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Trends Biotechnol. Jan;22(1) Transgenic microalgae as green cell- factories. León-Bañares R(1), González-Ballester D, Galván A, Fernández E.
Table of contents

Genetic manipulation of microalgae for the production of bioproducts. References Publications referenced by this paper. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors. Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer. Mesothelin variant 1 is released from tumor cells as a diagnostic marker. Conditions associated with antibodies against the tumor-associated antigen MUC1 and their relationship to risk for ovarian cancer.

Human tumor cell lysates as a protein source for the detection of cancer antigen-specific humoral immunity. Potential markers that complement expression of CA in epithelial ovarian cancer. Armin Rump , Yoshihiro. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer Zhanat E Muminova , Theresa V. Strong , Denise R. Martin W. Related Papers. Green algae, often referred to as chlorophytes, are highly abundant and are estimated to number as many as 8, species.

They are the most diverse group of algae, and include unicellular, colonial, coccoid, filamentous, and multicellular forms growing in a variety of habitats. Green algae are believed to share a common ancestor with higher plants, carrying the same photosynthetic pigments and having similar metabolic mechanisms. Generally, these algae use starch as their primary storage vehicle, however, in some strains large quantities of TAG accumulate under specific growing conditions.

Oleaginous green algae contain an average total lipid content of Chlamydomonas reinhardtii has been treated as a model organism for photosynthesis, and as a result has been studied extensively, because of its giant chloroplast and ability to control sexual reproduction, allowing detailed genetic analysis [ 32 ]. Indeed, Chlamydomonas was also the first alga to be genetically transformed and a draft sequence of the whole genome has recently been determined [ 33 ].

Although it does not typically accumulate lipids under ideal conditions, metabolic engineering can be used to transform this alga into an oleaginous factory [ 34 ]. Algal lipid metabolism from de novo fatty acid biosynthesis to the formation of complex glycerolipids is similar to that of the plant cells. Higher plants have differentiated organs, each of which performs specific physiological functions, and contains specific biochemical pathways.

Similarly to higher plants, algae process TAG into lipid droplets which are coated in a large number of proteins. Most of these are typical members of vesicular transport and signaling pathways such as RabGTPases, but a proteomics approach to algal lipid bodies has identified a protein called major lipid droplet protein MLDP which affecs the size of lipid droplets and may present a target for immunofluorescence imaging of algal lipid content [ 35 ]. Most of the algal lipids are glycerinated membrane lipids, with minor contributions to overall lipid content from TAG, wax esters, hydrocarbons, sterols, and prenyl derivatives [ 30 , 36 ].

Under unfavorable growing conditions many algae shift their metabolic pathways toward the biosynthesis of storage lipids or polysaccharides. TAG accumulation in response to environmental stress likely occurs as a means of providing an energy deposit that can be readily catabolized in response to a more favorable environment to allow rapid growth [ 27 ]. Nutrients, temperature, light, salinity and growing phase have been shown to influence the flux of algal cellular metabolism [ 37 ].

Since many of the algal lipid metabolism studies on environmental changes have been carried out in batch cultures, there is a lack of systematic, multi-factor monitored studies. This decreases the practicability of applying previous findings to large-scale algal cultures.

During the years of Aquatic Species Program, a 'silver bullet' was sought; a single species which could produce high levels of storage lipids without growth rate alteration. To maximize lipid production and growth efficiency for industrial scale culture, experiments with recombinant genetics and complex culture conditions multi-stage cultures, timed nutrient limitations may be required. Petroleum diesel or petrodiesel is a mixture of saturated and aromatic hydrocarbons with carbon atoms and is ignited in high-compression diesel engines. Most plant oils TAGs are too viscous to use in modern diesel engines, and eventually lead to engine failure caused by incomplete combustion.

Biodiesel is mono-alkyl usually methyl esters fatty acid methyl ester, or FAME made by the transesterification of TAGs from vegetable oils or animal fats, and has a similar viscosity to petrodiesel [ 38 ]. There are several advantages in addition to carbon neutrality when using biodiesel as a liquid fuel source. The cetane number, a measure of the delay between compression and ignition, can be higher for biodiesel than regular grade petrodiesel. This reflects the quality of the fuel and a higher number is associated with shorter delays in ignition, resulting in more complete combustion.

Burning biodiesel produces less carbon monoxide, particulate matter, sulfur, and aromatic compounds than burning petrodiesel. Furthermore, it has a higher flashpoint, allowing safer handling and storage and greater lubricity for engines than other fuels. It is made from renewable biomass and is biodegradable and "friendlier" to the environment than crude petroleum when fuel leakages do occur.

Currently only two major renewable liquid fuels are produced in large quantities, bio-ethanol and biodiesel. Additionally, ethanol has been shown to corrode pipelines, likely shortening their lifetimes [ 39 ]. Despite the many advantages, and increasing market share of biodiesel, there are limitations hindering its complete replacement of petrodiesel [ 38 ]. Negative biodiesel characteristics include poor cold-temperature properties, namely the tendency to solidify or gel, which can lead to fuel starvation and engine failure. The presence of polyunsaturated fatty acids in biodiesel also makes it susceptible to oxidation by atmospheric oxygen or hydrolytic degradation by water, which decrease the stability of biodiesel during long-term storage.

In addition, the emissions from biodiesel contain a higher concentration of nitrogen oxide NO x than do petrodiesel emissions, limiting its usage in areas under strict air quality standards. One of biodiesel's biggest limitations is cost and supply. As mentioned above, the use of oil crops for biodiesel production has already increased the cost of these commodities, and raised the 'food vs.

Although the oil supply problem may be relieved by switching from food plant to non-food plant feedstocks such as algae, the higher production costs of algal oil along with the lack of successful industry examples to date further hinders industry-scale adoption of algae-derived biodiesel. Unusual fatty acids produced by specific plant species contain unique functional groups giving them selective usages in industry [ 4 ]. The fatty acid composition determines the physical and chemical properties of the oil and its economic value.

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Traditionally, simple methods like blending or partial hydrogenation were applied to produce oils for specific applications. As the accumulating knowledge of plant lipid biosynthesis has been coupled with the development of advanced genetic technologies, various metabolic engineering methods have been performed to modify the fatty acid and lipid composition of several oleaginous plants [ 40 — 42 ].

Increasing oil content could be a straight-forward method to lower the high cost of biodiesel production, and may be applicable through genetic manipulation of lipid biosynthetic pathways. Table 1 shows an outline of genetic manipulations that have been performed in higher plants and the resulting changes in fatty acid composition and content. It has been proposed that lipid biosynthesis may be controlled by the availability of fatty acids, and that the production of fatty acids is regulated by acetyl CoA carboxylase ACCase [ 43 , 44 ].

Increasing the activity of ACCase may push excess substrate, malonyl-CoA, into the lipid biosynthesis pathway. Substantially increasing plastidial ACCase activity may prove quite complex due to the multigene-encoded enzyme complex and its post-translational regulation [ 45 ]. A successful example has been achieved by expressing a cytosolic version of the enzyme targeted to the rapeseed chloroplast [ 46 ].

Increasing malonyl-CoA substrate pools for de novo fatty acid biosynthesis resulted in only minor increases in seed oil yield. Fatty acid synthase has been suggested to be another rate-limiting regulator of lipid production and several studies have been performed where a single enzyme of the FAS complex is overexpressed. It seems unlikely that the up-regulation of any single enzyme will have a major positive effect on lipid biosynthetic flux. Multiple gene expression or activation of key regulators operating on the entire fatty acid biosynthetic pathway may have a more substantial effect on lipid production [ 48 ].

The second part of triacylglycerol biosynthesis is the Kennedy pathway, which depends on levels of glycerolphosphate.

Microalgae and Cyanobacteria as Green Molecular Factories: Tools and Perspectives

Other successful examples increasing plant oil levels have come by altering the acyltransferases of TAG biosynthesis. Arabidopsis thaliana has been transformed with a soluble safflower glycerolphosphate acyltransferase GPAT , where the plastidial targeting sequence was removed, and an Escherichia coli GPAT inserted. The resulting transgenic strain showed a substantial increase in seed oil content and an increase in the proportion of erucic acid [ 52 ].

Overexpression of the Arabidopsis DGAT1 gene in the wild-type strain led to increased seed oil deposition and average seed weight [ 54 ]. A functional DGAT homologue, the DGAT2 gene from the oleaginous fungus Mortieralla rammanniana was overexpressed in soybean, and resulted in small but significant increases in seed oil content in both greenhouse and field tests [ 55 ]. Together, these studies indicate that increased metabolic flux towards oil production may be achieved by manipulations targeted at later steps in the TAG biosynthetic pathway.

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A reasonable explanation is that the consequences of activating early biosynthetic steps may be slowed by later rate-limiting steps, and excess intermediate products may be utilized by other metabolic pathways sharing the same intermediates of TAG biosynthesis. Metabolic modeling networks that simulate flux of fatty acids through TAG biosynthetic pathways should play an important part in developing strategies for future genetic manipulation.

Actual values of the engineering results need to be properly calculated for whole organisms and total production costs, not just the oil itself. For example, increasing oil content of soybean usually comes at the expense of the reduction of high-value protein content used for animal feed. Rigorous field testing is necessary to determine whether oil content increases are reflected in an increased oil yield per hectare per year. These tests must prove that strains with lipid content increases are economically viable compared to elite, high-yield commercial varieties.

Beyond base supply, biodiesel has other limitations hindering its market competitiveness. The fuel properties of biodiesel are closely related to its fatty acid composition. Altering the fatty acid profile, for example the carbon chain length and number of double bonds, can lead to a better-quality, inexpensive biodiesel. The presence of methyl ester with saturated acyl chain longer than C12 significantly increases the cloud point of the biodiesel, the temperature at which crystals form [ 56 ].

The methyl esters derived from poly-unsaturated fatty acids are prone to oxidation and the hydroperoxides formed will eventually polymerize and form insoluble sediments capable of interfering with engine performance [ 57 ]. Highly saturated and longer carbon chain esters have lower NO x emissions relative to shorter, less conjugated chains [ 58 ].

In addition, biodiesel ignition quality is adversely effected by an increase in the number of double bonds [ 38 ]. When requirements for biodiesel quality are viewed together, it is clear no single fatty acid methyl ester FAME could fulfill every parameter. Therefore, down-regulation of the ER membrane-bound fatty acid desaturases should result in an increased percentage of oleic acid present, relative to total fatty acid content.

Several experiments have successfully enhanced the oleate concentration in various oleaginous plants [ 60 — 62 ]. In addition to oleic acid, other unusual monoenoic fatty acids from plants have potential for biodiesel production. The reason for the low levels of unusual monoene production in non-native plants may be lack of corresponding ACP, ferredoxin, 3-ketoacyl-ACP synthase, thioesterase, and acyltransferase present in the original strains [ 64 , 66 ].

Since fatty acid desaturases are highly conserved in their structure and amino acid sequences, several chimeric enzymes have been generated and shown to have broader substrate specificity [ 67 , 68 ]. These engineered desaturases may be more effective when designing transgenic plants to produce large amounts of monoenoic fatty acids [ 69 ]. As mentioned previously, fatty acyl chain length is another important factor that influences the viscosity and cold flow properties of biodiesel [ 38 ].

Short- to medium-chain fatty acids C8-C14 have lower viscosity and higher cloud points than common long-chain fatty acids CC Although cold-flow properties are superior, cetane numbers are lower, and overall NO x emissions higher for shorter chain fatty acids. However, increasing their proportion in market-available biodiesel still leads to better quality, more competitive fuel in terms of combustion performance.

Plants that accumulate short- to medium-chain C8 to C14 fatty acids in seed oil contain chain-length-specific acyl-ACP thioesterases that cleave the corresponding fatty acids from the growing acyl-ACP of de novo fatty acid biosynthesis [ 70 ]. The chain-length-specific acyl-ACP thioesterases were identified in both species as the cause of the unusual accumulation [ 71 , 72 ]. The reasons for lower production of short-chain fatty acids in transgenic hosts compared to donor species were further investigated. Additionally, structural analysis of TAG from the plants containing inserted medium-chain acyl-ACP thioesterase revealed that laurate was only present at sn -1 and sn -3 positions [ 74 ].

The high specificity of lysophosphatidic acid acyltransferase LPAAT from the hosts prevented laurate from being incorporated at the sn -2 position of TAG. These genes were induced with increasing levels of the lauric acid [ 78 ]. Obtaining significant amounts of short-chain fatty acids in TAG may require the engineering of multiple genes, including the short-chain-specific keto-synthase and thioesterase, as well as short-chain-specific acyltransferases, which assemble the novel fatty acids into TAG.

Production of unusual fatty acids in transgenic hosts can induce antagonistic pathways reducing the effects of genetic manipulation, which must be addressed to maximize production efficiency. Recently, direct use of low-molecular-weight TAG as fuel has been discussed and studied [ 59 , 79 ]. The lower cost of TAG fuels on the transesterfication and purification of FAMEs greatly enhances the market potential of such biodiesels.

Another interesting study involves the 1,2-diacylacetyl- sn -glycerols ac-TAG from the seeds of Euonymus alatus Burning Bush.

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This specific acetyl DAGAT has been isolated from Euonymus alatus , and data on the oil properties of transgenic plants are much anticipated [ 82 ]. During the years of ASP Aquatic Species Program , an extra-copy of the monomeric ACCase gene was introduced into the genome of the diatom Cyclotella cryptica , in an attempt to increase lipid accumulation in the transformed strains [ 83 ]. Unfortunately, a two to three-fold higher ACCase activity in the transformed algae did not result in any enhancement of lipid production [ 26 ].

A major reason very few positive engineering results have been achieved in algae lipid metabolism is the lack of a reliable nuclear transformation system like that used in higher plants. A more promising method of genetic engineering has been successfully established in the chloroplast of Chlamydomonas reinhardtii [ 84 ]. However, as the examples in vascular plants have shown, most of the critical enzymes controlling lipid biosynthesis and fatty acid modification reside in the cytoplasm.

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Several transformation techniques have been developed to genetically engineer C. General transformation protocols such as electroporation, particle bombardment, silicon carbide whisker agitation, and even Agrobacterium tumefaciens have been shown to transform a number of diverse microalgae including both green and red algae, diatoms, and dinoflagellates [ 85 — 89 ]. Expression levels vary greatly depending on a number of factors including auto-attenuation of exogenous sequences, codon usage bias, GC content, and proteasome mediated degradation [ 90 ]. Transformation and expression research in C.

Transformation of the nuclear genome allows for inducible gene expression, targeting to subcellular compartments, and protein secretion [ 93 ].

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  6. Insertion typically occurs via non-homologous recombination, though homologous recombination is known to occur at a very low frequency [ 94 ]. Optimizing homologous recombination conditions should allow for the directed knockout of enzymes diverting carbon usage away from lipid production, or for the directed replacement of lipid synthesizing enzymes with more effective isozymes. High levels of transgene expression can be selected for by using antibiotic resistance genes in combination with transgenic constructs. Addition of the ble gene to a transgenic construct confers resistance to phleomycin and zeocin in a drug:protein ratio and can be used to select for transformants with high expression levels [ 95 ].

    Although the nuclear genome does not yet robustly support protein production on a scale viable for harvesting protein therapeutics such as antibodies, expression of cytosolic enzymes or signaling proteins which promote the production of storage lipids may reach high enough activity levels to significantly alter the overall lipid profile of the host microalgae. High-throughput screening by insertional mutagenesis could be followed up with an RNAi based approach to investigate pathways for regulators of stress response, which may yield a genetic mechanism to increase lipid yield while minimizing growth arrest in large scale cultures.

    RNAi of protein members of pathways involved in lipid catabolism such as lipase and proteins of the beta oxidation, glyoxylate, and gluconeogenesis may represent important modifications which could increase overall TAG content [ ]. To date, there have been over 30 complete genome sequences of algae determined, with still more unpublished [ 30 , ]. With this primary sequence data and the functional characterization of homologous plant genes in hand, we can more precisely determine the key regulators of algal lipid biosynthesis in silico.

    Work has already started in this field, including the first gene-expression profile of C. RNA-seq analysis of C. Many of these genes were expected to be upregulated in lipid producing conditions, but more thorough bioinformatic analysis should yield new targets for genetic manipulation. More genomic, proteomic and metabolomic studies on algae lipid biosynthesis should also be nearing completion.

    The idea of algal oils as a potential biodiesel feedstock has been proposed and developed for years. The progresses in algal genetic engineering technology should accelerate any steps taken in achieving this goal. Renewable energy has become an important issue of recent political campaigns, and an increase in usage with less reliance on fossil energy will create substantial benefits for the global environment, economy, and industry. Biofuels are one of the few renewable energies proposed that have generated large public expectation as a real possibility for one of the fuels of the future.

    The use and production of plant oil as a source of biodiesel is expanding annually. Decades of studies have provided a general scheme of the plant lipid metabolism, and genetic engineering methods have provided valuable data and several field trials. However, more studies in organism-scale metabolic regulation will be necessary to understand how plants control their lipid biosynthetic pathways in response to physiological and environmental conditions. Elucidation of complex flux-control will hold great benefits for future biofuel production. Algae, the world's largest group of photosynthetic organisms, contribute a majority of the carbon fixation on earth, turning greenhouse gases into carbohydrates and lipids.

    Using algal oils as a biodiesel feedstock holds major advantages in comparison to plant oils. Algal cultures have long been studied, and already are used to produce several important value-added products for the agriculture and food industries, such as VLC-PUFA, carotenoids, and high-protein animal feeds. The carbohydrates and cellulosic cell wall of algae have the potential to be hydrolyzed and fermented into bioethanol, further increasing the utility of algae as a biofuel feedstock. Algae cells can also be used to synthesize important eukaryotic proteins or natural products for pharmaceutical applications.

    Further fundamental studies in algae metabolism hold the possibility of making the algae cell a multi-use feedstock and creating a true "green gold". Olsson L: Biofuels. Chiaramonti D: Bioethanol: role and production technologies, pp. Edited by: Ranalli P. Gerpen JV: Biodiesel production, pp.

    Murphy DJ: The biogenesis and functions of lipid bodies in animals, plants and microorganisms.

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      Energ Environ Sci. J R Soc Interface. J Theor Biol. Plant Molecular Biology. Harwood JL: Recent advances in the biosynthesis of plant fatty acids. Biochimica et Biophysica Acta. Plant Journal. Ohlrogge J, Browse J: Lipid biosynthesis. Plant Cell. Harwood JL: Fatty acid metabolism. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis. Plant Physiology.