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DOE OSTI2021

Mechanisms and functions of membrane lipid remodeling in plants

Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This focused review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2002

Opportunities for nutritional amelioration of radiation-induced cellular damage

The closed environment and limited evasive capabilities inherent in space flight cause astronauts to be exposed to many potential harmful agents (chemical contaminants in the environment and cosmic radiation exposure). Current power systems used to achieve space flight are prohibitively expensive for supporting the weight requirements to fully shield astronauts from cosmic radiation. Therefore, radiation poses a major, currently unresolvable risk for astronauts, especially for long-duration space flights. The major detrimental radiation effects that are of primary concern for long-duration space flights are damage to the lens of the eye, damage to the immune system, damage to the central nervous system, and cancer. In addition to the direct damage to biological molecules in cells, radiation exposure induces oxidative damage. Many natural antioxidants, whether consumed before or after radiation exposure, are able to confer some level of radioprotection. In addition to achieving beneficial effects from long-known antioxidants such as vitamins E and C and folic acid, some protection is conferred by several recently discovered antioxidant molecules, such as flavonoids, epigallocatechin, and other polyphenols. Somewhat counterintuitive is the protection provided by diets containing elevated levels of omega-3 polyunsaturated fatty acids, considering they are thought to be prone to peroxidation. Even with the information we have at our disposal, it will be difficult to predict the types of dietary modifications that can best reduce the risk of radiation exposure to astronauts, those living on Earth, or those enduring diagnostic or therapeutic radiation exposure. Much more work must be done in humans, whether on Earth or, preferably, in space, before we are able to make concrete recommendations.

Review↗

DOE OSTI2024

Arabidopsis trigalactosyldiacylglycerol1 mutants reveal a critical role for phosphtidylcholine remodeling in lipid homeostasis

Lipid remodeling plays a critical role in plant response to abiotic stress and metabolic perturbations. Key steps in this process involve modifications of phosphatidylcholine (PC) acyl chains mediated by lysophosphatidylcholine: acyl-CoA acyltransferases (LPCATs) and phosphatidylcholine: diacylglycerol cholinephosphotransferase (ROD1). To assess their importance in lipid homeostasis, we took advantage of the trigalactosyldiacylglycerol1 (tgd1) mutant that exhibits marked increases in fatty acid synthesis and fatty acid flux through PC due to a block in inter-organelle lipid trafficking. Here, we showed that the increased fatty acid synthesis in tgd1 is due to posttranslational activation of the plastidic acetyl-coenzyme A carboxylase. Genetic analysis showed that knockout of LPCAT1 and 2 resulted in a lethal phenotype in tgd1. In addition, plants homozygous for lpcat2 and heterozygous for lpcat1 in the tgd1 background showed reduced levels of PC and triacylglycerols (TAG) and alterations in their fatty acid profiles. We further showed that disruption of ROD1 in tgd1 resulted in changes in fatty acid composition of PC and TAG, decreased leaf TAG content and reduced seedling growth. Combined, our results reveal a critical role of LPCATs and ROD1 in maintaining cellular lipid homeostasis under conditions, in which fatty acid production largely exceeds the cellular demand for membrane lipid synthesis.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2001

[Modifications in myocardial energy metabolism in diabetic patients]]

The capacity of cardiac myocyte to regulate ATP production to face any change in energy demand is a major determinant of cardiac function. Because FA is the main heart fuel (although the most expensive one in oxygen, and prompt to induce deleterious effects), this process is based on a balanced fatty acid (FA) metabolism. Several pathological situations are associated with an accumulation of FA or derivatives, or with an excessive b-oxidation. The diabetic cardiomyocyte is characterised by an over consumption of FA. The control of the FA/glucose balance clearly appears as a new strategy for cytoprotection, particularly in diabetes and requires a reduced FA contribution to ATP production. Cardiac myocytes can control FA mitochondrial entry, but display weak ability to control FA uptake, thus the fate of non beta-oxidized FA appear as a new impairment for the cell. Both the trigger and the regulation of cardiac contraction result from membrane activity, and the other major FA function in the myocardium is their role in membrane homeostasis, through the phospholipid synthesis and remodeling pathways. Sudden death, hypercatecholaminemia, diabetes and heart failure have been associated with an altered PUFA content in cardiac membranes. Experimental data suggest that the 2 metabolic pathways involved in membrane homeostasis may represent therapeutic targets for cytoprotection. The drugs that increase cardiac phospholipid turnover (trimetazidine, ranolazine,...) display anti-ischemic non hemodynamic effect. This effect is based on a redirection of FA utilization towards phospholipid synthesis, which decrease their availability for energy production. A nutritional approach gave also promising results. Besides its anti-arrhythmic effect, the dietary docosahexaenoic acid is able to reduce FA energy consumption and hence oxygen demand. The cardiac metabolic pathways involving FA should be considered as a whole, precariously balanced. The diabetic heart being characterised by a different metabolic "status" with similarities to that of myocardium in coronary disease. Diabetes and other chronic cardiac diseases share common FA metabolism disorders leading to an altered energy balance, a decrease in long chain polyunsaturated Fas, and altered FA profiles in cardiac membranes. These disturbances, however, do not represent independent therapeutic targets, and should be considered as a whole.

Myocardium/metabolism↗

DOE OSTI2024

Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H 2 :CO feedstock ratios for enhancing carbon capture efficiency

Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO 2 ) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H 2 :CO to maximize CO 2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO 2 , we established controlled chemostats that facilitated a novel and high (11:1) H 2 :CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H 2 :CO condition where ~ 50% of the carbon in ethanol is derived from CO 2 . Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H 2 :CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H 2 :CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

09 BIOMASS FUELS↗

DOE OSTI2025

Data for Metabolic Engineering of β-Oxidation to Leverage Thioesterases for Production of 2-Heptanone, 2-Nonanone, and 2-Undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified 15 homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Metabolic Engineering↗

DOE OSTI2020

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. Here, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We further tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Finally, using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

2-Heptanone↗

DOE OSTI2020

The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

09 BIOMASS FUELS↗

DOE OSTI2021

Modular polyketide synthase contains two reaction chambers that operate asynchronously

Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo–electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co‐expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Nitrogen limitation causes a seismic shift in redox state and phosphorylation of proteins implicated in carbon flux and lipidome remodeling in Rhodotorula toruloides

Background: Oleaginous yeast are prodigious producers of oleochemicals, offering alternative and secure sources for applications in foodstuff, skincare, biofuels, and bioplastics. Nitrogen starvation is the primary strategy used to induce oil accumulation in oleaginous yeast as part of a global stress response. While research has demonstrated that post-translational modifications (PTMs), including phosphorylation and protein cysteine thiol oxidation (redox PTMs), are involved in signaling pathways that regulate stress responses in metazoa and algae, their role in oleaginous yeast remain understudied and unexplored. Results: Towards linking the yeast oleaginous phenotype to protein function, we integrated lipidomics, redox proteomics, and phosphoproteomics to investigate Rhodotorula toruloides under nitrogen-rich and starved conditions over time. Our lipidomics results unearthed interactions involving sphingolipids and cardiolipins with ER stress and mitophagy. Our redox and phosphoproteomics data highlighted the roles of the AMPK, TOR, and calcium signaling pathways in regulation of lipogenesis, autophagy, and oxidative stress response. As a first, we also demonstrated that lipogenic enzymes including fatty acid synthase are modified as a consequence of shifts in cellular redox states due to nutrient availability. Conclusions: We conclude that lipid accumulation is largely a consequence of carbon rerouting and autophagy governed by changes to PTMs, and not increases in the abundance of enzymes involved in central carbon metabolism and fatty acid biosynthesis. Our systems-level approach sets the stage for acquiring multidimensional data sets for protein structural modeling and predicting the functional relevance of PTMs using Artificial Intelligence/Machine Learning (AI/ML). Coupled to those bioinformatics approaches, the putative PTM switches that we delineate will enable advanced metabolic engineering strategies to decouple lipid accumulation from nitrogen limitation.

Lipid Signalling↗

DOE OSTI2026

Polyketide synthase-based controlled synthesis of polycyclopropanated fuel molecules

Reducing carbon emissions from aviation and long-distance transportation sectors requires the development of sustainable biofuels with suitable energy density, freezing point, and other physical properties. We previously demonstrated biological production of high energy polycyclopropanated fatty acids (POP-FAs, class I) using an iterative polyketide synthase (iPKS) pathway in a Streptomyces host. Here, we used a computational model of fuel properties to identify chain length and cyclopropanation control as critical steps to engineer this iPKS for biofuel applications. We next explored the natural diversity of POP biosynthesis by investigating homologous pathways. Then, by in vivo gene exchange, we determined cyclopropanase (CP) catalysis to be key for POP-FA engineering. Leveraging both natural and engineered pathway product diversity, we demonstrate targeted production of improved POP-FAs, namely shortened POP-FAs with predicted superior freezing point properties for aviation, as well as fully cyclopropane-saturated POP-FAs which should have superior energy-density. These precise and controllable modifications to POP-FA structure open the door for bioproduction of designer POP fuels.

Yin, Kevin↗

DOE OSTI2022

Data for Metabolic Engineering of Rhodotorula toruloides IFO0880 Improves C16 and C18 Fatty Alcohol Production from Synthetic Media

The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Conversion↗

DOE OSTI2022

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

Abstract Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets ( ACL1 and ACC1 , improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1 ) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1 Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1 , or the deletion of DGA1 combined with overexpression of ACC1 and ACL1 . Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Lepidopteran mevalonate pathway optimization in Escherichia coli efficiently produces isoprenol analogs for next-generation biofuels

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, in this study, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol’s seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Inhibition of DNA methylation in P. soloecismus alters algae productivity

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism’s molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (~1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 μM 5-aza-2'-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (~66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

5-aza-2'-deoxycytidine↗

DOE OSTI2025

Author Correction: Genome-guided isolation of the hyperthermophilic aerobe Fervidibacter sacchari reveals conserved polysaccharide metabolism in the Armatimonadota

Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-024-53784-3, published online 4 November 2024 In the version of this article initially published, Table 1 did not include the properties of the taxa being proposed or refer directly to another location in the main manuscript describing the properties. As such, the original manuscript did not comply with Rule 27 (2)(c) of the ICNP. Also, Table 1 listed the order Fervidibacterales as the nomenclatural type for the class Fervidibacteria, which violates latest emended version of Rule 15 stating that the nomenclatural type for a class must be a genus. Below we provide a modification of Table 1 containing protologues with these errors corrected. We have also changed the order of the taxa in the table to meet the most common ordering. (Table presented.) Taxon names proposed under the ICNP Proposed taxon Etymology Description Genus Fervidibacter Fer.vi.di.bac’ter. L. masc. adj. fervidus, hot, steaming; N.L. masc. n. bacter, a rod; N.L. masc. n. Fervidibacter, a hot rod Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic, with high-affinity and/or low-affinity terminal oxidases present in the genomes. The oxidative pentose phosphate pathway and the tricarboxylic acid cycle are complete in genomes belonging to the genus. Gram-stain-negative and diderm cell envelope structure. Ovoid- to rod-shaped morphology. Spores are not formed. The genus is a distinct phylogenetic lineage in the family Fervidibacteraceae, the order Fervidibacterales, and the class Fervidibacteria in the phylum Armatimonadota. The type species is Fervidibacter sacchariT. Species Fervidibacter sacchari sac’cha.ri. N.L. gen. n. sacchari, of sugar Hyperthermophilic, microaerophilic, facultatively anaerobic, and grows chemoheterotrophically on monosaccharides and polysaccharides. Cells are ovoid- to rod-shaped, Gram-stain negative, and are 0.9–1.3 µm in width and 1.6–3.6 µm in length. Grows between 65 and 87.5 °C and an optimum temperature of 80 °C, and a pH range of 6.5–8.6 with an optimum pH of 7.5. Grows at an optimum O2 concentration of 5–10%. Grows on D-arabinose, D-galactose, D-glucose, D-rhamnose, D-ribose, D-xylose, chondroitin sulfate, colloidal chitin, galactan, gellan gum, guar gum, karaya gum, locust bean gum, xantham gum, xyloglucan, β-glucan, glycogen, starch, AFEX-pretreated corn stover, miscanthus, sugarcane bagasse, acetate and casamino acids. Grows weakly on xyloglucan under fermentation conditions. The major fatty acids (>10%) are C16:0, C18:0 and/or cyclo-C17:0, and iso-C16:0. The major respiratory quinones (>10%) are MK-8 and MK-9. The isolate and genomes of the species have been recovered from geothermal springs in the Great Basin, Nevada, USA. GC content of genomes range between 51–52%. Subunits for both the high-affinity and low-affinity terminal oxidases are encoded in the genomes. Genomes also encode a Group 3d [NiFe] hydrogenase, which produces hydrogen as an electron sink for NAD+ regeneration. The type strain PD1T (= JCM 39283T = DSM 113467T) was isolated from Great Boiling Spring in Nevada, USA. Family Fervidibacteraceae Fer.vi.di.bac.te.ra’ce.ae. N.L. masc. n. Fervidibacter type genus of the family; L. suff. -aceae ending to denote a family; N.L. fem. pl. n. Fervidibacteraceae the family of the genus Fervidibacter Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic, with high-affinity and/or low-affinity terminal oxidases present in the genomes. The oxidative pentose phosphate pathway and the tricarboxylic acid cycle are complete in genomes belonging to the family. The family is a distinct phylogenetic lineage in the order Fervidibacterales and the class Fervidibacteria in the phylum Armatimonadota. The type genus is Fervidibacter. Order Fervidibacterales Fer.vi.di.bac.te.ra’les. N.L. masc. n. Fervidibacter type genus of the order; L. suff. -ales ending to denote an order; N.L. fem. pl. n. Fervidibacterales the order of the genus Fervidibacter Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic or strictly anaerobic. Phylogenomic placement of this lineage within the Fervidibacteria and relative evolutionary divergence supports delineation of this lineage as an order within the class Fervidibacteria and phylum Armatimonadota. The type genus is Fervidibacter. Class Fervidibacteria Fer.vi.di.bac.te’ri.a. N.L. masc. n. Fervidibacter type genus of the type order of the class; L. suff. -ia ending to denote a class; N.L. neut. pl. n. Fervidibacteria the class of the order Fervidibacterales Thermophilic or hyperthermophilic inhabitants of freshwater thermal environments. All members are likely polysaccharide-degrading chemoheterotrophs with numerous carbohydrate-active enzymes encoded in their genomes. Aerobic or strictly anaerobic. Phylogenomic placement of this lineage within the Armatimonadota and relative evolutionary divergence supports delineation of this lineage as a class within the Armatimonadota. The type genus is Fervidibacter. The error has not been corrected in the PDF or HTML versions of the Article.

Nou, Nancy O↗