Abstract:
Genetically engineered algae strains for biofuels and bioproduct production have improved iron utilization including iron uptake and storage, and exhibit improved growth characteristics, magnetic separation, and magnetic hysteresis induced cell lysis. Pond production algal strain embodiments with high iron scavenging capabilities limit iron availability to contaminating microorganisms and invading species. Accumulation of high iron and other paramagnetic elements content in the form of ferritin enhances the cells magnetic susceptibility improving efficiency of magnetic separation and magnetic hysteresis induced cell lysis. Several genes of the embodiments are capable of improving iron acquisition including the ferritin gene fer1, iron transport gene fea1, and iron reductase gene fre1. Other genes which improve growth in the high iron conditions permitting higher accumulation of iron include radical scavenging enzymes such as superoxide dismutase, peroxidase, catalase, glutathione peroxidase and the Ferritin like DPR/DPS genes which also bind iron and protect DNA from reactive oxygen species.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is based on and claims priority to U.S. provisional patent application Ser. No. 61/362,160 filed Jul. 7, 2010, the teachings of which are included herein by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The disclosed embodiments of the present invention are in the field of algal biomass and biofuel production. 
       BACKGROUND 
       [0003]    Biofuels will play an increasing role in the United States energy market as energy prices increase, political concerns of establishing energy independence intensify, and apprehension about climate change grows. The price of petroleum has fluctuated dramatically, reaching record highs of more than US$140 per barrel in 2008. In part, those price increases reflected economic, political, and supply chain uncertainties. Political concerns about the availability of petroleum supplies have led to the realization that the United States&#39; energy independence is of critical strategic importance, both economically and militarily. Additionally, the release of CO 2  from fossil fuel combustion may also substantially contribute to global warming and climate change and that could have environmental and societal importance. Many states and federal governments have responded by enacting renewable portfolio standards (RPS) mandating that electricity providers and other heavy energy users obtain a certain percentage of their power from renewable energy sources. As a result of these concerns, sustained high prices for fossil fuels, and the emerging RPS requirements, domestically produced biofuels have become an increasingly attractive alternative to foreign sourced fossil fuels. 
         [0004]    Microalgae are some of the most productive and therefore desirable sources of biofuel feedstocks. The Department of Energy (DOE) has determined that biofuel yield per acre from microalgae culture exceeds that of many organisms and land crops. Between the late 1970s and 1990s, the DOE&#39;s National Renewable Energy Laboratory (NREL, formerly SERI) evaluated the economic feasibility of producing biofuels from a variety of aquatic and terrestrial photosynthetic organisms (Sheehan et al., 1998). Biofuel production from microalgae was determined to have the greatest yield per acre potential of any of the organisms screened. Microalgal biofuel production was estimated to be 8 to 24 fold greater than the best terrestrial biofuel production systems. Current estimates of the potential productivity for algal biofuel production range from 3,000 to 10,000 gallons/acre. According to the DOE, microalgae yield “30 times more energy per acre than land crops such as soybeans.” Although promising, there is still a need for systems and methods that create even greater efficiencies in biofuel production from microalgae. 
         [0005]    One of the challenges faced by biofuel production from microalgae is that, traditionally, the organisms must be dewatered to reduce the overall processing volume and improve the efficiency of the extraction process. Currently available dewatering processes, however, have many drawbacks. For instance, centrifugation and filtration are costly and labor intensive, making them somewhat impractical for production of commodity products. Settling, in either holding tanks or plate type settling devices, is an inexpensive though time-consuming process (U.S. Pat. No. 3,431,200; Kothardaraman &amp; Evans 1972). This method is ill-suited for dewatering algae because of the small size and low specific gravity of algal cells. The settling rate for algae is therefore “too slow to permit the use of a settling as a routine procedure for harvesting algae cells” (Kothandaraman &amp; Evers 1972). The length of the settling process for algae poses several difficulties, especially in a production plant where the cells being collected will either be used in a downstream process or for extraction of oil. First, the long processing time decreases plant throughput. Second, the long processing time increases the likelihood of contamination that can compromise downstream processing (U.S. Pat. No. 3,431,200). The long term storage of the cells associated with settling can lead to decay or reabsorption of the product of interest (e.g., oil, pigments, secondary metabolites, and other co-products) for cell maintenance that can compromise oil extraction efficiency and yields. In addition, because of the large amount of water involved, centrifugation is cost prohibitive. 
         [0006]    Dissolved air flotation (DAF) is another separation method somewhat related to the settling method previously described (U.S. Pat. No. 3,780,471; U.S. Pat. No. 3,431,200; Kothandaram &amp; Evans 1972). Under this method, the cells are treated with flocculants and/or coagulating agents. The cells then aggregate and are exposed to a fine curtain of air bubbles that lift aggregated cells to the top of the holding tank where they are skimmed off and harvested. The utility of this method is limited because coagulated and flocculated cells can compromise downstream processing. Removing the coagulants or flocculants involves another processing step which increases total processing time and costs. Although faster than simple settling, this method still has the same drawbacks associated with any slow process for dewatering to downstream processing and extraction. 
         [0007]    A pitfall of all of these methods is that their dewatered products contain organisms other than algae. The presence of these organisms can compromise the efficiency of downstream processing. For instance, some of these organisms may out-compete algae, thereby decreasing the density of the targeted algae. 
         [0008]    Dewatering can be achieved magnetically if the material to be separated is sufficiently magnetized. Commercially available magnetic separators operate by allowing the magnetized target particle to attach directly to a rotating magnetic disc(s). As the disc is rotated, one section is continuously scrubbed by a cell scraper that removes the adhered biomass and transfers it to a conveyor belt or pipe where it is distributed for downstream processing. Contaminating species, nonmagnetic algae, and debris flow preferentially to waste or further treatment such that it can be sent back to the pond for further growth. An alternative use would be to use the magnetic properties of the production strain to positively select and return it to the production process while removing contaminating microorganisms from the culture on a continuous or periodic basis as a maintenance or emergency response protocol. 
         [0009]    In order for magnetic separation to be effective, however, the materials to be separated must be sufficiently magnetized. Existing methods of magnetization are limited to altering the exterior of the material to be separated. One such method is magnetic seeding, which involves the addition of magnetite to the outside of the material with or without the use of a sorption agent (U.S. Pat. No. 7,473,356; Niki et al.). Magnetic separation of this type of seeded material has drawbacks. This method causes all materials to coagulate, including undesirable materials such as fungi, protozoa, bacteria, archaea, and other species of algae. As mentioned previously, the presence of these contaminating materials may out-compete the desired algae, decreasing the yield and efficiency of the lipid production process. 
         [0010]    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary embodiments, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
         [0011]    Microalgae have been variously defined through the ages and it is prudent to describe the microalgae to which this invention could apply. For the purposes of this patent, microalgae include the traditional groups of algae described in Van Den Hoek et al. (1995). The subject application is applicable to, for example and without limitation, cultures of macroalgal seaweeds and all other algae which form filaments or other structures that could be helped during harvest by this invention. It is noted that this definition includes the cyanobacteria, traditionally referred to as bluegreen algae, which are prokaryotic in nature even though the majority of algal strains are eukaryotic in nature. 
         [0012]    The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the invention as a whole. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably. Furthermore, as used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such. The singular “alga” is likewise intended to be inclusive of the plural “algae.” 
         [0013]    The phrase “genetically modified algae,” as used herein, refers to algae whose genetic material has been altered using genetic engineering techniques so that it is no longer a “wild type” organism. An example of genetically modified algae is transgenic algae that possess one or more genes that have been transferred to the algae from a different species. Another example is an alga wherein endogenous genes have been rearranged such that they are in a different and advantageous arrangement or amplified so that specific sequences are increased. In this example, no foreign DNA remains in the modified cell. 
         [0014]    The phrase improved productivity or enhanced productivity of algae can refer to increases in oil content per cell, increased cell number per unit volume, increased cell size, increased areal productivity, various combinations of these, or other bio-product produced per unit area per time interval. 
       SUMMARY OF THE EMBODIMENTS 
       [0015]    These and other unmet needs of the prior art are met by exemplary compositions and methods as described in more detail below. 
         [0016]    Exemplary embodiments of the compositions, systems, and methods disclosed herein improve the process of producing biofuels and bioproducts from microalgae. This is achieved by enhancing the natural magnetic susceptibility of algal cells through genetic engineering and taking advantage of this magnetic property to improve the processing of algal biomass. These improvements include the separation of algal biomass from liquid media, and induction of cell lysis through magnetic hysteresis. The systems and methods provided include obtaining an algal strain(s) transformed by using an expression vector to improve iron utilization. Methods for utilization of said strains include improved growth conditions in open production ponds, improved dewatering through magnetic separation, and efficient product extraction through improved cell lysis via magnetic hysteresis. In one embodiment, for example, excess iron is stored in the form of ferritin engineered to be expressed in greater quantities than the wild-type strain which confers the enhanced magnetic susceptibility of the host cell. 
         [0017]    In one aspect, genetically engineered algae strains have resultant improved iron utilization for biofuels production for improved growth characteristics, magnetic separation, and magnetic hysteresis induced cell lysis. In open pond production, algal strains of the embodiments with high iron scavenging capabilities limit iron availability to contaminating microorganisms and invading species. Accumulation of high iron and other paramagnetic elements content in the form of ferritin enhances the cells magnetic susceptibility. Enhanced magnetic susceptibility improves the efficiency of magnetic separation and magnetic hysteresis induced cell lysis. Several genes are capable in the embodiments for improving iron acquisition including the ferritin gene Fer1, the iron transport gene Fea1, and the iron reductase gene Fre1. Other genes which improve growth in the high iron conditions thus permitting higher accumulation of iron in accordance with the embodiments include gene encoding, radical scavenging enzymes such as superoxide dismutase, peroxidase, catalase, glutathione peroxidase and the ferritin-like DPR/DPS genes which also bind iron and protect DNA from reactive oxygen species. Gill and Tetuja  Plant Physiol. Biochem.  2010 December; 48(12):909-30 and Chiacone and Ceci, Biochim. Biophys. Acta 2010 August; 1800(8):798-805. 
         [0018]    In a further aspect, embodiments of the present invention involve genetically modifying algal strains so that a gene encoding the ferritin protein is overexpressed and accumulates in the cell&#39;s cytoplasm, chloroplast(s), or mitochondria. 
         [0019]    Another embodiment of the present invention involves genetically modifying algal strains so that an iron transporter and iron reductase are overexpressed simultaneously, allowing more iron to be transported into the cell. 
         [0020]    In yet another embodiment of the present invention, contamination control and separation efficiency are enhanced through precise iron dosage and feeding strategies during the cultivation of algae genetically modified to over-accumulate ferritin complexes. 
         [0021]    In another embodiment of the present invention, the enhancement of magnetic susceptibility makes recombinant algae more susceptible to magnetic hysteresis. When exposed to a magnetic field of reversing polarity at high frequency (100-400 kHz), the magnetic poles on each paramagnetic element in the accumulated magnetic particles (Ferritin for example) switch orientation along with the field. Internal resistance to high frequency dipole switching can induce heat generation and may be sufficient to induce cell lysis, thus liberating the oil contained within the cell. Such an embodiment would provide significant cost advantages to the extraction of oil from these modified algae. 
         [0022]    In another embodiment of the present invention, paramagnetic or ferromagnetic particles/elements besides iron may be assimilated into biomass to improve the magnetic susceptibility of the cell as a whole. These may include but are not limited to: manganese, lithium, magnesium, aluminum, tin, calcium, titanium, cobalt, nickel, tungsten, and neodymium. These elements may be incorporated into functional biomass, locked into storage mechanisms, or transported into the vacuole. 
         [0023]    In another embodiment of the present invention, strains may be improved to tolerate high concentrations of ferromagnetic or paramagnetic particles present within the cell during photosynthetic growth. This may be achieved by overexpression of genes that provide resistance to the specific stresses induced by the accumulation of one or more of the various elements (or similar) described above. For example the overaccumulation of iron can have deleterious effects on algal growth by interacting with some components of the photosynthetic apparatus. Reactive oxygen species can be produced by the Photosystem I complex under high light conditions. When these reactive oxygen species interact with iron they become even more reactive as free radical compounds (example: Fe 2+ +H 2 O 2 →Fe 3+ +OH+OH − ) and more toxic to a broader variety of sites within the cell. Genes encoding proteins that protect against reactive oxygen species would thus enable growth under conditions such that iron can overaccumulate safely within the cell. Some examples of radical scavenging enzymes are catalase, peroxidase, superoxide dismutase, glutathione peroxidase and the ferritin like protein DPS or DPR which bind and store iron, and bind to DNA. The DPR/DPS complexes serve to protect DNA from damage by free radicals. 
         [0024]    In yet another embodiment of the present invention, strategies to reduce the fluidic drag on cells during magnetic separation can enhance magnetic separation. There are two forces competing against magnetic separation. The first is gravity. Gravity pulls on the cell in a constant direction causing settling at the bottom of any reactor or separation chamber. The second is fluidic resistance. As cells move in a column of water toward a magnetic pole or surface, the rate at which cells move is a function of the strength of the magnet, the paramagnetic moment of the cells and the drag forces of the fluid the cells are suspended in. Reducing these drag forces can improve the efficiency of magnetic separation, reduce the time of magnetic separation, and increase flow rates. This improved efficiency can be achieved by maintaining a high ratio of magnetic moment/cell volume. That is, a small cell will be easier to separate if it has the same iron content as a larger cell as the fluid drag on the larger cell is greater and works against magnetic separation. It may also be possible to engineer genetic elements that reduce the drag coefficient of the algal surface through modification of glycosylation patterns or altering the composition of the cell wall 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]    A better understanding of the exemplary embodiments of the invention will be had when reference is made to the accompanying drawings, and wherein: 
           [0026]      FIG. 1   a  shows an example construct of a gene encoding ferritin protein (fer1) linked to the RuBisCo (rbcL) promoter and ATP synthase β subunit (atpB) terminator for improving the magnetic properties of algae. 
           [0027]      FIG. 1   b  shows an example construct of a gene for iron transport (fea1) expressed from an actin promoter and terminator for improving the magnetic properties of algae. 
           [0028]      FIG. 2  is a schematic illustration showing iron homeostasis under normal conditions as controlled by ferritin, iron transporters, and iron reductases. 
           [0029]      FIG. 3  is a schematic illustration showing high iron homeostasis achieved through routes including over-expression of the ferritin gene and resulting complexes, over-expression of the iron transporter and required iron reductase; and increasing the availability of iron in the medium surrounding these genetically modified algae through controlled and precise iron dosing in accordance with example embodiments. 
           [0030]      FIG. 4  is a schematic illustration showing magnetic separation based on an increased magnetic susceptibility due to increased iron storage in ferritin complexes in accordance with an example embodiment. 
           [0031]      FIG. 4   a  is a chart illustrating mean velocities of algae cells in a uniform magnetic field such as the field of  FIG. 4 . 
           [0032]      FIG. 5  is a schematic illustration showing magnetic separator system in accordance with an example embodiment which operates by allowing the target magnetically susceptible particle to attach directly to a rotating magnetic disc(s). 
           [0033]      FIG. 6  is a schematic illustration showing magnetically induced cell lysis of algae genetically modified to over-accumulate ferritin complexes in accordance with the example embodiments. 
           [0034]      FIG. 7  is a graphical illustration showing enhancement of contamination control and separation efficiency through precise iron dosage and feeding strategies in accordance with the example embodiments. 
           [0035]      FIG. 8  is a graphical illustration showing isolates of iron replete cells grown in iron deficient medium in accordance with example embodiments. 
           [0036]      FIG. 9   a  is a chart illustrating relative growth of algae in accordance with example embodiments in iron replete and in iron depleted conditions compared to wild-type algae. 
           [0037]      FIG. 9   b  is a bar chart illustrating bacterial contamination in algal growth in accordance with example embodiments in iron replete and in iron depleted conditions and as against wild-type algae. 
           [0038]      FIG. 10  is a growth chart illustrating effects of strains upon transfer to low iron media. 
           [0039]      FIGS. 11   a  and  11   b  are growth charts showing an impact of overexpression of multiple genes involved in iron assimilation on growth in media containing elevated iron. 
       
    
    
     DETAILED DESCRIPTION 
       [0040]    With reference now to the drawings wherein the showings are for purposes of illustrating the example embodiments only and not for purposes of limiting same, using the variety of methods of the exemplary embodiments of the invention, the example embodiments described herein are directed to improved algal cells and at improving the extraction of desirable compounds from algal cells. The example embodiments are particularly applicable to magnetic separation of microalgae modified to safely assimilate iron in the form of paramagnetic particles from liquid media. 
       Genetic Modification of Microalgae to Enhance Magnetic Susceptibility 
       [0041]    Ferritin, a protein existing in nearly all living organisms that store iron, is expressed in microalgae. Ferritin is an iron storage protein complex consisting of &gt;4000 iron atoms arranged such that the complex becomes paramagnetic. However, the amount of ferritin naturally occurring in microalgae is conditionally variable but typically insufficient to permit magnetic separation of algae from liquid media under normal growing conditions. Cells containing sufficient amounts of ferritin bound iron (either internally or externally labeled) demonstrate a response when exposed to a magnetic field. In a suspension, this response is to move toward one pole or another of the magnetic field. As discussed above, magnetic separation of algae from their liquid medium is desirable because it is less costly and time- and labor-intensive than conventional separation methods. To employ this method of separation, however, the amount of iron in algal cells must be increased such that the cells are sufficiently magnetically susceptible. 
         [0042]    A specific range of intracellular iron levels is required for normal growth and reproduction. If iron levels fall outside this range, either above or below, growth is slowed. To buffer the bioavailability of intracellular iron, ferritin complexes capture and store excess iron, and subsequently release iron as necessary for optimal growth. Iron homeostasis under normal conditions is controlled by ferritin, iron transporters, and iron reductases. 
         [0043]    With reference now in particular to  FIGS. 1   a  and  1   b , ferritin is nuclear encoded in algae and transported to the chloroplast for incorporation into ferritin complexes. Photosynthetic iron requirements in the chloroplast are high, giving the storage of iron in the chloroplast an evolutionary advantage over nuclear, cytoplasmic or mitochondrial localization as it permits a faster response to homeostatic iron levels. The nuclear fer1 gene encodes ferritin in  Chlamydomonas reinhardtii  (Long &amp; Merchant, Photochemistry and Photobiology, 84: 1395-1403 (2008)). Increased storage of iron, over wild type cells, can be therefore achieved by expressing the fer1 gene from  Chlamydomonas reinhardtii  in the chloroplast genome under the control of the RuBisCo large subunit promoter and atpB 3′ UTR as seen in  FIG. 1   a.    
         [0044]    In this case, the described fer1 gene encoding ferritin is located on a plasmid adjacent to a selective marker. Together these two elements are flanked by regions of sequence homology to an insertion site in the chloroplast genome of interest enabling double crossover homologous recombination. This recombination will successfully insert the selectable marker and the targeted ferritin gene into the chloroplast genome. Other potential promoters and terminators may be used such as those for photosynthesis core proteins, 16S rRNA, and chlorophyll biogenesis. Further alternative promoters may be inducible such that the magnetic properties only present themselves when necessary to reduce the burden of the increased iron requirements. These inducible promoters may be activated by light, specific carbohydrates, salt shock, heat stress, or other signal molecules that could be applied to a production environment. The iron homeostasis is then balanced by increased activity of the iron transporter and coupled iron reductase. Ferritin production in cells can also be increased by overexpressing the genes for iron transporters and iron reductases from the constitutively active highly expressed actin promoter. Known  Chlamydomonas reinhardtii  iron transporters include FTR1, FEA1, FEA2, IRT1, and IRT2 (Fei et al., J. of Biomedicine and Biotechnology, 2010, 1-9 (2010); Allen et al., Eukaryotic Cell, Vol. 6, No. 10: p. 1841-1852 (2007)). Iron reductases include fre1 which may encode a ferrireductase that aides the transport of iron by reduction of Fe 3+ to Fe 2+ (Long et al., Genetics Soc. Of Am., 179: p. 137-147 (2008)). These genes can use their own native terminator or a terminator native to the target host. As shown in  FIG. 1   b , fea1 is expressed from the constitutively active highly expressed actin promoter. As with the FER1 example embodiment of  FIG. 1   a , alternative promoters, such as those that are activated by the presence of certain compounds or stressors, may make this system inducible. 
         [0045]    Chloroplast genomes of  Chlamydomonas reinhardtii  have been transformed by biolistic bombardment with gold particles containing linearized plasmids carrying at least two kinds of genes and two flanking elements. The two flanking elements are regions of DNA taken from the host strain that allow double crossover homologous recombination. This is a naturally occurring process in algal chloroplasts that permits the introduction or recombination of DNA elements into a targeted site. For instance these two flanking regions may come from a contiguous region between genes. In this case, the insertion of the genetic material between the two flanking regions on the plasmid will be inserted into a “neutral region” on the chromosome. In another scenario it may be necessary to interrupt or “knock out” a gene by using two flanking regions comprised of DNA sequence identical to sequence from a target gene or from flanking regions around a target gene to be displaced. Based on scientific literature, roughly 600 nt of identical sequence in both flanking regions is sufficient for efficient transformation. 
         [0046]    The first of the two genes used for inducing magnetic susceptibility is a selectable marker. This marker gene can be comprised of an antibiotic resistance gene such as the aadA gene conferring resistance to spectinomycin or it could be the reintroduction of a previously displaced gene such as psbA or other required native gene. This selectable marker allows the easy identification of colonies after transformation through growth on selective medium (containing spectinomycin or under photoautotrophic growth respectively for the two example marker genes). Regardless of the gene used as a marker it will be necessary to ensure moderate expression. This is achieved through the use of chloroplast appropriate 5′ and 3′ UTRs. Native genes expressed from a chloroplast genome use a typical prokaryotic promoter element with or without a −35 region. Typical 5′UTRs contain a −10 region (TATAATAT) around 10 nucleotides upstream of the +1 nt of the transcript (transcript initiation site) followed closely by a ribosome binding site (GGCC). A 5′UTR roughly 100 nt in length is sufficient for moderate expression. Typically 5′UTRs from native genes such as atpA, atpB, psbA, psbB, rpoA, or other specific gene are used as promoter elements. 3′ UTR regions contain a stop codon (or pair of stop codons) followed by an AT rich region of ˜20 nt and a hairpin forming region of DNA of roughly 25 nt in length. This stops translation and transcription and stabilizes the transcript for full activity. Stopping transcription is required when there are concerns of read through of downstream genes. 
         [0047]    The second gene used for this construct is the gene conferring the magnetic property. In this example that target gene is ferritin. The native ferritin from the  C. reinhardtii  nuclear genome is sufficient but will require trimming of the N terminus sequence to its mature form for expression from the chloroplast and codon optimization to that preferred by the target chloroplast. (Joanne C. Long, 2008). In this case the ferritin gene is expressed using the rbcL 5′ UTR and atpB 3′ UTR for expression at high levels in the chloroplast as seen in  FIG. 1 . Over-expression of the FER1 protein produces ferritin complexes in abundance allowing significantly enhanced iron storage capability. Use of native ferritin allows normal iron homeostasis, but with increased storage capacity. Iron stored in ferritin complexes are paramagnetic and thus confer the magnetic properties necessary for separation, and magnetic hysteresis. 
         [0048]    The DNA sequence used to generate the Fer1 over-expression strain 100 illustrated in  FIG. 1   a  and described above is, in the example embodiment, as set out as follows (in fasta format). 
         [0049]    &gt;Fer1 overexpression vector sequence for transformation of  Chlamydomonas reinhardtii  chloroplastic genome: 
         [0000]    
       
         
               
             
           
               
                 CCCTTTTGGCAGGACGTCCCTTTCCCCTTACGGGATATTTATATACTCC 
               
               
                 CATGTTTTGCCCTTTACGGGACGCCAGTGGACGTCCCCTTCCCCTTCCG 
               
               
                 GGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCCCTTA 
               
               
                 CGGGAATATAAATATTAGTGGCAGTTGCCTGCCAACTGCCGAGGCAAAT 
               
               
                 AAATTTTAGTGGACAAATTTATTTATTGTTGCCTGCCAACTGCCGATAT 
               
               
                 TTATATACTCCGAAGGAGGCAGTTGCCGGCCAACTGCCTGCCCCGCAGT 
               
               
                 ATTAACATAAGCAGTGTCGGTACCATTGCCACTGGCGTCCCGTTAGGTG 
               
               
                 TTCGTAGCAGGCAGTGTCCTGCCACTGCCTCCTGTGGAGTATATAAATA 
               
               
                 TCCCTAAGTGTACTTGCCCGTAAAGGGAAAGGGAAGGGTATTTTATTTA 
               
               
                 CTTTTTAGGTTTAGTAATAAATGAATCACAGGTAATTTCACTTTACATG 
               
               
                 CCATAAACTCTCTAATATACGTAAAGCTTCGCTCTGCGCAAGACTAAAG 
               
               
                 TGCCCAAGCTCAAAGAACCTTTTCAAGAGTAAATAAATTCCCTTAAGGG 
               
               
                 CTAATGAGCAAATTTAATGGTTATAGTTTTGTCAAGAAACGTTAGCACT 
               
               
                 GTTAGTACCAGGTTTAAAACATTTTTAATTTTTAATAATGCCAACAAAC 
               
               
                 AATGTCGATGTAAGAAATGGATTTATTTTTTTAATAAATTTAATTTTAT 
               
               
                 GCGTGAATCAATTCGAAGCGAATATGAAGTGAGCGGTTCAAAAAGAACA 
               
               
                 ACTAACTATGGCTTTAGATATGTGAAATATCTATATAACTAAATTGCAC 
               
               
                 ACCCTTTTGTAAATAGAACCTAAAAAGGAAAAAAATTGAATTTAATAAA 
               
               
                 TGACCCCTCAGCCAAAAGGTATGATTGGCAGTGCCTTTACTCGATAAAC 
               
               
                 CCTTTGGAGATTTAATTGCCCTTAAAAGAAAAGACTAAAAATGTTTGCT 
               
               
                 ACCCAGGCAGTAGCGGGATTTGAAATACTCCGTACAGCAGACGTCCCCT 
               
               
                 TACGGGACGTCAGTGGATATTTATATACTCCGAAGGAGGCAGTTGCCTG 
               
               
                 CCAACTGCCGATATTTATATACTGCGATAAACTTTAGTTGCCCGAAGGG 
               
               
                 GTTTACATACTAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTCCTTC 
               
               
                 GGAGTATGTAAACTACTTGCCCGAAGGGGAAGGAGGAAGCAGGCAGTGG 
               
               
                 CGGTACCACTGCCCCTTACGGGATCCAATTGACGGGATCCGAATTCCGT 
               
               
                 GAGCTCACGCGATCGCGAGGTACCCATATGACTAGTCATCTAGATAAAT 
               
               
                 TTGCTAGTTTACATTATTTTTTATTTCTAAATATATAATATATTTAAAT 
               
               
                 GTATTTAAAATTTTTCAACAATTTTTAAATTATATTTCCGGACAGATTA 
               
               
                 TTTTAGGATCGTCAAAAGAAGTTACATTTATTTATATAAATGGACAAAA 
               
               
                 TCACTGGCATTGTTGTTCAGCCTGCTGTTCAGTTCTCTGAAGTTCAGTC 
               
               
                 TGAACTTGCTACTGTTGATAAAACTAACCAGAACATCCAGTCACTTGCA 
               
               
                 CGTGTTGACTTCCACCCAGCTTGTGAAGCAGCTATCAACGAACAGGTAA 
               
               
                 ACATCGAATACAACGTTTCTTACCTATACCACGCTCTTTGGGCATACTT 
               
               
                 CGACCGTGACAACGTAGCTCTACCTGGCCTTGCTGCTTTCTTCAAAGCT 
               
               
                 GGCTCTGAAGAAGAACGTGAACACGCAGAACTTCTTATGGAATACCAGA 
               
               
                 ACCGTCGTGGCGGCCGTGTTGTTCTAGGCGCAATCTCTATGCCTGACCT 
               
               
                 AGACCTTTCTGCTTCTGAAAAAGGCGACGCTCTTTACGCAATGGAACTT 
               
               
                 GCTCTTAGCCTAGAAAAACTTAACTTCCAGAAACTTCGTCAGCTTCACT 
               
               
                 CTGTTGCAGACGAACACGGCGATGCTTCTATGGCTGACTTCGTAGAAGG 
               
               
                 CGAATTATTAAACGAACAGGTTGAAGCTGTTAAAAAAGTTTCAGAATAC 
               
               
                 GTATCTCAGCTTCGTCGTGTTGGCCAGGGCCTAGGCGTTTACCAGTTTG 
               
               
                 ACAAACAGCTTGCAGCTGAAGTAGCAGCTGGTGCTGCAGCTTAATAACT 
               
               
                 CGAGCCCGGGATGTGTAATTAAAATAAATATTTGGACACCATTAAGTTG 
               
               
                 TTTTCTTCTTAAAGAGCCAATTTATTTTAATTACACATACATTATATAT 
               
               
                 GTCATATTAGCTATTTAATCTATTTACTTTAAAATAAAGGACGCCGTTA 
               
               
                 AACGTGTACGCGTGCATCGGCTGGTACCATGCATGCGGCCGCAGTAGAC 
               
               
                 TTTATTAGAGGCAGTGTTTATATACCTAAACGTCAAAAGTCATTTTTAT 
               
               
                 AACTGGTCTCAAAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCT 
               
               
                 AAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAAT 
               
               
                 ACTATATAAATACATTTACCTTTTTATAAATACATTTACCTTTTTTTTA 
               
               
                 ATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAA 
               
               
                 ACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAG 
               
               
                 CAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCC 
               
               
                 CCCTTCCCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATA 
               
               
                 AATTTGTCCTCTTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGG 
               
               
                 GTGAACCATTACTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTG 
               
               
                 TTGAATTTTAAACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAA 
               
               
                 CTTTTTTAATCTTTTATTTATTTTTTCTTTTATGGCAATGCGTACTCCA 
               
               
                 GAAGAACTTAGTAATCTTATTAAAGATTTAATTGAACAATACACTCCAG 
               
               
                 AAGTGAAAATGTCCATGGCTCGTGAAGCGGTGATCGCCGAAGTATCGAC 
               
               
                 TCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACG 
               
               
                 TTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGC 
               
               
                 CACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGA 
               
               
                 AACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCC 
               
               
                 CCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGC 
               
               
                 ACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATT 
               
               
                 TGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCC 
               
               
                 ACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATA 
               
               
                 GCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCC 
               
               
                 TGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAAC 
               
               
                 TCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGT 
               
               
                 CCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGT 
               
               
                 CGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTC 
               
               
                 ATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGG 
               
               
                 CCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGA 
               
               
                 GATCACTAAGGTAGTTGGCAAATAACAGCTTGTACTCAAGCTCGTAACG 
               
               
                 AAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTG 
               
               
                 TAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATT 
               
               
                 AAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGA 
               
               
                 TGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAG 
               
               
                 GTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGT 
               
               
                 TATCTTTTGTTTAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGA 
               
               
                 AAAATAAAATCTTAGCTTTTTATTATAGAATTTATCTTTATGTATTATA 
               
               
                 TTTTATAAGTAATAAAAGAAATAGTAACATACTAAAGCGGATGTAACTC 
               
               
                 AATCGGTAGAGTGCGATCGCTAGCACTAGTCTCGAGGTTTAAACATGTA 
               
               
                 CAAGCTTGGATCAATTGATTGGCTGCTGTGCAGCCCCAAAGTAGAAGGG 
               
               
                 TACGCCAGTGGACGTCAGTGGCAGGCAACTGCCTCCTTCGGAGTATATA 
               
               
                 AATATTGGCAGTTGGCAGGCAACTGCCACTGACGTCCACTTAAATTTAT 
               
               
                 TTGCCCGAAGGGGACGTCCACTGGCGTACCGTAAGGGGAAGGGGACACC 
               
               
                 CACTGGCGTCCCGAAAGGATTAGCCAATGGGCGAGGCCACTGCTTCAGC 
               
               
                 TACTAAAGTTTTAAAGTGGTCTGTTGGACCATCAAATTCATATGGCATG 
               
               
                 TAATACTCCTAACGCTAATGCCTGGCTTTAGGGTCAGTTTACTGACCCA 
               
               
                 AAAACCTGTTAATAGCTTGTTAAACCCTTCTTTCCCTTTTGGAGTATAT 
               
               
                 CACTATCACAAAGGGGCGTCAGATGGAGTTAGTAAACTTTTTAAAGTGA 
               
               
                 ACAAGAATCACAGATGTAATAAGCTTATTAAGTAAAGCTTAGAAAAATT 
               
               
                 GTTTTAATAACTAACAAAACTTTAAAAGTTCAAAAATTTTAATGTTATG 
               
               
                 TATAATATTTTAATTAAAAAAACCATAACTATGTAAACCTTCTCCAATT 
               
               
                 AAATTTACACCTAAATAACAAAACCAAACAACTAAAAAACCAACAGCAG 
               
               
                 CAATAATTGCCGGTTTTTCACCTTCCCAACCA. 
               
             
          
         
       
     
         [0050]    With reference to  FIG. 1   b , the Fea1 over-expression construct for  Chlamydomonas reinhardtii  using the psaD promoter and terminator with aphVII as the selective marker in accordance with a further embodiment (in fasta sequence) is as set out below. The vector for over-expression of the fea1 gene in  Chlamydomonas reinhardtii  is based on pSL18 derived from the work of Dèpege et al. (Role of Chloroplast Protein Kinase Stt7 in LHCII Phosphorylation and State Transition in  Chlamydomonas in Science  7 Mar. 2003 pages 1572-1575). This vector contains an aphVII antibiotic resistance gene driven by the Hsp/RbcS2 promoter and RbcS2 terminator for selection on paromomycin. The fea1 gene was PCR amplified from cDNA of  Chlamydomonas reinhardtii  and cloned in a TOPO BluntII vector and subsequently sub-cloned in between the psaD promoter and terminator sequences of pSL18 as shown in  FIG. 1B . The plasmid was linearized and introduced into  Chlamydomonas reinhardtii  strain CC2137 through biolistic transformation. Transformants were selected on Modified High Salt Agar plates with paromomycin as the selective agent. Isolated colonies were picked and screened for the presence of the introduced fea1 gene (the introduced fea1 gene can be differentiated from the wild type by the lack of intronic sequences). Positive transformants were then assayed for growth under low iron conditions. The sequence for the two DNA elements contained within the transformation vector required for conferring fea1 expression and supplying a positive selection marker in  C. reinhardtii  is as follows below. 
         [0051]    &gt;Fea1 nuclear expression vector sequence for the two components required for transformation of  Chlamydomonas reinhardtii  102 is shown in  FIG. 1   b.    
         [0052]    &gt;Fea1 over-expression construct for  Chlamydomonas reinhardtii  using the psaD promoter and terminator with aphVII as the selective marker: 
         [0000]    
       
         
               
             
           
               
                 CTGGGTACCCGCTTCAAATACGCCCAGCCCGCCCATGGAGAAAGAGGCC 
               
               
                 AAAATCAACGGAGGATCGTTACAACCAACAAAATTGCAAAACTCCTCCG 
               
               
                 CTTTTTACGTGTTGAAAAAGACTGATCAGCACGAAACGGGGAGCTAAGC 
               
               
                 TACCGCTTCAGCACTTGAGAGCAGTATCTTCCATCCACCGCCGTTCGTC 
               
               
                 AGGGGGCAAGGCTCAGATCAACGAGCGCCTCCATTTACACGGAGCGGGG 
               
               
                 ATCCCAACGTCCACACTGTGCTGTCACCCACGCGACGCAACCCTACCCA 
               
               
                 GCCACCAACACCATCAGGTCCCTCAGAAGAACTCGTCCAACAGCCGGTA 
               
               
                 AAACGCCAGCTTTTCCTCCGATACCGCCCCATCCCACCCGCGCCCGTAC 
               
               
                 TCCCGCAGGAACGCCGCGGAACACTCCGGCCCGAACCACGGGTCCTCCT 
               
               
                 CGTGGGCCAGCTCGCGCAGCACCAGCGCGAGATCGGAGTGCCGGTCCGC 
               
               
                 ACGGCCGACCCGCCCCACGTCGATCAGCCCGGTCACCTCGCAGGTACGA 
               
               
                 GGGTCGAGCAGCACGTTGTCCGGGCACAGGTGACCGTGGCAAACCGCCA 
               
               
                 GATCCTCGTCCGCAGGCCGAGTCCGCTCCAGCTCGGCGAGAAGCCGCTC 
               
               
                 CCCCGACCACCCCTTCCGCTCCTCGTCCAGATCCTCCAAGTCGACGCTC 
               
               
                 CCTTCAGCGACAGCACGGGCCGCCTGCGGCACCGTCACCGCGAGACTGC 
               
               
                 GATCGAACGGACACCGCTCCCAGTCCAGCGCGTGCAGCGAACGAGCGAG 
               
               
                 CCCCGCGAGCGCCACCGCCACGTCCAGCCGCTGCTCCCGCGGCCACCGC 
               
               
                 GCACTGGCCGGACGCCCCGGAACCGCTTCGGTGACCAACCAGGCGACCC 
               
               
                 TCTCGTCCCCACCACCCTCCACAACACGAGGTACGGGAATCCCCACCTC 
               
               
                 CGCCAACCACACCAGCCGCTCAGCCTCACCCAACAAGCCCACCCCGGCC 
               
               
                 CCCAGAGCTGCCACCTTGACAAACAACTCCCGCCCACCACCCCGAAGCC 
               
               
                 GATAAACACCAGCCCCCGAGGCCCCATCCTCCACAACAACCCACTCACA 
               
               
                 ACCGGGATACCGACCCCGCAGTGCACGCAACGCATCGTCCATGCTTCGA 
               
               
                 AATTCTTCAGCACCGGGGAGGGCGGAGTGGCCATCCTGCAAATGGAAAC 
               
               
                 GGCGACGCAGGGTTAGATGCTGCTTGAGACAGCGACAGAGGAGCCAAAA 
               
               
                 GCCTTCGTCGACACAATGCGGGCGTTGCAAGTCAAATCTGCAAGCACGC 
               
               
                 TGCCTGATCCGCCGGGCTTGCTCGTCGACTCACCTGGCCATTTTAAGAT 
               
               
                 GTTGAGTGACTTCTCTTGTAAAAAAGTAAAGAACATAGGCCCCCTGGCC 
               
               
                 GGTTTATCAGGAGGGCACCGCTCCAGGGGCTGCATGCGAACTGCTTGCA 
               
               
                 TTGGCGCCTAGCCTTTGTGGGCCAGGGGGCTTCCGGATAAGGGTTGCAA 
               
               
                 GTGCTCAAATACCCCATCAAACATCATCCTGGTTTGGCTGCGCTCCTTC 
               
               
                 TGGCGCGCCCGGCATGCAAGCTTGATGGGATCTTAAGCTAGCTGAGTGG 
               
               
                 TTATGTATAGCGGCAGAATAGTCGCGTATGTATAAGTGCTCGTTTGTCG 
               
               
                 CTGAAAGTGGAGGTCACCGTTCGGGGTCGCGGGCTTTTATACCGGATGG 
               
               
                 GTGCCGCCAGCGGGCCGTATGGCGCCTTCTGGACGCCGCGCGCCCCATC 
               
               
                 GCGGCCCTTCCAGAGCTTCCCGCGCCCTCATAGCCCGCCAAATCAGTCC 
               
               
                 TGTAGCTTCATACAAACATACGCACCAATCATGTCAAGCCTCAGCGAGC 
               
               
                 TCCCCGCTCGAGCACACACCTGCCCGTCTGCCTGACAGGAAGTGAACGC 
               
               
                 ATGTCGAGGGAGGCCTCACCAATCGTCACACGAGCCCTCGTCAGAAACA 
               
               
                 CGTCTCCGCCACGCTCTCCCTCTCACGGCCGACCCCGCAGCCCTTTTGC 
               
               
                 CCTTTCCTAGGCCACCGACAGGACCCAGGCGCTCTCAGCATGCCTCAAC 
               
               
                 AACCCGTACTCGTGCCAGCGGTGCCCTTGTGCTGGTGATCGCTTGGAAG 
               
               
                 CGCATGCGAAGACGAAGGGGCGGAGCAGGCGGCCTGGCTGTTCGAAGGG 
               
               
                 CTCGCCGCCAGTTCGGGTGCCTTTCTCCACGCGCGCCTNCCACACCTAC 
               
               
                 CGATGCGTGAAGGCAGGCAAATGCTCATGTTTGCCCGAACTCGGAGTCC 
               
               
                 TTAAAAAGCCGCTTCTTGTCGTCGTTCCGAGACATGTTAGCAGATCGCA 
               
               
                 GTGCCACCTTTCCTGACGCGCTCGGCCCCATATTCGGACGCAATTGTCA 
               
               
                 TTTGTAGCACAATTGGAGCAAATCTGGCGAGGCAGTAGGCTTTTAAGTT 
               
               
                 GCAAGGCGAGAGAGCAAAGTGGGACGCGGCGTGATTATTGGTATTTACG 
               
               
                 CGACGGCCCGGCGCGTTAGCGGCCCTTCCCCCAGGCCAGGGACGATTAT 
               
               
                 GTATCAATATTGTTGCGTTCGGGCACTCGTGCGAGGGCTCCTGCGGGCT 
               
               
                 GGGGAGGGGGATCTGGGAATTGGAGGTACGACCGAGATGGCTTGCTCGG 
               
               
                 GGGGAGGTTTCCTCGCCGAGCAAGCCAGGGTTAGGTGTTGCGCTCTTGA 
               
               
                 CTCGTTGTGCATTCTAGGACCCCACTGCTACTCACAACAAGCCCATATG 
               
               
                 TCGGTCGGATTTCTGGTCCTCGCGCTGGGCGCGCTTGTCGTGGCGACGG 
               
               
                 CGGACTACAAGGACGACGACGACAAGCAGCCCACGACGACTGGCACTCG 
               
               
                 CTTCGAGGGTTTCTCTTACGCGGGCAATGTCATTGGCTATGTGAACATG 
               
               
                 ACGATGGACTACTGCGACATCAAGGCCGCCATGGCTGCTGGCAACTTCA 
               
               
                 CCGAGGCTCTGTCCATCTACTCTACTGGCAAGAACTCGTTCTCTGGCCT 
               
               
                 GGCGCGCCGCACCTTCTTCCGCTTCGCCTCGTACATCACCGCCAACGGC 
               
               
                 TCCGTGGAGCCGCTGCACGACTCCATCCTGGCCGGCAAGGACACGTCCT 
               
               
                 CCCTGGACGCCGCCATCCGGGCTGCCCTGGCCGACGGCAAGGCCACCCT 
               
               
                 GGCCGCCGGTCTGATCCAGGTGGGCACGCTCAAGTACCACCTGCACGAG 
               
               
                 GTGGATGAGGCCTACAACAAGATCAAGACCTACCTGGCTGACGGCACCG 
               
               
                 GCAACCTCACCAACCTGGTTTCTGACGCCTCGGGCGCACCCCACAACGT 
               
               
                 GGATGAGGCCTGGGCGCTGTGGGCCGGCGGCGCCGCCAACAACTGCGGC 
               
               
                 ACCCTGTCCGGCTGGGCCTCCTCCCTGGGCGCCGCCATGGGCACCACCT 
               
               
                 TCCTCGGCAAGAGCTATGTCAACACCGCCATGATCAACACCGTCAATGA 
               
               
                 GATGCTGGCCGCCGCCCGCCTGTCTACCCTGAACATCCAAGCCTACGAC 
               
               
                 GCCGCGCGCACGAACGAGGTCCGACTGCTGACCCTGCTGGGCCTGCAGG 
               
               
                 GCGTGTCCGTGGCCGCGTACACCGCTGACGCCGCCGCCGCCTGCAAGCG 
               
               
                 CCCCGCCGCCGAGGTGGAGGATGCCAAGACCATGATCGCCGTGCACTGG 
               
               
                 GCGTACCTGGAGCCTATGCTCAAGCTGCGCAACTTCAAGGCCTCCGCCG 
               
               
                 TGACCGAGCTGCACCACCAGCTCACCGCCTCCAAGCTGAGCTACAAGAA 
               
               
                 GGTGGCCGCCGCGGTGAAGGGCGTGCTGTCGGCTATGGGCCGCCGCTCC 
               
               
                 AGCGAGCTGGGTGCCCCGCAGTCCGCCATCATTGCCGCCAACTGGAAGT 
               
               
                 GCAGCTCCAAGACCCTGCGCAGCATTGCGTAATCTAGATGGCAGCAGCT 
               
               
                 GGACCGCCTGTACCATGGAGAAGAGCTTTACTTGCCGGGATGGCCGATT 
               
               
                 TCGCTGATTGATACGGGATCGGAGCTCGGAGGCTTTCGCGCTAGGGGCT 
               
               
                 AGGCGAAGGGCAGTGGTGACCAGGGTCGGTGTGGGGTCGGCCCACGGTC 
               
               
                 AATTAGCCACAGGAGGATCAGGGGGAGGTAGGCACGTCGACTTGGTTTG 
               
               
                 CGACCCCGCAGTTTTGGCGGACGTGCTGTTGTAGATGTTAGCGTGTGCG 
               
               
                 TGAGCCAGTGGCCAACGTGCCACACCCATTGAGAAGACCAACCAACTTA 
               
               
                 CTGGCAATATCTGCCAATGCCATACTGCATGTAATGGCCAGGCCATGTG 
               
               
                 AGAGTTTGCCGTGCCTGCGCGCGCCCCGGGGGCGCAGTTTAGCTGACCA 
               
               
                 GCCGTGGGATGATGCACGCATTTGCAAGGACAGGGTAATCACAGCAGCA 
               
               
                 ACATGGTGGGCTTAGGACAGCTGTGGGTCAGTGGACGGACGGCAGGGGA 
               
               
                 GGGACGGCGCAGCTCGGGAGACAGGGGGAGACAGCGTGACTGTGCAATG 
               
               
                 CGGCCGCCACCGCGGTGGA 
               
             
          
         
       
     
         [0053]    In addition to the above, to express the DPS gene (sync — 2856 from the  Synechococcus  strain CC9311) in the chloroplast of the green alga  Chlamydomonas reinhardtii  the gene sequence is positioned between the rbcL promoter and atpB terminator such as that described for the fer1 gene. This is then situated within a plasmid construct adjacent to a selective marker gene with a promoter and terminator sequence. These two elements are then situated between two regions of sequence of &gt;600 nt in length showing identical sequence to two closely linked sequences in the targeted chloroplast genome. These two regions of sequence homology permit site directed insertion of the DPS and selective marker gene (aada in this example)into the chloroplast genome of  Chlamydomonas reinhardtii.  An example of how this sequence could be assembled is included in fasta format: 
         [0054]    &gt;DPS (Synechococcus CC9311) expression vector for  Chlamydomonas reinhardtii  chloroplast 
         [0000]                    GCCCTTTTGGCAGGACGTCCCTTTCCCCTTACGGGATATTTATATACTC       CCATGTTTTGCCCTTTACGGGACGCCAGTGGACGTCCCCTTCCCCTTCC       GGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCCCTT       ACGGGAATATAAATATTAGTGGCAGTTGCCTGCCAACTGCCGAGGCAAA       TAAATTTTAGTGGACAAATTTATTTATTGTTGCCTGCCAACTGCCGATA       TTTATATACTCCGAAGGAGGCAGTTGCCGGCCAACTGCCTGCCCCGCAG       TATTAACATAAGCAGTGTCGGTACCATTGCCACTGGCGTCCCGTTAGGT       GTTCGTAGCAGGCAGTGTCCTGCCACTGCCTCCTGTGGAGTATATAAAT       ATCCCTAAGTGTACTTGCCCGTAAAGGGAAAGGGAAGGGTATTTTATTT       ACTTTTTAGGTTTAGTAATAAATGAATCACAGGTAATTTCACTTTACAT       GCCATAAACTCTCTAATATACGTAAAGCTTCGCTCTGCGCAAGACTAAA       GTGCCCAAGCTCAAAGAACCTTTTCAAGAGTAAATAAATTCCCTTAAGG       GCTAATGAGCAAATTTAATGGTTATAGTTTTGTCAAGAAACGTTAGCAC       TGTTAGTACCAGGTTTAAAACATTTTTAATTTTTAATAATGCCAACAAA       CAATGTCGATGTAAGAAATGGATTTATTTTTTTAATAAATTTAATTTTA       TGCGTGAATCAATTCGAAGCGAATATGAAGTGAGCGGTTCAAAAAGAAC       AACTAACTATGGCTTTAGATATGTGAAATATCTATATAACTAAATTGCA       CACCCTTTTGTAAATAGAACCTAAAAAGGAAAAAAATTGAATTTAATAA       ATGACCCCTCAGCCAAAAGGTATGATTGGCAGTGCCTTTACTCGATAAA       CCCTTTGGAGATTTAATTGCCCTTAAAAGAAAAGACTAAAAATGTTTGC       TACCCAGGCAGTAGCGGGATTTGAAATACTCCGTACAGCAGACGTCCCC       TTACGGGACGTCAGTGGATATTTATATACTCCGAAGGAGGCAGTTGCCT       GCCAACTGCCGATATTTATATACTGCGATAAACTTTAGTTGCCCGAAGG       GGTTTACATACTAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTCCTT       CGGAGTATGTAAACTACTTGCCCGAAGGGGAAGGAGGAAGCAGGCAGTG       GCGGTACCACTGCCCCTTACGGGATCCAATTGACGGGATCCGAATTCCG       TGAGCTCACGCGATCGCGAGGTACCCATATGACTAGTCATCTAGATAAA       TTTGCTAGTTTACATTATTTTTTATTTCTAAATATATAATATATTTAAA       TGTATTTAAAATTTTTCAACAATTTTTAAATTATATTTCCGGACAGATT       ATTTTAGGATCGTCAAAAGAAGTTACATTTATTTATATAAATGGCCCAA       AGCTCCGCCATCGATATCGGCATCACCAGCGCACAACGGGAGGAAATCG       CTGCTGAACTCAGCCGCCTCCTCGCAGATACGTACGTGTTGTACGGCAA       AACCCACGGTTTCCACTGGAACGTGACCGGGCCGATGTTCAACACATTG       CACCTGATGTTCATGGACCAGTACACCGAGTTGTGGAACGCCCTGGATG       TGATCGCTGAACGCATACGCGCCCTTGGCGTTTTGGCGCCCCATGGAGG       TTCCACCCTGGCCGGTTTGGCCTCGATCCAAGAAGCAGAGCAACAGCCT       GCCGCGCTCGACATGGTGCGTGAGCTGGTAACCGGCCATGAGGCCGTTG       CCCGTACGGCACGGAGCATCTTTCCTCTGGCAGAGGCTGCCAACGACGA       ACCCACTGCTGACCTGCTCACCCAACGACTTCAGATCCACGAAAAAACA       GCCTGGATGCTGCGCAGCCTGCTGGAAAACTAATAACTCGAGCCCGGGA       TGTGTAATTAAAATAAATATTTGGACACCATTAAGTTGTTTTCTTCTTA       AAGAGCCAATTTATTTTAATTACACATACATTATATATGTCATATTAGC       TATTTAATCTATTTACTTTAAAATAAAGGACGCCGTTAAACGTGTACGC       GTGCATCGGCTGGTACCATGCATGCGGCCGCAGTAGACTTTATTAGAGG       CAGTGTTTATATACCTAAACGTCAAAAGTCATTTTTATAACTGGTCTCA       AAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCTAAGAACAATCA       ATTTATAAATATATTTATTATTATGCTATAATATAAATACTATATAAAT       ACATTTACCTTTTTATAAATACATTTACCTTTTTTTTAATTTGCATGAT       TTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAAACCTTTAAAGG       ACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAGCAATCGGCGTC       ATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTCCCCTT       ACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTC       TTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTA       CTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAA       ACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAACTTTTTTAATC       TTTTATTTATTTTTTCTTTTATGGCAATGCGTACTCCAGAAGAACTTAG       TAATCTTATTAAAGATTTAATTGAACAATACACTCCAGAAGTGAAAATG       TCCATGGCTCGTGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAG       AGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGT       ACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGAT       ATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGC       GAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAG       CGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATC       ATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGC       AGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACAT       TGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTG       GTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATC       TATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGA       CTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGG       TACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACT       GGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGC       TAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCA       GATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACTAAGG       TAGTTGGCAAATAACAGCTTGTACTCAAGCTCGTAACGAAGGTCGTGAC       CTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTGTAAATGGTCTC       CAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATTAAATTCGAATT       TGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTTATGTGA       ATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAGGTTAAATACCT       AAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTT       TAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGAAAAATAAAATC       TTAGCTTTTTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTA       ATAAAAGAAATAGTAACATACTAAAGCGGATGTAACTCAATCGGTAGAG       TGCGATCGCTAGCACTAGTCTCGAGGTTTAAACATGTACAAGCTTGGAT       CAATTGATTGGCTGCTGTGCAGCCCCAAAGTAGAAGGGTACGCCAGTGG       ACGTCAGTGGCAGGCAACTGCCTCCTTCGGAGTATATAAATATTGGCAG       TTGGCAGGCAACTGCCACTGACGTCCACTTAAATTTATTTGCCCGAAGG       GGACGTCCACTGGCGTACCGTAAGGGGAAGGGGACACCCACTGGCGTCC       CGAAAGGATTAGCCAATGGGCGAGGCCACTGCTTCAGCTACTAAAGTTT       TAAAGTGGTCTGTTGGACCATCAAATTCATATGGCATGTAATACTCCTA       ACGCTAATGCCTGGCTTTAGGGTCAGTTTACTGACCCAAAAACCTGTTA       ATAGCTTGTTAAACCCTTCTTTCCCTTTTGGAGTATATCACTATCACAA       AGGGGCGTCAGATGGAGTTAGTAAACTTTTTAAAGTGAACAAGAATCAC       AGATGTAATAAGCTTATTAAGTAAAGCTTAGAAAAATTGTTTTAATAAC       TAACAAAACTTTAAAAGTTCAAAAATTTTAATGTTATGTATAATATTTT       AATTAAAAAAACCATAACTATGTAAACCTTCTCCAATTAAATTTACACC       TAAATAACAAAACCAAACAACTAAAAAACCAACAGCAGCAATAATTGCC       GGTTTTTCACCTTCCCAACC            
Enhanced Contamination Control and Separation Efficiency through Precise Iron Dosage and Feeding Strategies
 
         [0055]    Strategic periodic iron dosing can be used to both improve contamination control and enhance both magnetic and gravimetric separation techniques. Under this method, a minimal threshold iron requirement is temporarily exceeded through periodic dosing of soluble iron in chelated form. The soluble iron concentration in solution is periodically elevated above the minimal threshold until it is assimilated into algal biomass. The moderate to high cell density of algae with over-expressed high affinity iron transporters out-competes wild-type strains and contaminants for the brief period that iron is available. 
         [0056]    Over time, iron is assimilated into algal biomass and accumulated to increasing levels. Just prior to harvesting, iron is dosed to very high levels, and is assimilated into algal biomass. This induces the over-accumulation of ferritin complexes thus achieving the conditions for magnetic separation of the algal biomass. The process of harvesting the biomass removes the iron from solution (assimilated in algal biomass) and the process continues while maintaining low iron concentrations. This dosing strategy may also allow ferritin to accumulate in the genetically modified algae in sufficient amounts to permit separation using gravimetric techniques. 
         [0000]    Enhanced Lipid Extraction Using Heat Produced from Rapid Magnetic Field Polarity Reversal on Modified Microalgae 
         [0057]    The energy required for lipid extraction for algae genetically modified to over-accumulate ferritin complexes can be reduced through the use of magnetic hysteresis. In the presence of a field of reversing magnetic polarity the dipole moment of paramagnetic particles switches orientations. At increased frequencies the internal resistance to this switching results in heat generation. At a frequency of or around 100 kHz, magnetic particles contained within or on the surface of algae could produce enough heat to cause cell deterioration and lysis. This is illustrated in  FIG. 6 . Cell lysis aids in the lipid extraction process as it liberates the oil from within the cell. Cell lysis through magnetic hysteresis would reduce the energy requirements for lipid extraction. This is achieved by focusing the energy expenditure on the target magnetically susceptible cells and not on the rest of the water and contaminating species. 
         [0000]    Enhancement of Ma g netic Susce s tibilit and Contamination Control Throu h Iron Dosin Strategies 
       Induced Magnetic Susceptibility During Growth in Excess Iron 
       [0058]    Iron homeostasis is a significant constraint on the welfare of all forms of life. It is absolutely required in several protein complexes and is toxic when exposed to oxygen in the presence of a reduced metabolic state where many forms of reactive oxygen species can be produced. As a result, a mechanism for storing iron and controlling its availability is highly desirable. Surprisingly few mechanisms have been identified. Instead a nearly ubiquitous mechanism of storing iron in ferritin or bacterioferritin complexes is present. A variety of iron transporters and reductases act to contribute to the iron pool, but ferritin and bacterioferritin are largely responsible for regulating the availability of that iron pool. 
         [0059]    One way to take advantage of the iron homeostasis mechanism for improved algal biofuels production is to grow the algae in excess iron. Iron concentrations at or in excess of 200 micromolar are inhibitory to photoautrophic growth of  C. reinhardtii  under full sunlight. However, it is not inhibitory to growth under low light or heterotrophic growth. Growth conditions where iron is spiked into the medium in the evening when the light levels are low permit optimal growth. The natural process of capturing iron and storing it enables photoautrophic growth when iron levels return to low levels. This condition enhances iron storage in the form of ferritin complexes. This increase in ferritin enhances magnetic susceptibility of the cell which enables magnetic manipulation. 
         [0060]    With reference next to  FIG. 2 , an algal cell  200  is surrounded by chelated ferric/ferrous iron molecules  202  in solution  204 . A ferric iron transporter  210  is illustrated. Ferritin complexes  220  and ferric reductases  222  are illustrated schematically. As shown, iron homeostasis under normal conditions is controlled by the ferritin, the iron transporters  210 , and the iron reductases  222 . The embodiments of the disclosure assist in promoting elevated iron homeostasis by enhancing the storage of iron in ferritin, and improving the iron transporters  210  and the iron reductases  222  mechanisms in manners to be described below. 
         [0061]    In particular, with reference next to  FIG. 3 , a schematic representation of iron homeostasis of an algal cell  300  in accordance with an example embodiment having enhanced magnetic susceptibility is shown. In accordance with the embodiments, high iron homeostasis is preferably achieved through at least three (3) routes, namely an over-expression of the ferritin gene  350  and resulting complexes, an over-expression of the iron transporter  360  and required iron reductase  370 , and an increased availability of iron  380  in the surrounding media either continuously or in sporadic dosing. 
         [0062]      FIG. 4  presents a schematic illustration of a process  400  for separation of algae  402  with enhanced magnetic susceptibility from liquid media  404  and other organisms  406 . As shown, the algae with enhanced magnetic susceptibility  402  are disposed in a flow  410  of a liquid such as, for example, a water flow. A magnetic field  420  is generated in a direction substantially transverse to a direction  412  of the water flow. Under these conditions, high ferritin algae  430  formed in accordance with any of the embodiments herein are reactive to the magnetic field and migrate in a direction transverse to the flow. On the other hand, under these conditions, low ferritin algae  440  not being formed in accordance with any of the embodiments herein, together with any other contaminants  450  are non-reactive to the magnetic field and therefore follow along without significant deviation with the fluid flow  410 . Accordingly, by this process and system, high ferritin algae  430  formed in accordance with any of the embodiments herein are easily and efficiently separated from low ferritin algae  440  not being formed in accordance with any of the embodiments herein, together with any other contaminants  450 . 
         [0063]      FIG. 4   a  is a chart illustrating mean velocities of algae cells in a uniform magnetic field such as the field of  FIG. 4 . With reference in particular to  FIG. 4   a , algae strains in accordance with the embodiments are placed in a fluid bath and the mean velocity of the modified algae cells when exposed to a uniform magnetic field is measured. In accordance with another embodiment, an associated Cell Tracking Velocimetry (CTV) instrument is preferably used to help characterize the magnetic moment of selected one or more particle types in the solution. (Chalmers, et. al. Quantification of Non-Specific Binding of Magnetic Micro- and Nanoparticles Using Cell Tracking Velocimetry: Implication for Magnetic Cell Separation and Detection, Biotechnology and Bioengineering, Vol. 105, No. 6, Apr. 15, 2010). As illustrated in the chart, it is demonstrated in accordance with data collected that all of the cells move in the direction of the magnetic field. However only the Fer1 strain in accordance with the example embodiment herein moved fast enough to overcome their inherent random motility and gravitational settling. In the plots of  FIG. 4   a , it is to be appreciated that the error bars are greater than the mean velocity, except for the Fer1 strain  460  grown in elevated iron. While any of these strains could be separated using a stronger magnet than what is in this particular system, the most magnetically susceptible strain and condition was Fer1 grown in 3× the iron content of normal MHS. 
         [0064]      FIG. 5  shows a schematic illustration of a magnetic separator  500  in accordance with a further embodiment. As shown there, material  502  from a growth pond (not shown) is introduced to a rotating magnetic wheel  510  via a fluid chute  504 . The high ferritin algae  530  are attracted to the wheel  510  because of the mutual magnetic properties of the wheel and algae. As discussed in connection with the embodiment of  FIG. 4 , high ferritin algae  530  formed in accordance with any of the embodiments herein are easily and efficiently separated from low ferritin algae  540  not being formed in accordance with any of the embodiments herein, together with any other contaminants  550  wherein the contaminants and low ferritin algae  540  pass by the wheel and continue on to a discharge chute  560  to be carried to a treatment plant or otherwise returned to the pond. In addition to the separation technology described above it is also potentially feasible in accordance with the embodiments to use other forms, systems or technologies of magnetic separation including for example superconducting electromagnetic filter separation or high intensity rare earth magnetic drum systems which can handle the high volumes and small particle sizes required for algal biofuels production. 
         [0065]      FIG. 6  is a schematic comparative illustration  600  showing magnetically induced cell lysis  602  of algae  610  genetically modified in accordance with the embodiments herein to over-accumulate ferritin complexes  620  in accordance with the example embodiments. 
         [0000]    Iron dosing strategy for control of contaminants 
         [0066]      FIG. 7  illustrates a graph  700  showing enhancement of contamination control and separation efficiency through precise iron dosage and feeding strategies. A minimal threshold iron requirement  710  (dashed line) is temporally exceeded through periodic dosing of soluble iron in chelated form. The soluble iron concentration in solution  720  (black line) is periodically elevated above the minimal threshold until it is assimilated into biomass. The moderate to high cell density of algae with over-expressed high affinity iron transporters outcompetes wild type strains and contaminants for the brief period that iron is available. Iron is assimilated into algal biomass  720  (gray line) and accumulated to increasing levels. Just prior to harvesting, iron is dosed to very high levels, and is assimilated into algal biomass inducing the over-accumulation of ferritin complexes thus achieving the conditions for magnetic separation of the algal biomass. The process of harvesting the biomass removes the iron from solution (assimilated in algal biomass) and the process continues while maintaining low iron concentrations. This dosing strategy may also allow ferritin to accumulate in the genetically modified algae in sufficient amounts to permit separation using gravimetric techniques. 
         [0067]    With reference next to  FIG. 8 , it is to be appreciated that the availability of iron in the growth media is a common constraint for both algae and contaminants in an open pond algal production scenario. The development of algal strains with enhanced iron uptake and storage makes it possible to inhibit growth of contaminants by limiting the bioavailability of iron. Algal strains with improved iron uptake or storage can be produced through the over-expression of ferritin, or iron transporters, and their corresponding reductase components. This is demonstrated in  FIG. 8  where, as shown, several fea1 over-expression isolates were screened for growth under low iron conditions. In particular, it is to be noted that over-expression of the fea1 gene permits enhanced growth under low iron and low light conditions and can approximate growth of the wild-type under normal iron conditions with low illumination. 
         [0068]    In this example, the high affinity iron storing algae are grown in medium with iron concentrations below the threshold required to maintain growth of the contaminating species. Periodically, additional iron is added to the medium to supply enough iron for algal growth. In a relatively short amount of time, however, this iron is biologically removed from the medium and assimilated into biomass as iron storage particles (ferritin complexes). Due in part to the high abundance of algae in the medium and largely to the high activity iron transport and storage of the engineered strain, it is able to out-compete contaminating strains and bacteria. The result is a scenario diagrammed in  FIG. 7  where genetically modified algae are able to scavenge the trace iron and the temporarily excessively available iron that enables their growth while limiting the growth of competitors. 
         [0069]    As demonstrated in  FIGS. 9   a  and  9   b , the modified strain over-expressing the fea1 gene under control of the psaD promoter and terminator shows improved growth under iron limiting conditions with reduced bacterial contamination when in open containers grown in a greenhouse environment (full sunlight). In this case, both strains were grown in open top vessels for a period of nine days. Samples were taken to measure optical density at 750 nm to demonstrate growth. After day six, samples were serially diluted and spread plated on tryptic soy agar to isolate single colonies. After one day&#39;s incubation, the colonies formed on the surface of the agar plates were counted (cfu/mL). Error bars represent the standard deviation across three replicate cultures of each strain at each condition. 
         [0070]    In  FIG. 9   b , as shown, bacterial contamination is reduced by growing cultures under low iron conditions. Further improvements result when the algal strain is over-expressing the iron transporter gene fea1 whose gene product scavenges any iron introduced through airborne dust particle contamination or other methods. 
         [0000]    Cell Lysis through Magnetic Hysteresis 
         [0071]    With reference to  FIG. 6 , engineered strains of algae formed in accordance with any of the example embodiments herein are capable of producing excess ferritin complexes and storing iron in abundance inside these complexes making them paramagnetic. This can be achieved through any one or more of the examples provided above. Magnetically susceptible cells are then exposed to a magnetic field of reversing polarity of around 100 kHz. This field induces magnetic hyperthermia and cell damage. The result is damaged cell walls, which liberate the lipids contained within the cell. When used in combination with magnetic separation, this method reduces the energy requirements for cell lysis as energy is only applied to the target algae species, not contaminating species and excess water. Ferritin complexes of the novel algae of the embodiments are susceptible to magnetic hysteresis induced heat generation. Under conditions where ferritin complexes are present at high levels, it is possible to induce cell lysis from the heat generated from their resistance to rapid magnetic dipole switching. Cell lysis aids the lipid extraction process in the example embodiment by liberating the oil within the algal cells. 
         [0072]      FIGS. 10 ,  11   a , and  11   b  are charts with examples of one measure of increased productivity of modified strains grown under conditions of depleted iron and under excess iron. In both cases growth enhancement is demonstrated by increases in optical density which is often accepted as increases in biomass on a per volume basis. In  FIG. 10  the fer1 overexpression strain  1003  tolerates the switch to low iron media and grows for several generations before inernal iron stores are depleted and growth becomes limited while the wild type strains growth  1004  is limited immediately upon transfer to low iron media. Note that the greater lag demonstrated by the wild-type strain  1002  is likely a result of the EDTA washes used to remove iron from the external surface of the cells. The actual growth maximum approximates that of the fer1 overexpression strain  1001 . The impact of an extended lag period in growth is uncertain, whereas the impact of maximal growth rate is a certain impact on productivity. 
         [0073]      FIGS. 11   a  and  11   b  demonstrate the additive or even synergistic impact of overexpression of multiple genes involved in iron assimilation on growth in media containing elevated iron.  FIG. 11   a  illustrates a strain comparison graph  1100  in media containing 28 micromolar iron and  FIG. 11   b  illustrates a strain comparison graph  1102  in media containing 96 micromolar iron. The FF7 strain  1110  co-expresses both fea1 and fer1 and confers greater tolerance to growth at high iron concentrations than either of the fea1  1112  or fer1  1114  overexpression lines, both of which are arguably better than Wild-type  1116  at growth under elevated iron conditions.