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Background: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. Results: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. Conclusions: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels. [ABSTRACT FROM AUTHOR]

Background: Biofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC). Results: Among nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01). Conclusions: Based on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or –NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced. [ABSTRACT FROM AUTHOR]

Background: Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. Results: Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. Conclusions: The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application. [ABSTRACT FROM AUTHOR]

Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogenreplete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. [ABSTRACT FROM AUTHOR]

Background: The extraction of lipids from microalgae requires a pretreatment process to break the cell wall and subsequent extraction processes to obtain the lipids for biofuels production. The multistep operation tends to incur high costs and are energy intensive due to longer process operations. This research work applies the combination of radicals from hydrogen peroxide with an organic solvent as a chemical pretreatment method for disrupting the cell wall of microalgae and simultaneously extracting lipids from the biomass in a one-step biphasic solution. Result: Several parameters which can affect the biphasic system were analyzed: contact time, volume of solvent, volume ratio, type of organic solvent, biomass amount and concentration of solvents, to extract the highest amount of lipids from microalgae. The results were optimized and up to 83.5% of lipid recovery yield and 94.6% of enhancement was successfully achieved. The results obtain from GC-FID were similar to the analysis of triglyceride lipid standard. Conclusion: The profound hybrid biphasic system shows great potential to radically disrupt the cell wall of microalgae and instantaneously extract lipids in a single-step approach. The lipids extracted were tested to for its comparability to biodiesel performance. [ABSTRACT FROM AUTHOR]

The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don't need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology. [ABSTRACT FROM AUTHOR]

Background: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. Results: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. Conclusions: Growth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds. [ABSTRACT FROM AUTHOR]

Background: The regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. DNA binding with one finger (DOF) is a plant-specific transcription factor in higher plants, where it regulates carbon and nitrogen metabolic pathways by regulating key genes involved in these pathways. Overexpression of DOF can increase lipid production in plants; however, it is not clear whether overexpression of DOF can increase lipids in microalgae. Results: In this study, we cloned a DOF transcription factor, crDOF, from Chlamydomonas reinhardtii. The sequence of this transcription factor is 1875 bp and encodes a peptide of 624 amino acids with a conserved DOF domain. Overexpression of crDOF in C. reinhardtii significantly increased the intracellular lipid content. The content of total fatty acids in the transgenic algae line Tranc-crDOF-12 was 126.01 μg/mg (dry weight), which was 23.24% higher than that of the wild type. Additionally, the content of unsaturated fatty acids in the transgenic Tranc-crDOF-12 line increased significantly. Fluorescence quantitative PCR analysis showed that in the transgenic line Tranc-crDOF-12, the expression levels of BCC1, FAT1, SQD1, MGD1, DGD1 and PGP1 genes were significantly upregulated, while the expression levels of ACP1, ACS1, CIS1 and SQD2 were downregulated. Conclusions: Our results confirm that crDOF increases intracellular lipids in C. reinhardtii by regulating key genes involved in lipid metabolism. According to these findings, we propose that enhancing the lipid content in microalgae by overexpressing DOF may be achieved in other industrial strains of microalgae and be employed for the industrial production of biodiesel. [ABSTRACT FROM AUTHOR]

Background: Several abundant proteins have been identified in lipid body of an alkenone-producing marine haptophyte alga Tisochrysis lutea. The gene expression patterns of these proteins were investigated to better understand their roles in alkenone biosynthesis. For this purpose, T. lutea was first cultured in nitrogen-sufficient medium for biomass production and then shifted to nitrogen-deprived medium to induce lipid body formation. Results: There were remarkable increases in the volume of alkenone body (AB) and alkenone content in the alga after they were exposed to nitrogen depletion medium. Relative mRNA levels of the genes coding for the identified proteins V-ATPase subunit VA, V-ATPase subunit Vd, hypothetical protein EMIHUDRAFT_465,517, coccolith scale associated protein-1, cycloartenol-c-24-methyltransferase 1-like and SPFH domain-containing protein were investigated over the culture period. RT-PCR data showed that the expression of all these genes except the gene coding for SPFH domain-containing protein was up-regulated during the transition period from nitrogen-sufficient to nitrogen-deficient medium. Among them, the expression of the coccolith scale associated protein-1 gene was up-regulated 50–650 folds. These up-regulations were consistent with the increased alkenone production in nitrogen-deprived medium, suggesting that these proteins are involved in alkenone biosynthesis in T. lutea. Conclusions: Expression analysis of the lipoprotein genes suggests that five out of the six genes are up-regulated and are therefore likely to code for the identified lipoproteins associated with alkenone biosynthesis in T. lutea. These data would help better understand alkenone metabolism and engineer for improved biofuel production in T. lutea. [ABSTRACT FROM AUTHOR]

Background: Amelioration of biofuel feedstock of microalgae using sustainable means through synthetic ecology is a promising strategy. The co-cultivation model (Tetraselmis striata and Pelagibaca bermudensis) was evaluated for the robust biofuel production under varying stressors as well as with the selected two-stage cultivation modes. In addition, the role of metabolic exudates including the quorum-sensing precursors was assessed. Results: The co-cultivation model innovated in this study supported the biomass production of T. striata in a saline/marine medium at a broad range of pH, salinity, and temperature/light conditions, as well as nutrient limitation with a growth promotion of 1.2–3.6-fold. Hence, this developed model could contribute to abiotic stress mitigation of T. striata. The quorum-sensing precursor dynamics of the growth promoting bacteria P. bermudensis exhibited unique pattern under varying stressors as revealed through targeted metabolomics (using liquid chromatography–mass spectrometry, LC–MS). P. bermudensis and its metabolic exudates mutually promoted the growth of T. striata, which elevated the lipid productivity. Interestingly, hydroxy alkyl quinolones independently showed growth inhibition of T. striata on elevated concentration. Among two-stage cultivation modes (low pH, elevated salinity, and nitrate limitation), specifically, nitrate limitation induced a 1.5 times higher lipid content (30–31%) than control in both axenic and co-cultivated conditions. Conclusion: Pelagibaca bermudensis is established as a potential growth promoting native phycospheric bacteria for robust biomass generation of T. striata in varying environment, and two-stage cultivation using nitrate limitation strategically maximized the biofuel precursors for both axenic and co-cultivation conditions (T and T-PB, respectively). Optimum metabolic exudate of P. bermudensis which act as a growth substrate to T. striata surpasses the antagonistic effect of excessive hydroxy alkyl quinolones [HHQ, 4-hydroxy-2-alkylquinolines and PQS (pseudomonas quorum signal), 2-heptyl-3-hydroxy-4(1H)-quinolone]. [ABSTRACT FROM AUTHOR]

Background: Microalgae, being a phototroph, grow in the presence of light, and utilizing photons in narrow and specific range of wavelengths. There have been numerous attempts to take advantage of this trait of wavelength-dependent growth for the purpose of increasing biomass productivity. One potential option involves wavelength conversion of sunlight. In the present study, three fluorescent dyes with blue, red, and green emission spectra were employed with the aim of improving sunlight utilization efficiency and thus enhancing biomass and lipid productivity of Nannochloropsis gaditana. Results: When DPA and R101 were used to enrich blue and red spectra, biomass productivity of Nannochloropsis gaditana was increased by 35.1 and 40.3%, respectively. The maximum quantum yield values were higher than 0.6 at the early stage of growth for the cultures grown under DPA- and R101-modified solar radiation. Chlorophyll a content was also 57.0 and 32.3% higher than the control at the early growth stage under DPA- and R101-modified solar radiation, respectively. This stimulation of photosynthetic activity at the early growth stage correlated well with rapid growth under DPA- and R101-modified light during the first 4 days of cultivation. Lipid productivity consequently increased by 26.9 (DPA) and 39.4% (R101) after 10 days of cultivation. An immediate effect on lipid induction was observed in cultures under modified light, which exhibited 19.1% improvement in lipid content at the cost of some degree of impaired growth. Conclusion: Fluorescent dyes with the capability of enriching wavelengths of light favored by the algal photosystem could indeed be an effective means of promoting growth of Nannochloropsis gaditana. This strategy would be particularly powerful for mass cultivation where sunlight is the only economically viable option for illumination. [ABSTRACT FROM AUTHOR]