Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Differential Cellular Isotope Effects of Deuterium on Photosynthetic Metabolism of Carbon in Chlorella ellipsoidea

Isotope effects of deuterium on photosynthetic metabolism ofcarbon in Chlorella ellipsoidea were investigated. Photosyntheticfixation of ¹⁴C in D₂O was about a half of that in H₂O. Eachstep in the photosynthetic metabolism of carbon was affecteddifferently by D₂O in the medium and constitutive D. (Received June 15, 1989; Accepted October 23, 1989)     

Studies on frost hardiness in Chlorella ellipsoidea I. Development of frost hardiness of Chlorella ellipsoidea in synchronous culture

Cells of Chlorella ellipsoidea Gerneck (IAM C-27) were synchronouslygrown under a 28-hr light-14-hr dark regime at 25°C. Thealgal cells at different stages during the cell cycle were hardenedat 3°C for 48 hr. The survival rate of hardened cells wasmaximum (70%) at the L₂ stage(ripening phase) in the life cycle.The average cell volume of L₂ cells increased during hardening,but the process of nuclear division scarcely advanced. The hardinessof L₂ cells increasedwith prolongation of hardening time upto 48 hr. Their viability decreased upon increasing the ratof cooling and lowering the final freezing temperature. Butthe hardened cells, which had been prefrozen stepwise, showeda survival rate above 50% even at –196°C when thawedrapidlyin a bath at 25°C. Although L₂ cells were somewhathardened in the dark, illumination was the more effective whenused with bubbling gas. Under illumination, bubbling of 1% CO₂-airincreased the hardiness more than CO₂-free air, but in the dark,this relation was reversed. The hardiness was lowest with nitrogengas bubbling under both conditions. (Received December 3, 1975; )     

Two new sterols from Chlorella ellipsoidea

Eight sterols were observed in Chlorella ellipsoidea and the four major components were identified as ergosterol, 5α-ergost-7-en-3β-ol, 22-trans-ergosta-5,8(9),22-trien-3β-ol and ergosta-5,8(9)-dien-3β-ol. This is the first report of the latter two sterols from green plants.     

Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea

The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 μg/g triparanol. Triparanol appears to inhibit the removal of 14α-methyl group, the second alkylation at C-24, Δ⁷-reductase, and Δ⁸ → Δ⁷-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals.     

Action Spectra of Photosynthetic Activities of Chlorella ellipsoidea

• 1) Suspensions of Chlorella show an even stronger light scattering than suspensions of chloroplasts of spinach. The bands of absorption are thus broadened and, at higher concentrations, moved to lower wave-lengths. The intensity of the photosynthesis closely follows the curves of light scattering, a fact partly explaining the high efficiency of green light. Calculated per unit thermoelectrically measured incident energy the action spectrum shows bands at 660–670 nm and c. 500 nm and a comparatively high level of the whole region 500–560 nm.
• 2) Flash experiments show the existence of a steady state carotene/xanthophyll that is moved to reduction (c/x > 1) in blue and green light and to oxidation (c/x < 1) in red light. All experiments point to the existence of two light reactions, the first one involving excitation of carotenoids, with ferredoxin-TPN as acceptor, the second one involving excitation of chlorophyll, with the cytochrome system of the chloroplasts acting as donors of electrons and thus completing an energy converting circulation between pigments and enzyme systems.
• 3) The operation of combined light reactions appears also from experiments with simultaneous or succedaneous illumination with monochromatic light of different wave-lengths. Some effects may be explained from separate excitations of carotenoids and chlorophylls, others may depend on still unknown photic reactions.
• 4) The action spectrum in ultrared shows a positive band at c. 900 nm but no or only very small effects in the region 950–1400 nm. Ultrared radiation has on the other hand an enhancing effect on the light excitation in the visible spectrum. A combination of infrared and visible radiation shows a roughly linear relation between incident energy and photosynthetic effect.
• 5) All experiments were performed in the region of linear relation between intensity of incident light and O₂-production. Induced effects of combined monochromatic regions show a very rapid initial change in the steady states that in one or two minutes simmers down to a balanced state of continued photosynthesis. No change was observed in the total quantity of the pigments.

     

Superoxide dismutase and chilling injury in Chlorella ellipsoidea

The relationship between superoxide dismutase (SOD) and chilling injury was examined in chilling-sensitive and chilling-resistant strains of Chlorella ellipsoidea. The sensitive strain contained less SOD than the resistant strain. Moreover, all of the SOD in the sensitive strain was the H2O2-sensitive, iron-containing SOD, whereas most of the SOD in the resistant strain was the H2O2-resistant, manganese-containing SOD. Illumination further enhanced the disparity in SOD content between the sensitive and resistant strains since the SOD in the former declined during illumination, whereas the SOD in the latter strain did not. It was possible to elevate the SOD content of the sensitive strain and to increase the proportion of MnSOD by prior growth in the presence of 50 microM paraquat. The SOD content of the cultures after 5 h of illumination at 4 degrees C fell in the order sensitive strain less than paraquat-induced sensitive strain less than resistant strain. The resistance of these cultures to chilling injury was related to SOD content. This was the case whether resistance was assessed in terms of growth rate after chilling, bleaching of chlorophyll during chilling, or loss of viability during chilling. It thus appears likely that O2- is an agent of chilling injury.     

Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea

A circular DNA molecule was isolated from chloroplasts of Chorella ellipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, HpaII, MspI, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA.     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Alterations in the Heat Response of Chlorella ellipsoidea by Deuteration

For the elucidation of the isotope effect on cell functionsof deuterium (D) incorporated into cell constituents, alterationsin the heat response of D-exchanged Chlorella ellipsoidea (D-Chlorella)were investigated. D-Chlorella cells obtained by culture inmedium that contained 60 mol% D₂O were assayed for their responseto heat in H₂O medium to rule out the solvent isotope effectof D₂O. Upon heating at 41–45?C, the heat sensitivityof D-Chlorella was greater than that of ordinary (H-Chlorella)cells; at 43?C, the heat sensitivity of D-Chlorella was 1.5–1.6times higher than that of H-Chlorella. For the induction ofresistance to heating, preheating of the cells at a lower temperaturethan that used for heat treatment was effective in the caseof both D- and H-Chlorella. However, the optimum temperaturefor preheating of D-Chlorella (34?C) was lower than for H-Chlorella(36–37?C). With preheating at 34?C, heat-shock proteins(HSPs), in particular proteins of 62 and 79 kDa, were inducedsimilarly in both types of cell. However, the gel-electrophoreticpatterns of HSPs induced at 37?C were differed somewhat betweenD- and H-Chlorella. These results suggest that the responseof cells to heat, in particular the induction of resistanceto heating and the synthesis of HSPs, was altered by deuterationof cell constituents. (Received June 11, 1990; Accepted November 24, 1990)     

Optimization of pressurized liquid extraction of zeaxanthin from Chlorella ellipsoidea

The total zeaxanthin level in Chlorella ellipsoidea, a green microalga, was more than nine times that of red pepper, a plant source of zeaxanthin. Additionally, the zeaxanthin in C. ellipsoidea consisted of the free form, while those in other plants exist as zeaxanthin mono- and diesters. Pressurized liquid extraction (PLE) was used to extract zeaxanthin from C. ellipsoidea. Both the extraction temperature and extraction time, the two main factors in PLE, were optimized with a central composite design to obtain the highest extraction efficiency of zeaxanthin. Hexane, ethanol, and isopropanol were used as PLE extraction solvents. Ethanol extracted zeaxanthin most efficiently from C. ellipsoidea. Temperature was the parameter with the strongest influence on the extraction of zeaxanthin. The optimum extraction temperature and time for zeaxanthin were 115.4°C and 23.3?min, respectively. The maximum predicted value of 4.28?mg?g^?1 agreed with the experimental value of 4.26?mg?g^?1, supporting the quality of the fitted model. These results indicate that PLE using ethanol may be a useful method for extracting zeaxanthin from C. ellipsoidea.     

Accumulation of Free Fatty Acids during Hardening of Chlorella ellipsoidea

Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown, and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3°C for 48 hours. A nonpolar lipid which increased greatly during hardening was analyzed by gas-liquid chromatography. Palmitic, oleic, linoleic, and linolenic acids were the main components of the lipid. Electron micrographs revealed the appearance of lipid bodies in hardened cells. When formation of free fatty acids and lipid bodies was inhibited with cycloheximide, oligomycin, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the development of a high level of hardiness was always inhibited. However, the converse results were not always realized. Cells hardened in the dark in the absence of glucose developed a measurable hardiness in spite of their failure to form free fatty acids. The appearance of lipid bodies was invariably accompanied by the formation of the fatty acids. In pulse-labeling with [¹⁴C]NaHCO₃ for 4 minutes at zero time and at the 12th hour of hardening, initial incorporation rates of ¹⁴C into total lipids of whole cells and the cellular membrane fraction were significantly higher than that into free fatty acids. These results suggest that, although fatty acids are inserted into membrane lipids during hardening, the accumulation of free fatty acids and the appearance of lipid bodies per se are not involved in the development of frost hardiness.     

利用椭圆小球藻硝酸还原酶缺失突变体为生物反应器表达兔防御素NP-1蛋白

利用转基因小球藻为生物反应器生产兔防御素NP-1蛋白具有重要的应用价值。本研究利用椭圆小球藻(Chlorella ellipsoidea)硝酸还原酶(nitrate reductase)缺失突变体为受体, 构建了包含NPTII基因和硝酸还原酶基因两个筛选标记的兔防御素蛋白表达载体, 采用电激法将目的基因转入椭圆小球藻硝酸还原酶缺失突变体nrm-4, 获得了正确表达防御素蛋白的转基因藻, 从而表明通过硝酸还原酶作为筛选标记基因并结合硝酸还原酶缺失突变体可作为较好的小球藻生物反应器生产模式。     

以异养细胞作为种子的椭圆小球藻产油脂光自养培养优化

异养细胞种子/光自养培养方法是一种可异养培养的能源微藻培养的有效方法,但已有文献尚未从工艺优化角度考察其发展潜力。为了获得较高细胞密度的用于光自养培养的种子和提高光自养培养的细胞密度与油脂产率,对异养细胞种子/光自养培养的培养基和培养条件进行了优化。结果表明,采用优化后的培养基,椭圆小球藻在摇瓶中异养培养的最高藻细胞密度可达11.04 g/L,比在初始培养基条件下提高了28.0%,在5 L发酵罐中异养培养的藻细胞密度达到73.89 g/L;在2 L柱式光生物反应器中光自养培养的藻细胞密度、油脂含量和油脂产率分别达1.62 g/L、36.34%和6.1 mg/(L·h),油脂成分主要为含C16-C18碳链的脂肪酸,是制备生物柴油的理想原料。经过优化,异养细胞种子/光自养培养这一方法能够显著地提高椭圆小球藻产油脂的能力,这进一步表明异养细胞种子/光自养培养方法有望成为可异养的能源微藻的高效培养方式。     

The metabolism of cycloartenol,lanosterol, 24-methylenecholesterol and fucosterol in Chlorella ellipsoidea

Lanosterol and cycloartenol labelled with tritium at C-2, and 24-methylenecholesterol and fucosterol labelled with tritium at C-2 and C-4 were fed to actively growing cultures of Chlorella ellipsoidea. Lanosterol and cycloartenol were converted to each of the five desmethyl sterols of C. ellipsoidea. Lanosterol was more efficiently incorporated than cycloartenol.Although there was some evidence for the reduction of the 24-methylene group, it was apparent that 24-methylene-cholesterol was converted primarily to the C₂₉ sterols, clionasterol and poriferasterol. Labelled fucosterol was reduced at the 24(28) double bond, producing clionasterol.     

Detection and first characterization of a cell-wall lytic activity in Chlorella ellipsoidea C-27

Cell wall lytic activity was detected in Chlorella ellipsoidea Gernick IAM C-27 using the [¹⁴C]-labeled cell wall as substrate. The highest activity was obtained at pH 8. and the solubilized product was a polysaccharide of high molecular weight. The lytic activity appeared to be a protease and did not hydrolyse glycosidic bonds of the cell wall polysaccharides. The activity probably solubilizes the cell wall by cleaving the peptide bonds that interconnect the polysaccharides of the cell wall.