Lipid degradation processes are important in microalgae because survival and growth of microalgal cells under fluctuating environmental conditions require permanent remodeling or turnover of membrane lipids as well as rapid mobilization of storage lipids. Lipid catabolism comprises two major spatially and temporarily separated steps, namely lipolysis, which releases fatty acids and head groups and is catalyzed by lipases at membranes or lipid droplets, and degradation of fatty acids to acetyl-CoA, which occurs in peroxisomes through the β-oxidation pathway in green microalgae, and can sometimes occur in mitochondria in some other algal species. Here we review the current knowledge on the enzymes and regulatory proteins involved in lipolysis and peroxisomal β-oxidation and highlight gaps in our understanding of lipid degradation pathways in microalgae. Metabolic use of acetyl-CoA products via glyoxylate cycle and gluconeogenesis is also reviewed. We then present the implication of various cellular processes such as vesicle trafficking, cell cycle and autophagy on lipid turnover. Finally, physiological roles and the manipulation of lipid catabolism for biotechnological applications in microalgae are discussed.
Nitrogen (N) starvation-induced triacylglycerol (TAG) synthesis, and its complex relationship with starch metabolism in algal cells, has been intensively studied; however, few studies have examined the interaction between amino acid metabolism and TAG biosynthesis. Here, via a forward genetic screen for TAG homeostasis, we isolated a Chlamydomonas (Chlamydomonas reinhardtii) mutant (bkdE1a) that is deficient in the E1a subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. Metabolomics analysis revealed a defect in the catabolism of branched-chain amino acids in bkdE1a. Furthermore, this mutant accumulated 30% less TAG than the parental strain during N starvation and was compromised in TAG remobilization upon N resupply. Intriguingly, the rate of mitochondrial respiration was 20% to 35% lower in bkdE1a compared with the parental strains. Three additional knockout mutants of the other components of the BCKDH complex exhibited phenotypes similar to that of bkdE1a. Transcriptional responses of BCKDH to different N status were consistent with its role in TAG homeostasis. Collectively, these results indicate that branched-chain amino acid catabolism contributes to TAG metabolism by providing carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae.
Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, longterm growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.KEYWORDS clostridia, evolution, genomics F ermentation of lignocellulosic biomass by bacteria like Clostridium phytofermentans is central to the function of soil, aquatic, and intestinal microbiomes. In addition, industrial fermentation of lignocellulosic biomass into fuels and chemicals could contribute significantly to global energy needs without impacting food production (1). Plant biomass is primarily composed of a macromolecular network of polysaccharides linked with lignin, a polymer of phenylpropanoid subunits with aromatic rings of various degrees of methoxylation (2). Thus, when microorganisms hydrolyze lignocel-
Ocean acidification increases pCO2 and decreases pH of seawater and its impact on marine organisms has emerged as a key research focus. In addition to directly measured variables such as growth or calcification rate, stable isotopic tracers such as carbon isotopes have also been used to more completely understand the physiological processes contributing to the response of organisms to ocean acidification. To simulate ocean acidification in laboratory cultures, direct bubbling of seawater with CO2 has been a preferred method because it adjusts pCO2 and pH without altering total alkalinity. Unfortunately, the carbon isotope equilibrium between seawater and CO2 gas has been largely ignored so far. Frequently, the dissolved inorganic carbon (DIC) in the initial seawater culture has a distinct 13C/12C ratio which is far from the equilibrium expected with the isotopic composition of the bubbled CO2. To evaluate the consequences of this type of experiment for isotopic work, we measured the carbon isotope evolutions in two chemostats during CO2 bubbling and composed a numerical model to simulate this process. The isotopic model can predict well the carbon isotope ratio of dissolved inorganic carbon evolutions during bubbling. With help of this model, the carbon isotope evolution during a batch and continuous culture can be traced dynamically improving the accuracy of fractionation results from laboratory culture. Our simulations show that if not properly accounted for in experimental or sampling design, many typical culture configurations involving CO2 bubbling can lead to large errors in estimated carbon isotope fractionation between seawater and biomass or biominerals, consequently affecting interpretations and hampering comparisons among different experiments. Therefore, we describe the best practices on future studies working with isotope fingerprinting in the ocean acidification background.
<p>Carbonate clumped isotope thermometry, based on the temperature-dependence of clumping of <sup>13</sup>C and <sup>18</sup>O in the carbonate molecule (&#916;<sub>47</sub>) is a promising tool for paleoclimate reconstruction. In the last few years many discrepancies among &#916;<sub>47</sub>-temperature calibrations have been resolved across the range of relevant paleoclimate temperatures (Meinicke et al., 2020; Anderson et al., 2021). However, there might be other environmental effects on biogenic carbonates from parameters such as the pCO<sub>2</sub> and growth rates of the organisms that are still unresolved. We provide a new assessment of the temperature dependence of clumped isotopes in laboratory grown biogenic carbonate at well-constrained experimental conditions, with results from three species of coccolithophores across a growth temperature range of 6-27&#176;C. The three cultured species cover a range of growth rates, growth conditions and species-specific carbon and oxygen vital effects. Because variations in pCO<sub>2</sub> and media carbon chemistry are known to trigger vital effects in carbon and oxygen isotopes in coccoliths, we decoupled the temperature solubility effect on CO<sub>2</sub> by manipulating culture CO<sub>2</sub> independently. Three pCO<sub>2</sub> levels at reduced, present day and elevated levels; 200, 400 and 1000 ppm respectively, were kept constant for at least two different temperatures through a continuous culturing set-up. Our new multi-parameter comparison, using updated standardization approaches, provides a critical test of previous conclusions (Katz et al., 2017) that coccolithophore clumped isotopes show little to no vital effects and are close to abiotic equilibrium. Thus, we have performed the first calibration of coccolith calcite and clumped isotopes combining different temperature and pCO<sub>2</sub> conditions.</p> <p>References:</p> <p>Anderson, N. T., J. R. Kelson, S. Kele, M. Da&#235;ron, M. Bonifacie, J. Horita, T. J. Mackey, et al. 2021. "A Unified Clumped Isotope Thermometer Calibration (0.5&#8211;1,100&#176;C) Using Carbonate&#8208;Based Standardization." <em>Geophysical Research Letters</em> 48 (7).</p> <p>Katz, A., M. Bonifacie, M. Hermoso, P. Cartigny, D. Calmels. 2017. &#8220;Laboratory-grown coccoliths exhibit no vital effect in clumped isotope (&#916;47) composition on a range of geologically relevant temperatures.&#8221; <em>Geochimica et Cosmochimica Acta </em>208: 335-353.</p> <p>Meinicke, N., S.L. Ho, B. Hannisdal, D. N&#252;rnberg, A. Tripati, R. Schiebel, and A.N. Meckler. 2020. "A robust calibration of the clumped isotopes to temperature relationship for foraminifers." <em>Geochimica et Cosmochimica Acta</em> 270: 160-183.</p>
Ocean acidification increases pCO2 and decreases pH of seawater and its impact on marine organisms has emerged as a key research focus. In addition to directly measured variables such as growth or calcification rate, stable isotopic tracers such as carbon isotopes have also been used to more completely understand the physiological processes contributing to the response of organisms to ocean acidification. To simulate ocean acidification in laboratory cultures, direct bubbling of seawater with CO2 has been a preferred method because it adjusts pCO2 and pH without altering total alkalinity. Unfortunately, the carbon isotope equilibrium between seawater and CO2 gas has been largely ignored so far. Frequently, the dissolved inorganic carbon (DIC) in the initial seawater culture has a distinct 13C/12C ratio which is far from the equilibrium expected with the isotopic composition of the bubbled CO2. To evaluate the consequences of this type of experiment for isotopic work, we measured the carbon isotope evolutions in two chemostats during CO2 bubbling and composed a numerical model to simulate this process. The isotopic model can predict well the carbon isotope ratio of dissolved inorganic carbon evolutions during bubbling. With help of this model, the carbon isotope evolution during a batch and continuous culture can be traced dynamically improving the accuracy of fractionation results from laboratory culture. Our simulations show that, if not properly accounted for in experimental or sampling design, many typical culture configurations involving CO2 bubbling can lead to large errors in estimated carbon isotope fractionation between seawater and biomass or biominerals, consequently affecting interpretations and hampering comparisons among different experiments. Therefore, we describe the best practices on future studies working with isotope fingerprinting in the ocean acidification background.
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