Neutral lipid production in <Emphasis Type="Italic">Dunaliella salina</Emphasis> during osmotic stress and adaptation

The salt-tolerant green microalga Dunaliella salina can survive both hyper- and hypo-osmotic shock. Upon osmotic shock, the cells transiently and rapidly decreased or increased in size within minutes and slowly over hours acquired their original cell size and volume. Cell size distribution differs significantly in the cultures grown in the salinity range from 1.5 to 15 % NaCl. By using Nile Red fluorescence to detect neutral lipids, it became clear that only hyper-osmotic shock on cells induced transient neutral lipid appearance in D. salina, while those transferred from 9 to 15 % NaCl stimulated the most neutral lipid accumulation. These cells grew well in 9 % NaCl, but they cannot recover a shift to 15 % NaCl and cell division is accordingly slowed down. The transient appearance of neutral lipid could be dependent on the inhibition of cell division experiencing the NaCl shift. Moreover, the effect of nutrient limitation slows down cell division and photosynthesis as a secondary result, which triggers the cells to accumulate neutral storage lipids when they entered the stationary phase, which is seen in all the batch cultures of D. salina grown in the salinity range of 3–15 %. The changes in salt concentration did not significantly influence the overall fatty acid composition in D. salina cells. Although there shows both increased amounts of total lipids and neutral lipids in the cells grown in salinity higher than 9 % NaCl, lipid productivity is however compromised by the slower cell growth rate and lower cell density under this condition.     

Cardiolipin deficiency causes triacylglycerol accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In yeast, the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE) occurs mainly in mitochondria. CL and PE have overlapping functions, and they are required for mitochondrial function. PE is physiologically linked with triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, involving an acyl-CoA-independent pathway through the phospholipid:diacylglycerol acyltransferase activity of the Lro1 protein. There is no report on the physiological link between CL and TAG metabolism. Here we report a metabolic link between CL and TAG accumulation in the S. cerevisiae. Our data indicated that CL deficiency causes TAG accumulation, involving an acyl-CoA-dependent pathway through the diacylglycerol acyltransferase activity of the Dga1 protein with no changes in the TAG molecular species. The DGA1 gene deletion from the CL-deficient strains reduced the TAG levels. Data from in vitro and in vivo analyses showed that CL did not affect the enzymatic activity of Dga1. Our data also showed that CL deficiency leads to the up-regulation of acetyl-CoA synthetase genes (ACS1 and ACS2) of the cytosolic pyruvate dehydrogenase bypass pathway. This study establishes a physiological link between CL and TAG metabolism in S. cerevisiae.     

Role of autophagy in triacylglycerol biosynthesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revealed by chemical inducer and inhibitors

Autophagy mediates degradation and recycling of cellular components and plays an important role in senescence and adaptive responses to biotic and abiotic stresses. Nutrient deprivation has been shown to trigger triacylglycerol (TAG) accumulation and also induces autophagy in various green algae. However, the functional relationship between TAG metabolism and autophagy remains unclear. To gain preliminary evidence supporting a role of autophagy in TAG synthesis, Chlamydomonas reinhardtii CC-2686 was grown in Tris-acetate phosphate medium with or without nitrogen and treated with an autophagy inducer (rapamycin) or inhibitors (wortmannin, 3-methyladenine, and bafilomycin A1). Fluorescence microscopic analysis of Nile red-stained cells following 72-h treatments showed that rapamycin induced accumulation of subcellular lipid droplets which are storage sites of TAG. Rapamycin treatment in combination with nitrogen starvation led to a greater abundance of lipid droplets. Wortmannin and bafilomycin A1, but not 3-methyladenine, inhibited lipid droplet accumulation in rapamycin-treated cells and to a less extent in nitrogen-depleted cells. These results suggested that autophagy contributes to TAG synthesis in C. reinhardtii, but is not a necessary process. Autophagy induction may also be used to further enhance TAG accumulation in microalgae under nutrient deprivation.     

Changes in the fatty acid composition of symbiotic dinoflagellates from the hermatypic coral <Emphasis Type="Italic">Echinopora lamellosa</Emphasis> during adaptation to the irradiance level

The composition of fatty acids (FAs) of symbiotic dinoflagellates isolated from the hermatypic coral Echinoporal lamellosa adapted to the irradiance of 95, 30, 8, and 2% PAR was studied. Polar lipids and triacylglycerols (TAG) differed between them in FA composition. Polar lipids were enriched in unsaturated FAs, whereas TAG, in saturated FAs. Light exerted a substantial influence on the FA composition in both polar lipids and TAG. The elevation of irradiance resulted in the accumulation of 16:0 acid in both lipid groups and 16:1(n-7) acid in TAG. It seems likely that de novo synthesis of 16:0 acid occurred actively in the cells of symbiotic dinoflagellates in high light. Since these processes are energy-consuming ones, they utilize excessive energy. When light intensity declined, 18:4(n-3) and 20:5(n-3) acids accumulated in polar lipids, which was accompanied by the increase in the content of chlorophyll a in the cells of zooxanthellae, whereas the levels of 22:6(n-3) and 20:4(n-6) acids reduced. Although the relative content of particular FAs varied substantially in dependence of irradiance, the balance between the sum of saturated and unsaturated FAs changed insignificantly. We concluded that the role of photoadaptation could not be limited only to changes in the degree of lipid unsaturation and membrane fluidity. It is supposed that light-induced changes in the FA composition reflect the interrelation between photosynthesis and FA biosynthesis.     

High temperature enhances lipid accumulation in nitrogen-deprived <Emphasis Type="Italic">Scenedesmus obtusus</Emphasis> XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 °C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 °C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 °C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.     

<Emphasis Type="Italic">Rhodococcus</Emphasis> bacteria as a promising source of oils from olive mill wastes

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2–2.7 g/l) and lipids (77–83% of CDW, 1.8–2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1–1.6 g/l of cell biomass and 7.1–14.0% of CDW, 0.1–0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.     

Optimization of environmental parameters for <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> growth and lipid content using the response surface method and invading organisms

Algae biofuel has the potential to replace fossil fuels. However, cultivation and productivity of target algae need improvement, while controlling undesired organisms that can lower the efficiency of production systems. A central composite design and response surface model were utilized to predict cultivation optima of marine microalga, Nannochloropsis salina, under a suite of environmental parameters. The effects of salinity, pH, and temperature and their interactions were studied on maximum sustainable yield (MSY, a measure for biomass productivity), lipid content of N. salina, and invading organisms. Five different levels of each environmental predictor variable were tested. The environmental factors were kept within ranges that had previously been determined to allow positive N. salina growth (14.5–45.5 PSU; pH 6.3–9.7; 11–29 °C). The models created for this experiment showed that N. salina’s MSY and lipid content are not strongly affected over the broad range of salinity and temperature values. Calculated optima levels were 28 PSU/20 °C for MSY and 14.5 PSU/20 °C for lipid accumulation, but neither value significantly influenced the model. However, pH was the most important factor to influence algae productivity, and pH optimum was estimated around 8. Both MSY and lipid content were strongly reduced when pH deviated from the optimum. Occurrence of invading organisms seemed stochastic, and none of the environmental factors studied significantly influenced abundance. In conclusion, pH should be kept around 8 for maximum productivity of N. salina. Temperature and salinity should be kept around 20 °C and 28 PSU; however, moderate variations are not too much of a concern and might enhance lipid content of N. salina.     

Complete mitochondrial genome of <Emphasis Type="Italic">Ampittia dioscorides</Emphasis> (Lepidoptera: Hesperiidae) and its phylogenetic analysis

The complete mitochondrial genome of Ampittia dioscorides (Lepidoptera: Hesperiidae) was determined. The sequenced genome is a circular molecule of 15313 bp, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T-rich region. The gene arrangements and transcribing directions are identical to those in most of the reported lepidopteran mitogenomes. The base composition of the whole genome and genes or regions are also similar to those in other lepidopteran species. All the PCGs are initiated by typical ATN codons; the exception being COI, which begins with a CGA codon. Eight genes (ND2, ATPase8, ATPase6, COIII, ND5, ND4L, ND6, and Cytb) end with a TAA stop codon, and two genes (ND1 and ND3) end with TAG. The remaining three genes (COI and COII, which end with TA-, and ND4, which ends with T-) have incomplete stop codons. All tRNAs have the typical clover-leaf structure of mitochondrial tRNAs, with the exception of tRNA^Ser(AGY). On the basis of the concatenated nucleotide and amino acid sequences of the 13 PCGs and wingless gene of 22 butterfly species, maximum parsimony (MP) and Bayesian inference (BI) trees were constructed, respectively. Both MP and BI trees had the same topological structure: ((((Nymphalidae + Danaidae) + Lycaenidae) + Pieridae) + Papilionidae) + Hesperiidae). The results provide support for Hesperiidae as a superfamily-level taxon.     

Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca²⁺) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.     

Changes in growth and composition of the marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> in response to changing sodium bicarbonate concentrations

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L^?1 NaHCO₃ added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO₃ is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly (p?<?0.05) higher in the NaHCO₃-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO₃] during the GP. During the LP, cell densities were generally higher in the NaHCO₃-treated cells compared with controls. F _V/F _M (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO₃ and significantly different values between controls and 5.0 g L^?1 NaHCO₃ at the end of the GP (p?<?0.05). F _V/F _M was variable between treatments in P. tricornutum (0.3–0.65) but less so in N. salina for (0.5–0.7) regardless of [NaHCO₃]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2?±?6.5 and 4.4?±?2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L^?1 NaHCO₃ treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L^?1 NaHCO₃. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO₃]; there was three times more crude protein in the highest NaHCO₃ (5.0 g L^?1) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03?±?0.12 and 10.16?±?0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO₃] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO₃] (t tests; p?<?0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO₃ treatments. These findings reveal the importance of considering NaHCO₃ as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels.     

<Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> as a source of fatty acids and phytosterols: potential nutritional and biological implications

The main aim of this work was to describe the effect of light-induced stress on bioactive lipid accumulation in Haematococcus pluvialis CCALA 1081. To this end, the lipid profile was analyzed in order to determine triacylglyceride (TAG) and phytosterol content, the fatty acid profile of TAGs and the composition of the sterol fraction. After 3 days of light-induced stress, the content of both bioactive lipids significantly increased compared to controls. Palmitic, linoleic, and α linolenic fatty acid content was higher whereas caproic acid content diminished in H. pluvialis under stress. High irradiance also reduced β-sitosterol content whereas chlerosterol, brassicasterol, and Δ7-campesterol content was higher in the phytosterol fraction. No significant levels of lipid peroxidation were detected after 3 days of light-induced stress. In addition, H. pluvialis phytosterols displayed radical scavenging capacity and also a cytotoxic effect associated with the induction of apoptosis in human IMR-32 neuroblastoma cells. Our results show that high light stress induced the accumulation of commercially valuable compounds in H. pluvialis that are of potential benefit to human health.     

Analysis of metabolic responses of <Emphasis Type="Italic">Dunaliella salina</Emphasis> to phosphorus deprivation

It has been reported that phosphorus deprivation can induce β-carotene and triacylglycerol accumulation in Dunaliella salina cells. In this study, we aimed to elucidate the metabolic responses of D. salina to phosphorus deprivation, using gas chromatography-mass spectrometry as analytical tool. A total of 79 metabolites were identified in cells cultured in either phosphorus-deprived or replete media, including 18 amino acids, 28 other acids, 16 sugars, 12 alcohols, and 5 amino compounds. Hierarchical clustering was used to sort these metabolites into three groups with different change trends. Most amino acids and sugars, including the abiotic stress-related metabolites lysine, proline, trehalose, talose, and tagatose, increased, whereas N,N-dimethylglycine, L-serine, D-erythro-pentose, and D-ribose remained constant upon phosphorus deprivation. Multivariate statistical partial least squares and principal component analyses indicated that metabolite profiles were significantly changed upon phosphorus deprivation, and 18 biomarkers which can be used to distinguish the two culture conditions were identified. Stress-related polyamines such as cadaverine, antioxidants such as L-ascorbic acid, and L-methionine, as well as the osmolytes proline, mannitol, and arabitol, also increased. Furthermore, phosphorus deprivation resulted in increases of both saturated and unsaturated fatty acids in D. salina cells. These results suggest that phosphorus deprivation triggers comprehensive metabolic responses in D. salina which may be useful for future bioprocesses.     

Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous <Emphasis Type="Italic">Rhodococcus</Emphasis> strains

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.