Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell ...wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, ...deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (~75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.
Polysaccharides are a major carbon/energy-reservoir in microalgae, yet their relationship with another form of carbon/energy storage, triacylglycerol (TAG), is poorly understood. Here employing ...oleaginous microalga Nannochloropsis oceanica as a model, we probed the crosstalk between carbohydrate metabolism and TAG accumulation by tracking the temporal dynamics of lipidomes, monosaccharides and polysaccharides and transcripts of selected genes over 14days under nitrogen-depleted (N−) and nitrogen-replete (N+) conditions. Glucose, galactose and mannitol were the main monosaccharides in IMET1, and laminarin may be the storage polysaccharide that competes for carbon precursors with TAG. Transcriptional expression analysis revealed that the β-1,3-glucan degradation and pyruvate dehydrogenases pathways were the main regulatory components involved in driving carbon flow to TAG synthesis. Furthermore, temporal changes of lipidomes and transcripts of glycerolipid metabolism genes were indicative of possible conversion of membrane lipids to TAG, especially under an early stage of nitrogen deprivation conditions. A carbon partitioning model for N. oceanica was proposed, in which β-1,3-glucan metabolism, acetyl–CoA synthesis and membrane lipid turnover/degradation, in addition to de novo fatty acid synthesis, all contributed to TAG synthesis.
•Glucose, galactose and mannitol are the main mono-saccharides in IMET1.•Laminarin, the possible storage polysaccharide, competes for carbon precursors with TAG.•Beta-1,3-glucan degradation and PDH pathways drive carbon flow to TAG synthesis.•Membrane lipids are possibly converted to TAG at an early stage under N− conditions.•A model of photosynthetic carbon partitioning towards TAG was proposed.
Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this ...study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera
, and
, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m
/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m
/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.
In this study, a rational combination of 200 pre-selected Carbohydrate-Active enzymes (CAZymes) and sulfatases were tested, individually or combined, according to their ability to degrade Chlorella ...vulgaris cell wall to access its valuable nutritional compounds. The disruption of microalgae cell walls by a four-enzyme mixture (Mix) in comparison with the control, enabled to release up to 1.21 g/L of reducing sugars (p < 0.001), led to an eight-fold increase in oligosaccharides release (p < 0.001), and reduced the fluorescence intensity by 47% after staining with Calcofluor White (p < 0.001). The Mix treatment was successful in releasing proteins (p < 0.001), some MUFA (p < 0.05), and the beneficial 18:3n-3 fatty acid (p < 0.05). Even if no variation was detected for chlorophylls (p > 0.05), total carotenoids were increased in the supernatant (p < 0.05) from the Mix treatment, relative to the control. Taken together, these results indicate that this four-enzyme Mix displays an effective capacity to degrade C. vulgaris cell wall. Thus, these enzymes may constitute a good approach to improve the bioavailability of C. vulgaris nutrients for monogastric diets, in particular, and to facilitate the cost-effective use of microalgae by the feed industry, in general.
The ratio of carbon to nitrogen (C/N) in media plays a crucial role in the production of microbial carotenoids. However, the effects of a high C/N ratio on carotenoid production are ambiguous, and ...the mechanism of how C/N ratio affects astaxanthin accumulation in
X. dendrorhous
is unclear. In this study, the influence of C/N ratio on astaxanthin biosynthesis in
X. dendrorhous
at a fixed nitrogen concentration was investigated, and comparative proteomics were applied to address how C/N ratio affects cell growth and astaxanthin accumulation in
X. dendrorhous
. The results showed that cell growth and astaxanthin accumulation in
X. dendrorhous
were strongly related to the ratio of carbon to nitrogen with increasing C/N ratio in medium. However, the astaxanthin content per cell showed an inverse relationship, decreasing with an increasing C/N ratio. Differential proteomics showed the proteins with highest degree of change in expression under varying C/N ratios were mainly involved in carbohydrate metabolic pathways and carotenogenesis metabolism. In addition, several redox- and stress-associated proteins were up-regulated along with the carotenogenesis proteins, implying the environmental stress may affect metabolism and astaxanthin synthesis. A possible regulatory mechanism in response to glucose in
X. dendrorhous
is discussed.
Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell ...wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various
Chlorella
strains tested and also inhibited
Nannochloropsis
and
Nannochloris
strains.
Chlorella
is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing
N
-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated
Chlorella vulgaris
indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with ...better-prognosis disomy 3 (D3). We show that BAP1 loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-UM. Poor-prognosis M3-UM divide into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. We report change-of-function SRSF2 mutations. Within D3-UM, EIF1AX- and SRSF2/SF3B1-mutant tumors have distinct somatic copy number alterations and DNA methylation profiles, providing insight into the biology of these low- versus intermediate-risk clinical mutation subtypes.
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•Both D3 and M3-UM divide into molecularly distinct subsets with different outcomes•Poor-prognosis M3-UM are characterized by a global DNA methylation pattern•Poor-prognosis M3-UM subsets have distinct genomic, signaling, and immune profiles•EIF1AX and SRSF2/SF3B1 mutant D3-UM have different genomic/DNA methylation profiles
Robertson et al. analyze 80 uveal melanomas (UM) and divide poor-prognosis monosomy 3 UM into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. Somatic copy number changes and DNA methylation profiles separate better-prognosis disomy 3 UM into low or intermediate risk.
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