen-limiting conditions. Given that R. palustris primarily expresses AmtB2 in these conditions, AmtB2 plays a more important role in NH4+ uptake compared to AmtB1. In addition, transcriptomic analysis showed that the deletion of the amtB1 and amtB2 genes resulted in the upregulation of many transporter genes, providing potential targets for future investigation of alternative NH4+ uptake systems.

Predator-induced defense decreases growth rate and photoprotective capacity in a nitrogen-limited dinoflagellate, Alexandrium minutum

Some dinoflagellates produce toxic secondary metabolites that correlate with increased resistance to grazers. The allocation costs of toxin production have been repeatedly addressed, but with conflicting results. Few studies have considered the potential costs of this defense to the photosystem, even though defense toxins (e.g., karlotoxins and brevetoxins) are closely linked to the photoprotective process. Here, we used chemical cues from copepods to induce paralytic shellfish toxin (PST) production in resource-limited Alexandrium minutum and quantitatively determined the growth rate and potential trade-offs with the photosystem process. The results show that grazer-induced, more toxic A. minutum had larger cell volume, lower cell division rate, and lower pigment content under nitrogen-limited conditions than control cells. In addition, predator cues led to a lower relative abundance of photoprotective xanthophylls and a reduced de-epoxidation efficiency of the xanthophyll cycle under high light conditions, reducing the ability of the cells to resist photodamage. Decreased photoprotective capacity may reflect an overlooked defense cost of toxin production.

Mycelial mass, microbial lipids and γ-linolenic acid (GLA) by Cunninghamella elegans cultivated on agro-industrial residues

In the current study, the Zygomycetes fungus Cunninghamella elegans NRRL Y-1392 was evaluated for its ability to grow in extracts derived from dried and ground agricultural residues, such as mushroom stalks and roots from hydroponically cultivated lettuces and produce poly-unsaturated fatty acids (PUFA) and γ-linolenic acid (GLA) rich lipids. Initially, the compositions of stalks and lettuce roots were analysed, and the fungus was batch-flask cultivated on six different commercial semi-defined substrates containing different sugars detected in stalks and roots to evaluate its catabolic ability. C. elegans was capable to assimilate all sugars, but at a lower rate in the case of arabinose. Subsequently, C. elegans was cultivated on tailor-made semi-defined commercial substrates, resembling hydrolysates containing carbohydrates found in mushroom stalks, under both nitrogen-excess and nitrogen-limited conditions, and resembling that of hydrolysates of roots, under nitrogen-excess conditions. Based on the results, under nitrogen-excess conditions, in the case of media resembling stalks hydrolysates, higher production values for biomass, PUFAs, and GLA were observed (20.3 g/L, 1906 mg/L, 668 mg/L), accompanied by high productivity values due to short cultivation periods, while under nitrogen limitation, high lipid accumulation (lipid in dry cell weight =48%, w/w) was presented, and lipids rich in oleic acid were produced. Finally, the fungus was cultivated on a medium derived from hot water-extraction applied to mushroom stalks, enriched with organic nitrogen sources. The fungus was successfully grown on the sugar-rich water-extract derived from mushroom stalks, resulting in dry biomass of 14.5 g/L, lipids of 1.8 g/L, with 15% (w/w) of GLA in cellular lipids.

Microbial ecophysiology: Shedding light on the re-greening of chlorotic cyanobacteria

Cyanobacteria, key contributors to aquatic CO2 fixation, employ sophisticated acclimation strategies for survival under nitrogen-limited conditions. A new study reveals that red light influences the resuscitation of chlorotic cells through mechanisms involving the dark-operative protochlorophyllide reductase, which facilitates chlorophyll biosynthesis and re-greening under low-light conditions.

The evaluation of Starmerella magnoliae X3 as a biodiesel feedstock based on triacylglycerol (TAG) production, lipid productivity, and fatty acid profile under nitrogen limitation and acidic pH conditions.

The effects of four initial culture pH values (3, 4, 5, and 6) and nitrogen limitation on growth, TAG accumulation, lipid production, fatty acid profile, and estimated biodiesel quality of Starmerella magnoliae X3 were investigated. TAG and lipid levels were measured by Nile Red fluorescence and sulfo-phospho-vanilin (SPV) techniques, respectively. The results showed that a combination of nitrogen limitation and acidic pH significantly (p < 0.05) increased TAG accumulation, total lipid contents, and lipid productivity in Starmerella magnoliae X3 compared to the control group. Under nitrogen limitation, the highest TAG accumulation was achieved at initial pHs of 3 and 5 after 72h of cultivation, and the highest lipid productivity (0.306g L-1 d-1) was observed after 48h at pH 3; the major fatty acids at the four pH values were oleic acid (63.6%-64%), palmitoleic acid (11.3%-12.5%), stearic acid (9.7%-11.4%), and palmitic acid (9.4%-10%). In addition, both stresses were associated with lower iodine value and higher cetane number of the biodiesel compared to the control. These findings suggest that cultivation in a low-nitrogen medium at an initial pH of 3 or 5 holds promise in increasing TAG production in Starmerella magnoliae X3.

Optimizing vertical farm cultivation of Cichorium spinosum L.: White Light's influence and nutrition management

The objective of this study was to examine the integration of a wild leafy vegetable, Cichorium spinosum L., in vertical farms. This research comprises two experiments focusing on different “white” light products and nutrient solutions. During both experiments, the temperature varied between 25 and 28 °C, relative humidity ranged from 50 to 70 %, carbon dioxide was at 450 ppm, and light intensity was set at 300 μmol m−2 s−1 respectively. In the lighting experiment, the three spectra used had the commercial names Neutral, Full and a SunLike™, and their spectral composition (blue:green:red:far-red) were 14:32:43:10, 16:36:40:8, and 21:34:36:7 respectively. The photoperiod was set to 12 h and the plant density was 50 plants m−2. Results showed no significant impact on agronomical parameters and leaf anatomy. The stomatal length and width decreased as the red:blue ratio of the light sources decreased, being greater in the Neutral treatment (red:blue ratio of 3.1) compared to the Full and SunLike™ (red:blue ratios of 2.5 and 1.7 respectively). Based on these results the preferable “white light” product was the one with the highest efficiency and lowest market price at the time of the experiment. In the nutrient solution experiment, the agronomical and nutritional attributes of stamnagathi plants supplied with a control nutrient solution, “N10-Fe15” were compared to plants cultivated under limited nitrogen, “N4-Fe15” and elevated iron, “N10-Fe48”, EC was 1.5 ds m−1, and pH was 5.6–6.5. The experiment simulated commercial practices by increasing the photoperiod to 15 h and plant density to 100 plants per square meter. The results did not demonstrate significant effect of the nutrient solution differences on the agronomical characteristics except from a decrease in total Kjeldahl nitrogen under limited nitrogen conditions. Notably, leaf tissue phosphorus content increased under elevated iron conditions. The nitrate content remained within safe for consumption thresholds for all treatments. Based on these results, stamnagathi can be integrated in vertical farms under limited nitrogen conditions. Stamnagathi's resilience to elevated iron in the nutrient solution demonstrated its potential for future biofortification experiments.

Characterization of the conversion system of natural rubber to poly(3-Hydroxyalkanoate) in Piscinibacter gummiphilus strain NS21T

Poly(3-hydroxyalkanoate) (PHA), a bacteria-synthesized biodegradable polyester, is a useful alternative to fossil resources, and current systems for its production rely predominantly on edible resources, raising concerns about microbial competition for nutrients. Therefore, we investigated mechanisms underlying PHA production from non-edible resources by Piscinibacter gummiphilus strain NS21T. Strain NS21T can utilize natural rubber as a carbon source on solid media and potentially produces PHA. Gas chromatography and nuclear magnetic resonance analyses of NS21T cell extracts revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate) from natural rubber and glucose, respectively. Transcriptional analysis suggested that phaC is involved in PHA production. An increased PHBV accumulation rate under nitrogen-limiting conditions indicates the potential of this strain to be used as a PHBV production enhancement strategy. Furthermore, the disruption of PHA depolymerase genes resulted in enhanced PHA production, indicating the involvement of these genes in PHA degradation. These findings highlight the potential of NS21T for PHBV production from natural rubber, a non-edible resource.

Effects of Temperature and Nitrogen Limitation on Growth and Lipid Production of Oleaginous Microalgae from Hot Springs

Oleaginous microalgae have gained increasing attention as an alternative feedstock for biodiesel production due to the increasing demand of fuel, climate change, and global warming. This study aimed to isolate and screen robust microalgal strains from hot springs for cultivation in subtropical and tropical areas. The newly isolated oleaginous microalgae were cultivated at 30, 35 and 40 °C. Among mesophilic and thermophilic strains tested, Chlamydomonas sp. HP59 is considered the most robust strain as it showed high cell growth in a broad range of temperatures (30 - 40 °C), with the maximum dried cell weight at 40 °C. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. To increase lipid production, the 2-stage cultivation, in which nitrogen-rich was applied to promote cell growth in the 1st stage and nitrogen-limitation was applied to stimulate lipid accumulation in the 2nd stage, was performed. The high temperature combined with nitrogen-limitation did improve lipid production by all microalgae. With this strategy, Chlamydomonas sp. HP59 showed the highest dried cell weight of 4.33 g/L and lipid production of 1.72 g/L. This study has shown the potential use of the newly isolated oleaginous microalgae from hot springs to be cultivated at high temperatures and under nitrogen-limited conditions for the production of biodiesel feedstocks. HIGHLIGHTS Mesophilic and thermophilic oleaginous microalgae were isolated from hot springs. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. Combined effect of high temperature and nitrogen limitation effectively increased lipid content. Two-stage cultivation gave high dried cell weight and hence overall high lipid production. GRAPHICAL ABSTRACT

Modeling Bibb Lettuce Nitrogen Uptake and Biomass Productivity in Vertical Hydroponic Agriculture

Global fertilizer production and mismanagement significantly contribute to many harmful environmental impacts, revealing the need for a greater understanding of crop growth and nutrient uptake, which can be used to optimize fertilizer management. This study experimentally adapts first-principles microbial modeling techniques to the hydroponic cultivation of Bibb lettuce (Lactuca sativa) under nitrogen-limited conditions. Monod and Michaelis–Menten’s approaches are applied to predict biomass productivity and nutrient uptake and to evaluate the feasibility of reclaimed wastewater as a nutrient source of nitrogen. Experimental and modeling results reveal significantly different kinetic saturation constants (Ks = 1.331 and Km = 17.887 mg L−1) and a corresponding cell yield strongly dependent on nutrient concentration, producing visually and compositionally distinct tissue between treatments receiving ≤26.2 and ≥41.7 mgN L−1. The resulting Monod model overestimates dry mass predictions during low nutrient conditions, and the collective results support the development of a dynamic Monod curve that is temporally dependent during the plants’ lifecycle. Despite this shortcoming, these results support the feasibility of reclaiming nitrogen from wastewater in hydroponic agriculture, expecting to produce lesser biomass lettuce exhibiting healthy tissue. Furthermore, this study provides a mathematical foundation for agricultural simulations and nutrient management.

Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Responses in species diversity in the Hulunbuir grassland to phosphorus addition under nitrogen-limiting and non-limiting conditions.

The phenomenon of nitrogen deposition resulting in species loss in terrestrial ecosystems has been demonstrated in several experiments. Nitrogen (N) and phosphorus (P), as major nutrients required for plant growth, exhibit ecological stoichiometric coupling in many ecosystems. The increased availability of nitrogen can exacerbate the ecological effects of phosphorus. To reveal the ecological effects of phosphorus under nitrogen-limiting and non-limiting conditions, we conducted a controlled N-P interaction experiment over 5 years in the Hulunbuir meadow steppe, where two nitrogen addition levels were implemented: 0g N·m-2·a-1 (nitrogen-limiting condition) and 10g N·m-2·a-1 (nitrogen-non-limiting condition), together with six levels of phosphorus addition (0, 2, 4, 6, 8, and 10g P·m-2·a-1). The results showed that nitrogen addition (under nitrogen-non-limiting conditions) significantly decreased species diversity in the steppe community, which was exacerbated under phosphorus addition. Under nitrogen-limiting conditions, phosphorus addition had no marked impact on species diversity compared to the control; however, there were substantial differences between different levels of phosphorus addition, exhibiting a unimodal change. Under both experimental nitrogen conditions, the addition of 6g P·m-2·a-1 was the threshold for affecting the community species diversity. Nitrogen addition reduced the relative biomass of legumes, bunch grasses, and forbs, but substantially increased the relative biomass of rhizomatous grasses. In contrast, phosphorus addition only markedly affected the relative biomass of forbs and rhizomatous grasses, with the former showing a unimodal pattern of first increasing and then decreasing with increasing phosphorus addition level, and the latter exhibiting the opposite pattern. The different responses of rhizomatous grasses and other functional groups to nitrogen and phosphorus addition were observed to have a regulatory effect on the changes in grassland community structure. Phosphorus addition may increase the risk of nitrogen deposition-induced species loss. Both nitrogen and phosphorus addition lead to soil acidification and an increase in the dominance of the already-dominant species, and the consequent species loss in the forb functional group represents the main mechanism for the reduction in community species diversity.

ROS-mediated thylakoid membrane remodeling and triacylglycerol biosynthesis under nitrogen starvation in the alga Chlorella sorokiniana.

Many microbes accumulate energy storage molecules such as triglycerides (TAG) and starch during nutrient limitation. In eukaryotic green algae grown under nitrogen-limiting conditions, triglyceride accumulation is coupled with chlorosis and growth arrest. In this study, we show that reactive oxygen species (ROS) actively accumulate during nitrogen limitation in the microalga Chlorella sorokiniana. Accumulation of ROS is mediated by the downregulation of genes encoding ROS-quenching enzymes, such as superoxide dismutases, catalase, peroxiredoxin, and glutathione peroxidase-like, and by the upregulation of enzymes involved in generating ROS, such as NADPH oxidase, xanthine oxidase, and amine oxidases. The expression of genes involved in ascorbate and glutathione metabolism is also affected under this condition. ROS accumulation contributes to the degradation of monogalactosyl diacylglycerol (MGDG) and thylakoid membrane remodeling, leading to chlorosis. Quenching ROS under nitrogen limitation reduces the degradation of MGDG and the accumulation of TAG. This work shows that ROS accumulation, membrane remodeling, and TAG accumulation under nitrogen limitation are intricately linked in the microalga C. sorokiniana.

Root plasticity improves maize nitrogen use when nitrogen is limiting: an analysis using 3D plant modelling.

Plant phenotypic plasticity plays an important role in nitrogen (N) acquisition and use under nitrogen-limited conditions. However, this role has never been quantified as a function of N availability, leaving it unclear whether plastic responses should be considered as potential targets for selection. A combined modelling and experimentation approach was adopted to quantify the role of plasticity in N uptake and plant yield. Based on a greenhouse experiment we considered plasticity in two maize (Zea mays) traits: root-to-leaf biomass allocation ratio and emergence rate of axial roots. In a simulation experiment we individually enabled or disabled both plastic responses for maize stands grown across six N levels. Both plastic responses contributed to maintaining a higher N uptake, and plant productivity as N availability declined compared with stands in which plastic responses were disabled. We conclude that plastic responses quantified in this study may be a potential target trait in breeding programs for greater N uptake across N levels while it may only be important for the internal use of N under N-limited conditions in maize. Given the complexity of breeding for plastic responses, an a priori model analysis is useful to identify which plastic traits to target for enhanced plant performance.

Dynamic Energy Budget model for E. coli growth in carbon and nitrogen limitation conditions

The simulations and predictions obtained from mathematical models of bioprocesses conducted by microorganisms are not overvalued. Mechanistic models are bringing a better process understanding and the possibility of simulating unmeasurable variables. The Dynamic Energy Budget (DEB) model is an energy balance that can be formulated for any living organism and can be classified as a structured model. In this study, the DEB model was used to describe E. coli growth in a batch reactor in carbon and nitrogen substrate limitation conditions. The DEB model provides a possibility to follow the changes in the microbes’ cells including their elemental composition and content of some important cell ingredients in different growth phases in substrate limitation conditions which makes it more informative compared to Monod’s model. The model can be used as an optimal choice between Monod-like models and flux-based approaches.Key points• The DEB model can be used to catch changes in elemental composition of E. coli• Bacteria batch culture growth phases can be explained by the DEB model• The DEB model is more informative compared to Monod’s based models

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

New Solutions in Single-Cell Protein Production from Methane: Construction of Glycogen-Deficient Mutants of Methylococcus capsulatus MIR

The biotechnology of converting methane to single-cell protein (SCP) implies using fast-growing thermotolerant aerobic methanotrophic bacteria. Among the latter, members of the genus Methylococcus received significant research attention and are used in operating commercial plants. Methylococcus capsulatus MIR is a recently discovered member of this genus with the potential to be used for the purpose of SCP production. Like other Methylococcus species, this bacterium stores carbon and energy in the form of glycogen, particularly when grown under nitrogen-limiting conditions. The genome of strain MIR encodes two glycogen synthases, GlgA1 and GlgA2, which are only moderately related to each other. To obtain glycogen-free cell biomass of this methanotroph, glycogen synthase mutants, ΔglgA1, ΔglgA2, and ΔglgA1ΔglgA2, were constructed. The mutant lacking both glycogen synthases exhibited a glycogen-deficient phenotype, whereas the intracellular glycogen content was not reduced in strains defective in either GlgA1 or GlgA2, thus suggesting functional redundancy of these enzymes. Inactivation of the glk gene encoding glucokinase also resulted in a sharp decrease in glycogen content and accumulation of free glucose in cells. Wild-type strain MIR and the mutant strain ΔglgA1ΔglgA2 were also grown in a bioreactor operated in batch and continuous modes. Cell biomass of ΔglgA1ΔglgA2 mutant obtained during batch cultivation displayed high protein content (71% of dry cell weight (DCW) compared to 54% DCW in wild-type strain) as well as a strong reduction in glycogen content (10.8 mg/g DCW compared to 187.5 mg/g DCW in wild-type strain). The difference in protein and glycogen contents in biomass of these strains produced during continuous cultivation was less pronounced, yet biomass characteristics relevant to SCP production were slightly better for ΔglgA1ΔglgA2 mutant. Genome analysis revealed the presence of glgA1-like genes in all methanotrophs of the Gammaproteobacteria and Verrucomicrobia, while only a very few methanotrophic representatives of the Alphaproteobacteria possessed these determinants of glycogen biosynthesis. The glgA2-like genes were present only in genomes of gammaproteobacterial methanotrophs with predominantly halo- and thermotolerant phenotypes. The role of glycogen in terms of energy reserve is discussed.

Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides

BackgroundThe oleaginous yeast Rhodotorula toruloides is a promising chassis organism for the biomanufacturing of value-added bioproducts. It can accumulate lipids at a high fraction of biomass. However, metabolic engineering efforts in this organism have progressed at a slower pace than those in more extensively studied yeasts. Few studies have investigated the lipid accumulation phenotype exhibited by R. toruloides under nitrogen limitation conditions. Consequently, there have been only a few studies exploiting the lipid metabolism for higher product titers.ResultsWe performed a multi-omic investigation of the lipid accumulation phenotype under nitrogen limitation. Specifically, we performed comparative transcriptomic and lipidomic analysis of the oleaginous yeast under nitrogen-sufficient and nitrogen deficient conditions. Clustering analysis of transcriptomic data was used to identify the growth phase where nitrogen-deficient cultures diverged from the baseline conditions. Independently, lipidomic data was used to identify that lipid fractions shifted from mostly phospholipids to mostly storage lipids under the nitrogen-deficient phenotype. Through an integrative lens of transcriptomic and lipidomic analysis, we discovered that R. toruloides undergoes lipid remodeling during nitrogen limitation, wherein the pool of phospholipids gets remodeled to mostly storage lipids. We identify specific mRNAs and pathways that are strongly correlated with an increase in lipid levels, thus identifying putative targets for engineering greater lipid accumulation in R. toruloides. One surprising pathway identified was related to inositol phosphate metabolism, suggesting further inquiry into its role in lipid accumulation.ConclusionsIntegrative analysis identified the specific biosynthetic pathways that are differentially regulated during lipid remodeling. This insight into the mechanisms of lipid accumulation can lead to the success of future metabolic engineering strategies for overproduction of oleochemicals.

Optimization of continuous astaxanthin production by Haematococcus pluvialis in nitrogen-limited photobioreactor

The industrial production of astaxanthin from Haematococcus pluvialis is mainly operated following a two-stage cultivation strategy in photobioreactors (PBRs) operating in batch mode. This study provides a strategy for optimizing continuous astaxanthin production in a one-stage approach, by optimizing both astaxanthin accumulation and biomass productivity of H. pluvialis under nitrogen-limited condition. To achieve the good balance in culture conditions to maintain growth under nitrogen-limited conditions, while triggering significant astaxanthin accumulation in reddish vegetative cells, the role of light absorption, as represented by the mean rate of photon absorption (MRPA), and nitrate concentration was especially investigated. For that purpose, H. pluvialis cultures were grown in a flat-panel PBR with constant dilution rate D of 0.015 h−1, different photon flux densities PFDs ranging from 75 to 750 μmolhν·m−2·s−1 and different nitrogen concentration levels in the feeding medium [NO3−] of 1, 3 and 8.8 mM. A parabolic relationship between MRPA and astaxanthin production rate was obtained. This indicated that astaxanthin synthesis was limited by the MRPA, opening further optimization by adjusting incident light intensity, nitrogen concentration in the feeding medium or culture dilution rate. After optimization of operating conditions, we demonstrated that a large quantity of astaxanthin can be produced in continuous mode, following then a one-stage strategy which may be advantageous for industrial use. A maximum astaxanthin productivity of 1.27 ± 0.03 g·m−2·d−1 with an astaxanthin accumulation of 3.9 ± 0.2 % DW was reached for the culture featuring MRPA of 9000 μmolhν·kgx−1·s−1. The process was also proved reversible, allowing to tailor the biomass composition and physiological state (from green to red cells enriched in astaxanthin) by adjusting operating conditions, opening perspectives from an optimized coupling with downstream processing steps.