Accelerated triacylglycerol production and altered fatty acid composition in oleaginous microalga Neochloris oleoabundans by overexpression of diacylglycerol acyltransferase 2

BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.ResultsThe integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.ConclusionsOur results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.

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title: Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization
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title: Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii
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title: Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter
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title: Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2.
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title: A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica.
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title: Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum
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title: Taxonomic status of the species of the green algal genusNeochloris
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title: Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans
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title: Neochloris oleabundans UTEX #1185: a suitable renewable lipid source for biofuel production
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title: Microalgae as a raw material for biofuels production
abstract: Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO2, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulinamaxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.

title: Lipid composition of the nitrogen starved green alga Neochloris oleoabundans
abstract: Neochloris oleoabundans has been cultivated in mineral medium deficient in nitrogen. The yield of lipids was 35–54% of cell dry weight. Triglycerides comprised 80% of the total lipids. Aliphatic hydrocarbons, sterols, pigments, glycolipids and phospholipids comprised the remaining lipid fraction. Saturated, monounsaturated and diunsaturated octodecanoic acid represented approximately one-half of the total fatty acids.

title: Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation
abstract: Biodiesel from microalgae is technically feasible, but not yet economically viable. A potential approach to improve microalgae as an economically viable biodiesel feedstock is to increase microalgal lipid content via genetic engineering. Genetic manipulation of microalgae requires the accessibility to stable nuclear transformation. In this study, we describe a strategy for developing a stable nuclear transformation system of the oleaginous microalga Neochloris oleoabundans using electroporation. The hygromycin B-resistant gene Hyg3, which was used as a positively selectable marker, consisted of aph7” gene encoding aminoglycoside phosphotransferase of Streptomyces hygroscopicus and intron1 of Chlamydomonas reinhardtii rbcS2 gene, under the control of C. reinhardtii HSP70A-RBCS2 hybrid promoter. The transformation frequency was 5.2 × 10−4 transformants mg−1 DNA. The transformants showed stable hygromycin B-resistant phenotype for at least 6 months in the absence of the antibiotic selection. Co-transformation frequency of unselectable green fluorescent protein gene (Gfp) adapted to C. reinhardtii codon usage (ChGfp) and selectable Hyg3 gene was 2.6 × 10−4 transformants mg−1 DNA; up to 90 % of the transformants exhibited green fluorescent protein (GFP) activity. The ChGfp and Hyg3 gene were integrated into the nuclear genome of N. oleoabundans. The GFP fluorescence signal of the transformants under confocal laser scanning microscope was visible. The successful stable nuclear transformation system not only provides a basis for molecular genetics study, but also enables subsequent genetic engineering in the microalga to increase lipid content for biodiesel production. The strategy for developing the stable nuclear transformation system presented in this study may be applicable to other microalgal species without sequenced genome.

title: Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red
abstract: The fluorophore Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) has been used to determine neutral lipid in microalgal cells. Cellular fluorescence of stained cells and gravimetrically or chromatographically determined lipid were linearly correlated when Nile Red was excited at 488 – 525 nm and the fluorescent emision measured at 570 – 600 nm. Nile Red is a vital stain which allowed flow cytometric sorting of live microalgal populations based on their lipid content.

title: Lipid productivity as a key characteristic for choosing algal species for biodiesel production
abstract: Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production. This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The data available in the literature are far from complete and rigorous comparison across experiments carried out under different conditions is not possible. However, the collated information provides a framework for decision-making and a starting point for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid productivity as a selection parameter over lipid content and growth rate individually is demonstrated.

title: The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas.
abstract: The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, beta 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A-RBCS2 promoter fusion as compared to the RBCS2 promoter alone.

title: Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.
abstract: The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

title: A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.
abstract: The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

title: An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.
abstract: We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.

title: Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2[C][W]
abstract: With the aim of improving the energy density of plant vegetative tissues, this work uses algal diacylglycerol acyltransferase type two enzymes to alter acyl carbon partitioning in Arabidopsis vegetative tissues, increasing acyl-CoA–dependent triacylglycerol synthesis and thereby increasing the nutritional value of leaves. Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.

title: Influence of fatty acid composition of raw materials on biodiesel properties.
abstract: The aim of this work was the study of the influence of the raw material composition on biodiesel quality, using a transesterification reaction. Thus, ten refined vegetable oils were transesterificated using potassium methoxide as catalyst and standard reaction conditions (reaction time, 1h; weight of catalyst, 1 wt.% of initial oil weight; molar ratio methanol/oil, 6/1; reaction temperature, 60 degrees C). Biodiesel quality was tested according to the standard [UNE-EN 14214, 2003. Automotive fuels. Fatty acid methyl esters (FAME) for diesel engines. Requirements and test methods]. Some critical parameters like oxidation stability, cetane number, iodine value and cold filter plugging point were correlated with the methyl ester composition of each biodiesel, according to two parameters: degree of unsaturation and long chain saturated factor. Finally, a triangular graph based on the composition in monounsaturated, polyunsaturated and saturated methyl esters was built in order to predict the critical parameters of European standard for whatever biodiesel, known its composition.

title: Technical aspects of biodiesel production by transesterification—a review
abstract: Biodiesel is gaining more and more importance as an attractive fuel due to the depleting fossil fuel resources. Chemically biodiesel is monoalkyl esters of long chain fatty acids derived from renewable feed stock like vegetable oils and animal fats. It is produced by transesterification in which, oil or fat is reacted with a monohydric alcohol in presence of a catalyst. The process of transesterification is affected by the mode of reaction condition, molar ratio of alcohol to oil, type of alcohol, type and amount of catalysts, reaction time and temperature and purity of reactants. In the present paper various methods of preparation of biodiesel with different combination of oil and catalysts have been described. The technical tools and processes for monitoring the transesterification reactions like TLC, GC, HPLC, GPC, 1H NMR and NIR have also been summarized. In addition, fuel properties and specifications provided by different countries are discussed.

title: Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester
abstract: The cetane number, a widely used diesel fuel quality parameter related to the ignition delay time (and combustion quality) of a fuel, has been applied to alternative diesel fuels such as biodiesel and its components. In this work, the cetane numbers of 29 samples of straight-chain and branched C1 –C 4 esters as well as 2-ethylhexyl esters of various common fatty acids were determined. The cetane numbers of these esters are not significantly affected by branching in the alcohol moiety. Therefore, branched esters, which improve the cold-flow properties of biodiesel, can be employed without greatly influencing ignition properties compared to the more common methyl esters. Unsaturation in the fatty acid chain was again the most significant factor causing lower cetane numbers. Cetane numbers were determined in an ignition quality tester (IQT) which is a newly developed, automated rapid method using only small amounts of material. The IQT is as applicable to biodiesel and its components as previous cetane-testing methods.

title: Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing.
abstract: Numerous reports describe phenomena of transgene silencing in plants, yet the underlying genetic and molecular mechanisms are poorly understood. We observed that regeneration of Arabidopsis thaliana plants transgenic for the rolB gene of Agrobacterium rhizogenes results in a selection for transgene silencing. Transgene silencing could be monitored in this system by reversion of the visible RolB phenotype. We report a phenotypic, molecular, and genetic characterization of a meiotically reversible transgene silencing phenomenon observed in a rolB transgenic line. In this line, the rolB gene is expressed strongly and uniformly in seedlings, but in the course of further development, the rolB gene is silenced erratically at a frequency that depends on the dosage of rolB. The silenced state is mitotically stable, while complete resetting of rolB gene expression occurs in seedlings of the following generation. The silencing of rolB correlates with a dramatic reduction of steady-state rolB transcripts, while rolB nuclear run-off transcripts are only moderately reduced. Therefore, rolB gene silencing seems to act predominantly at the posttranscriptional level. The process of rolB gene silencing was found to be affected by two extragenic modifier loci that influence both the frequency and the timing of rolB gene silencing during plant development. These genetic data demonstrate a direct involvement of defined plant genes in this form of gene silencing.

title: RNA degradation and models for post-transcriptional gene silencing
abstract: Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are `aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.

title: Biofuels from algae: challenges and potential
abstract: Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality.

title: Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor.
abstract: The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m(-2) day(-1) was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m(-2) day(-1). Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m(-2) day(-1)) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m(-2) day(-1)). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).

title: A rapid method of total lipid extraction and purification.
abstract: Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.