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Transgenic microalgae as green cell-factories

Rosa Leon-Banares1, David Gonzalez-Ballester2, Aurora Galvan2 and 2 Emilio Fernandez
1 2

Departamento de Qumica, Area de Bioqumica, Universidad de Huelva, Avda de las Fuerzas Armadas s/n, 21071 Huelva, Spain Departamento de Bioqumica y Biologa Molecular, Campus de Rabanales, Edif Severo Ochoa, Universidad de Cordoba, 14071 Cordoba, Spain

There is increasing interest in the use of microalgae for biotechnological applications and as plant model systems. Although biotechnological processes based on transgenic microalgae are still in their infancy, researchers and companies are considering the potential of microalgae as green cell-factories to produce valueadded metabolites and heterologous proteins for pharmaceutical applications. New molecular biology tools are needed to standardize genetic modications of microalgae. Here, we outline methods and strategies for efcient nuclear transformation of microalgae, and discuss the main difculties associated with stable expression of transgenes. Most progress in the eld has been made with Chlamydomonas reinhardtii, but other species have now been successfully transformed, and many others will be transformed in the near future. Microalgae constitute a diverse group of prokaryotic and eukaryotic organisms with great ecological importance. They account for , 50% of global organic carbon xation [1]. Many species are a source of natural products and have been exploited for biotechnological applications [2,3] (see Table 1). In contrast to the large number of genetically modied bacteria, yeast and even higher plants, only a few species of microalgae have been successfully transformed with efciency. Efcient transformation systems in

microalgae are therefore necessary to enhance their potential; this has motivated researchers and companies to work towards this goal. Development of molecular tools for genetic engineering has mainly focused on Chlamydomonas reinhardtii, for which stable genetic transformation at both the chloroplast [4] and nuclear [5,6] level was rst reported. Since then, numerous selectable markers, promoters and procedures for the efcient insertion of DNA into the microalgal nucleus have been developed, and transformation efciency has increased dramatically. The main characteristics and potential uses of nuclear and chloroplast transformation are summarized in Box 1. The feasibility of microalgae to be genetically modied and express heterologous genes opens up the possibility of enhancing the productivity of traditional algal compounds and producing new bioactive products for industrial and pharmaceutical applications through metabolic engineering. In addition, microalgae can be used as model eukaryotic hosts, especially in cases where specic genes cannot be expressed in yeast (i.e. genes related to agellar function, photosynthesis and photoreception [7,8]). Furthermore, microalgae can be engineered to express highquality mammalian proteins, such as hormones or antibodies. Transgenic algae are especially suitable for containment and controlled growth in bioreactors both under phototrophic and heterotrophic conditions [3,9].

Table 1. Companies exploiting or researching microalgae-based products

Microalgae-based product Food additives (carotenoids, food dyes and dietary supplements) Company Cyanotech (http://www.cyanotech.com/) Mera Pharmaceuticals (http://www.aquasearch.com/) Nikken Sohonsha Corporation NBT (http://www.chlostanin.co.jp/) Cognis (http://www.cognis.com/cognis.html) Subitec (http://www.subitec.com/) Far East Microalgae Ind. Co. (http://www.allproducts.com.tw/manufacture11/fareast/supplier.html) Nikken Sohonsha Corporation Cyanotech Phycotransgenic (http://www.phycotransgenics.com) PharmaMar (http://www.pharmamar.com/language.cfm) Cyanotech Earthrise Farms (http://www.earthrise.com/ERFarms.html) Entelechon (http://www.entelechon.com/) Subitec

Polyunsaturated fatty acids

Polysaccharides Fluorescent pigments (phycobiliproteins) Bioactive compounds (anti-tumour and vaccines) Biomass (animal feed, aquaculture, nutritional and healthy products) Eukaryotic expression systems for mammalian proteins

Corresponding author: Rosa Leon-Banares ([email protected]).

http://tibtec.trends.com 0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.11.003

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Box 1. Nuclear versus chloroplastic transformation

Integration of transgenes into the chloroplast has important advantages. It enables controlled site-directed recombination of constructs and results in high expression levels with no silencing drawbacks (Table I). However, nuclear transformation might enable a wider range of possibilities both for transgenic protein expression (e.g. excretion, different cell-compartment expression, and glycosylation) and for manipulation of algal metabolism (gene inactivation or overexpression, and gain of additional pathways) (Table I).

Table I. Main characteristics of nuclear and chloroplastic transformations

Nuclear Cell compartment of expression Recombination machinery for integration of exogenous DNA Gene silencing Inheritance of integrated gene Level of expression (gene copy number) Co-transformation of different markers Versatility to express genes from different organisms Glycosylation pattern of proteins Extracellular, cytosol and chloroplast, among others Mostly non-homologous Probable Mendelian Low to intermediate High Intermediate to low Similar to plants and animals Chloroplastic Chloroplast Homologous Not probable Maternal High High High None

Most reviews documenting the genetic transformation of microalgae have focused exclusively on Chlamydomonas sp. [1012]. No general reviews about the transformation of eukaryotic algae have been published since a review of the eld in 1997 [13]. Here, we provide a general perspective of the progress achieved since then. We examine the methods and strategies presently used for efcient nuclear transformation of microalgae, including those species that have been successfully transformed, the constructions (promoters and marker genes) used, and the main difculties in establishing a standardized method for such transformations. The scope of this review does not include multicellular algae, although it should be noted that efcient transformation and molecular techniques have been developed in species such as Volvox carteri [14]. Particular focus is given to the problems associated with stable expression of transgenes because gene silencing together with the lack of appropriate promoters and codon usage might hinder the transformation of the majority of microalgal species. Biotechnological applications of transgenic microalgae Although the use of transgenic microalgae for commercial applications has not yet been reported, several examples of engineered microalgae for biotechnological applications show signicant promise. The contribution of Dunahay and colleagues to the manipulation of microalgae lipid production by genetic engineering was one of the rst reported approaches for the production of bio-diesel by transgenic diatoms [15]. Sayre and colleagues reported transgenic microalgae with an enhanced ability to bind heavy metals through expression of a foreign metallothionein [16], or expression of the mothbean pyrroline-5-carboxylate synthetase (P5CS) gene, which induces the accumulation of free proline in Chlamydomonas [17]. These pioneering works highlight the potential applications of transgenic microalgae in bio-remediation. A collaboration between the same research group and the US biotechnological company Phycotransgenic (http://www.phycotransgenics.com) investigated the development of transgenic microalgae for industrial and pharmaceutical applications. Transgenic Chlamydomonas expressing an extracellular antigenic protein of the pathogenic bacteria Rennibacterium
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salmoninarum (which causes kidney disease in salmonids) was used to feed trouts and rabbits, which then generated antibodies against the antigenic protein. Expression of the protein was achieved by chloroplast transformation (US patent application 20030022359). Other examples of recombinant vaccines obtained through antigenic protein expression in the chloroplast of Chlamydomonas have recently been reported [18]. Apt and colleagues devised a promising technique for the heterotrophic large-scale culture of the diatom Phaeodactylum tricornutum. They modied this obligate photoautotrophic diatom to live in the dark with glucose as the only carbon source through expression of the glucose transporter gene of human erythrocytes [9]. Studies by Melis and colleagues focused on the biological production of hydrogen by genetically modied Chlamydomonas. This is an exciting approach for the biological generation of hydrogen gas, which is thought to be a cheap, clean and safe fuel. Chlamydomonas was genetically modied to downregulate the production of chloroplastic sulphate permease by insertion of an antisense CrcpSulp sequence. The absence or limitation of sulphur causes the reduction of normal oxygen-producing photosynthesis. When the algae are in a substantially oxygen-free system in the presence of light, they begin photosynthesizing through an alternative cellular pathway, which leads to the production of hydrogen (US patent application 20030162273). Most of the studies involving the genetic transformation of microalgae aim to optimize new transformation methods and promoter or reporter genes, or analyse mechanisms for stable expression of the introduced gene. Currently, these aspects are the main limitations for microalgae transformation; the following sections deal with each of these aspects individually. Methods for nuclear transformation The basis of traditional methods used to transform microalgae is to cause, by various means, temporal permeabilization of the cell membrane, enabling DNA molecules to enter the cell while preserving viability. One method of permeabilization involves the use of glass beads; cells are vortexed in the presence of DNA,

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glass beads and polyethylene glycol (PEG) [10]. This method has been successfully used for the transformation of cell-wall-decient mutants, or wild-type cells of Chlamydomonas following enzymatic degradation of the cell wall, and is routinely used owing to its simplicity and efciency. It will be interesting to investigate whether this method can be applied to other species of microalgae. A similar method using silicon carbide (SiC) whiskers was described for Chlamydomonas transformation without removal of the cell wall [19]. SiC-mediated transformation has been used for the genetic manipulation of several higher plants and some microalgae species such as Amphidinium and Symbiodinium [20]. However, because SiC whiskers are difcult to purchase and can be a health hazard, this method is not recommendable as a rst choice. The introduction of genes by electroporation has been carried out in many types of cells and organisms. Stable transformants of both wall-less and walled strains of Chlamydomonas have been obtained in this way with high efciency [21,22]. Another method used for the transformation of microalgae is the particle-gun method, also known as particle bombardment. This method uses DNA-coated particles of gold or tungsten that are accelerated into the target cells by a helium-driven gun. It is routinely used for the transformation of plant cells and tissues, and also for transformation of prokaryotes and eukaryotic organelles. This method has been particularly successful in the transformation of diatoms [23 26]. Viruses that infect Chlorella-like algae and brown algae are being considered for the development of cloning and expression systems [27]. The large size (150 330 kbp) of these double-stranded DNA viral genomes might permit the insertion of large sequences of foreign DNA into algae [28]. However, more extensive studies are required before this attractive possibility can be developed. The use of articial transposons originally designed for in vitro mutagenesis is now expanding to encompass other applications such as genetic transformation. Articial mini-transposons are obtained from natural transposons and only contain elements essential for transposition. Incubation of the mini-transposon with an adequate transposase, and transfer of the mixture into the target cell, results in the integration of the mini-transposon into the chromosome of the target cell. Kojima and Kawata [29] successfully introduced a mini-transposon transposase complex into the cyanobacterium Spirulina. Furthermore, these authors suggest that the transposase method might also be applicable to eukaryotic microalgae because the transposition requires no host factor; however, there are no reports to-date of eukaryotic microalgae being transformed in this way. Transformation of microalgae groups Eukaryotic algae are phylogenetically heterogeneous. Stable nuclear transformations have been reported in only three eukaryotic microalgal groups: chlorophytes, diatoms and dinoagellates. Recently, chloroplast transformation of the unicellular red algae Porphyridium sp. has been reported [30].
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The freshwater alga Chlamydomonas is the rst and best-studied transformation system of the chlorophytes or green algae group [5,6,10]. Along with the recent complete sequencing of its genome [31], this has made Chlamydomonas an excellent model system for diverse areas of research. In addition, Chlorella has been transformed with a luciferase (Luc) construction (which showed transient expression [32]) and with a homologous nitrate reductase gene [33]. However, other chlorophytes with important economical value, such as Dunaliella sp. or Haematococcus sp., have not yet been stably transformed [13]. Diatoms belong to the division chrysophyta and are of biotechnological interest because they have silica-based cell walls and can be used to obtain pharmaceutical products, or as a food source in aquaculture. Five different species have been transformed: P. tricornutum [23], Cyclotella cryptica [24], Navicula saprophila [24], Cylindrotheca fusiformis [34] and Thalassiosira weissogii [25]. The microparticle bombardment method was used for transformation of all of these species. Dinoagellates or pyrrophyta are a group of unicellular eukaryotic alveolar algae that constitute an important part of marine phytoplankton. Some species of this division produce luminescent compounds; others produce compounds that are toxic to vertebrates, including humans. Species of this group have typical eukaryotic cytoplasmic features but the characteristics of their nucleus are relatively unique. They lack histones and combine typical prokaryote characteristics, such as permanently condensed chromosomes, with eukaryotic features, such as the presence of nucleolus or introns. Amphidinium and Symbiodinium are the two species of this group that have been successfully transformed [20]. Constructions and strategies Once the transgene has entered the cell and has been integrated into the nuclear chromosome, it has to be expressed. To ensure its transcription, a promoter region that is adequately recognized by the RNA polymerase of the host must precede the transgene; the mRNA is then translated. Similarity between codon usage of the transcript and that of the host organism is another important aspect that should be considered. Adequate availability of marker and reporter genes is key for selection of the transformed microalgae. For several years, the only adequate selection method available for microalgae transformation used homologous genes (Arg7, Nit1, Oee1 or AtpC) [10] for complementation of specic mutants. In all of these cases, a mutant defective in the chosen gene was isolated and transformed with a functional copy of the required gene from the same organism. However, this strategy is not applicable to wild-type or diploid microalgae (i.e. diatoms) owing to the difculties involved in generating mutants in which both alleles of a gene are defective. Nowadays, a large collection of reporter genes and selectable markers are available, some of which are summarized in Table 2. The most powerful selectable markers are those that confer resistance against antibiotics or herbicides, for example, Ble, NptII and AphVIII. It is important to note, however, that bleomycin could have mutagenic effects, and this

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Table 2. Marker and reporter genes used in microalgal constructs

Gene AphVIII Description Aminoglycoside 30 phosphotransferase (resistance to paromomycin, kanamycin and neomycin) Bleomycin resistance protein (resistance to tallysomycin and related antibiotics) Marker/reporter Marker Gene source Streptomyces rimosus Streptoalloteichus hindustanus Expressed in Chlamydomonas sp. Refs [50]

Ble

Marker

Chlamydomonas Phaeodactylum tricornutum Chlamydomonas

[40] [23] [45]

NptII

Neomycin phosphotransferase II (resistance to G418)

Marker

Transposon Tn5 from Escherichia coli

Als Cry1-1 Oee-1 Cat

Acetolactate synthase (resistance to sulphonylurea herbicides) Ribosomal protein S14 Oxygen evolving enhancer protein Chloramphenicol acetyltransferase (chloramphenicol resistance) Hygromycin B phosphotransferase Adenylyl transferase (spectinomycin resistance) Nourseothricin resistance Nourseothricin resistance Modied green uorescent protein

Marker Marker Marker Marker

Chlamydomonas Chlamydomonas Chlamydomonas Transposon Tn9

Amphidinium sp. and Symbiodinium sp. P. tricornutum Cyclotella cryptica and Navicula saprophila Chlamydomonas Chlamydomonas Chlamydomonas Chlamydomonas P. tricornutum Amphidinium and Symbiodinium Chlamydomonas P. tricornutum P. tricornutum P. tricornutum

[20] [26] [24] [51] [62] [42] [46] [23] [20] [53] [26] [26] [9]

Hpt AadA Nat Sat-1 eGfp

Marker Marker Marker Marker Reporter

E. coli Eubacteria Streptomyces noursei E. coli Synthetic (adapted to human codon usage) Synthetic (adapted to Chlamydomonas codon usage) E. coli

ChGfp

Modied green uorescent protein

Reporter

Chlamydomonas

[41]

Gus

b-Glucuronidase

Reporter

Glut1 Hup1

Glucose transporter Hexose transporter Calcium-binding glycoprotein Arylsulphatase Luciferase

Marker or reporter Marker or reporter Reporter Reporter Reporter

Human Chlorella kessleri Navicula pelliculosa Chlamydomonas Horatia parvula

e -frustulin
Ars Luc

Amphidinium and Symbiodinium P. tricornutum P. tricornutum P. tricornutum Cylindrotheca fusiformis C. fusiformis Chlamydomonas P. tricornutum

[20] [26] [9] [9] [34] [34] [36] [25]

must be taken into account, especially during insertional ndez et al., unpublished). mutagenesis experiments (E. Ferna Some reporter genes, such as Gus and adapted versions of the gene encoding green uorescent protein (Gfp), can be used to test protein localization when fused to the desired
Box 2. Main problems associated with foreign gene expression in microalgae
- Inadequate method of DNA delivery - No integration into the chromosome - Inadequate recognition of the promoter region - Biased codon usage - Lack of adequate regulatory sequences - Incorrect polyadenylation - Inappropriate nuclear transport - Instability of mRNA - Positional effects - Silencing by methylation Epigenetic silencing mechanisms
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protein [12,35]. The Ars gene provides an easy and rapid assay for studies of promoter activity, as shown in Chlamydomonas [36,37]. As in higher plants, most nuclear transformations observed in chlorophytes, diatoms and dinoagellates appear to involve random integration into the genome of one to several copies of the introduced DNA, depending on the DNA concentration used [10]. Nuclear homologous recombination has also been observed in Chlamydomonas, although at a much lower frequency [10]. Small, linearized plasmids tend to be inserted into the nuclear genome preferentially through their ends, causing deletions (5 20 kb) and/or rearrangements at the integration site. However, such characteristics could be exploited as a method for insertional mutagenesis [10,38]. Transformation with linearized DNA seems to be more efcient, and integration events are more predictable than with circular DNA [10]. Nevertheless, it is important that

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the chosen cleavage-site is not close to the 50 or 30 untranslated regions of the introduced gene, thereby preserving its integrity [39]. One of the difculties in expressing heterologous genes in microalgae is selection of the transformants. This problem can be solved by co-transformation with two different plasmids, one of which contains an easily selectable marker, although frequency of expression of both genes can be relatively low (10 50%) [10,40]. Transformation efciency increases dramatically if the two genes are in the same construction and if the construction is relatively small [20,41]. A cosmid vector containing the Ble cassette as selectable marker has been constructed for cloning Chlamydomonas genes by complementation [42]. Another construct of a shuttle vector with the Arg7 gene under the control of a chimeric promoter, rbcS2::T7, enables selection in Escherichia coli and Chlamydomonas [39]. Marker and reporter genes are expressed efciently under the control of strong constitutive homologous promoters (Table 3), or promoters from very close species [24]. The upstream fusion of a Hsp70A promoter fragment enhances the activity of neighbouring promoters [43] and decreases the probability of transcriptional silencing [44]. In diatoms, the promoters fcp-A and -B have been used with great success [26]. The use of inducible promoters, such as Nia1 from Chlamydomonas, provides a better control of expression level [36,37]. Attempts to express genes fused to heterologous promoters were unsuccessful in the diatom P. tricornutum [23]; in Chlamydomonas, transformation frequency was low [45] or expression was unstable [46]. The only report of stable transformation of microalgae with heterologous genes under the control of heterologous promoters is the expression of Gus driven by either the cauliower mosaic
Table 3. Promoters used in microalgal constructs
Promoter Nopaline synthase (Nos) Gene source Agrobacterium tumefaciens

virus 35S promoter or the p10 20 Agrobacterium promoter in the dinoagellates Amphidinium and Symbiodinium [20]. The unique nuclear characteristics of these microalgae can inuence their ability to express genes under the control of heterologous promoters. A certain frequency of transformation has been achieved with promoter-less heterologous genes in Chlamydomonas, probably as a result of in vivo gene-fusion with endogenous promoter regions [39,47]. Identication of promoters (promoter trapping) was achieved in Chlamydomonas with Arg7 as selectable marker, and a promoter-less reporter gene (Rsp3), which was expressed in 2 3% of the transformants [48]. However, it is generally accepted that, in chlorophytes and diatoms, stable expression of heterologous genes can only be optimally achieved when adequate homologous promoters and other regulatory regions are included. The presence of introns is an important factor for efcient expression. Ble and AphVIII genes, under the control of the rbcS2 promoter, increased their expression through inclusion of the rst intron of rbcS2 [49,50]. Expression of the Als gene under the control of the rbcS2 promoter was also optimal when all Als introns were present [51]. However, introns are not absolutely required for gene expression, as exemplied by experiments with the cDNA of Arg7 [39]. Expression vectors that have promoters from genes lacking introns, such as PsaD, are useful in enabling high-expression of endogenous and exogenous cDNAs [52]. The insertion of 30 untranslated regions increased expression in Chlamydomonas probably owing to increased mRNA stability [49,53] and their possible role as a reverse promoters [50]. Homologous 30 untranslated regions of rbcS2 and fcpA from Chlamydomonas and Phaeodactylum, respectively, are two of the most frequently used untranslated regions.

Expressed in Chlamydomonas sp., Amphidinium sp. and Symbiodinium sp. Chlamydomonas Amphidinium and Symbiodinium P. tricornutum

Fused gene NptII Nia1 NptII Cat Gus Ble Glut1/Hup1 Ble/NptII/Gfp/Gus e -frustulin Ble Luc Gfp Ble AphVIII Ble Cry1-1 Als AadA Arg7 cDNA Ars Ars Ble/Arg7/PsaF Gfp Cop AphVIII NptII Hpt Nia1

Refs [45] [20] [46] [20] [23] [9] [26] [34] [25] [41] [50] [40] [62] [51] [53] [39] [36,37] [43] [52] [41] [63] [24] [20] [64]

Cauliower mosaic virus 35S Fucoxanthin chlorophyll-a or -c binding protein

Cauliower mosaic virus Phaeodactylum tricornutum

RbcS2 (Rubisco)

Chlamydomonas

Chlamydomonas

Nia1(Nit1) Heat shock protein (Hsp)70B Hsp70A Photosystem I complex protein (PsaD) Chlamyopsin (Cop) b2-Tubulin Acetyl CoA carboxylase (Acc1) p10 20 Chlorophyll-ab binding (CabII-1)
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Chlamydomonas Chlamydomonas Chlamydomonas Chlamydomonas Chlamydomonas Cyclotella cryptica A. tumefaciens Chlamydomonas

Chlamydomonas Chlamydomonas Chlamydomonas Chlamydomonas Chlamydomonas C. cryptica and Navicula saprophila Amphidinium and Symbiodinium Chlamydomonas

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(a) Codon abundance (%) 60 50 40 30 20 10 0

Chlamydomonas reinhardtii

Ble

NptII

Aph VIII

CrGfp

(b) Codon abundance (%) 60 50 40 30 20 10 0

Chlamydomonas reinhardtii

Gfp

Gus Phaeodactylum tricornutum

Luc

(c) Codon abundance (%) 14 12 10 8 6 4 2 0

Ble

NptII

Glut1

Gus

eGfp

(d) Codon abundance (%) 14 12 10 8 6 4 2 0

Phaeodactylum tricornutum

Gpf

mGfp4

Hxt1

Hxt2

Hxt4

Extremely rare (03%) Rare (710%)

Very rare (46%) Total rare (010%)

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organisms is rarely used in others. This causes differences in tRNA abundance and signicantly inuences translation efciency and expression levels. The extremely high GC content found in Chlamydomonas and other chlorophytes (61% in Chlamydomonas and 71% in Monoraphidium sp.) [54] was the rst indication of biased codon usage in certain microalgae. In some diatoms, such as P. tricornutum, GC content is , 48% a typical value for eukaryotic organisms. Codon usage in Phaeodactylum is also biased, with a preference for C or G in the third position of the codon [55]. Comparisons of the codon bias of typical heterologous markers and those of Chlamydomonas and P. tricornutum genes are shown in Figure 1. Interestingly, in Chlamydomonas, unsuccessfully expressed genes (Gfp and Luc) show very high percentages of rarely used codons, whereas the genes that are successfully expressed (AphVIII, NptII and Ble) have a codon usage similar to that of Chlamydomonas genes. However, the Gus gene, which shows a percentage of rare codons similar to NptII, has not been successfully expressed in Chlamydomonas. These data suggest that, in addition to favourable codon usage, other factors are also important for expression (Box 2). In P. tricornutum, the abundance of rare codons is similar in both expressed and non-expressed genes (Figure 1c,d), although the percentage of extremely rare codons is much higher in those that are not expressed. Fuhrmann and colleagues [12,41] synthesized DNAs encoding Gfp and Luc with the codon preference of Chlamydomonas. These engineered genes were successfully expressed in Chlamydomonas and have been commercialized by the German company BioCat (http://www.biocat.de). Attempts to express Gfp in P. tricornutum [9,26] have helped to understand the importance of codon bias in the successful expression of heterologous genes. The expression of several Gfp genes under the control of the fucoxanthin chlorophyll binding protein (Fcp) promoter was studied in P. tricornutum. It was found that eGfp (adapted to human codon usage), which has a codon bias similar to that of P. tricornutum genes, was the only version of Gfp that was signicantly expressed. Low expression of wild-type Gfp and mGfp4 (developed for use in vascular plants) was attributed to the effect of codon bias. Stability of transgenes Expression of an exogenous gene can be very low or nonexistent, even though all the elements required for optimal transcription and translation (promoters, introns and other regulatory regions) have been included in the chimeric gene construction. Furthermore, when transgenic algal clones are not maintained under selection conditions, expression of the exogenous gene might be suppressed. This gene silencing has been attributed to a variety of epigenetic mechanisms similar to those observed in plants and other eukaryotic cells, and is thought to be related to the control of development and to the response of a cell to viruses, transposable elements, or transgenes [53,56]. Epigenetic processes are dened as heritable changes in gene expression without modication of DNA sequences. These changes might occur at the level of transcription or post-transcription by mechanisms that are not yet fully

Figure 1. Comparison of codon usage of several heterologous genes with that of Chlamydomonas reinhardtii and Phaeodactylum tricornutum. Percentage indicates the abundance of rare codons in the cited genes. Rare, very rare and extremely rare codons are those with fractions of use in the reference organisms from 7 to 10%, 4 to 6% and 0 to 3%, respectively. Total rare refers to the total percentage of codons in the reference organism with a fraction of use ,10%, and is equal to the sum of the number of rare, very rare and extremely rare codons. (a) Successfully and (b) unsuccessfully expressed genes in C. reinhardtii. (c) Successfully and (d) unsuccessfully expressed genes in P. tricornutum. Data are relative to full-length coding sequences. GenBank accession numbers are as follows: Ble, X52869; Npt II, L11017; AphVIII, AF182845; Gfp, M62653; CrGfp (Gfp adapted to C. reinhardtii), AF188479; eGfp (GFP adapted to humans), U57609; mGfp4 (Gfp adapted to Arabidopsis thaliana), U87625; Luc, L39929; Glut1, K03195; Gus, S69414. Hxt genes from Saccharomyces cerevisiae: Hxt1, L07079; Hxt2, M33270; Hxt4, M81960. Comparisons of codon usage performed using the Graphical Codon Usage Analyser (Version 1.0) (http://gcua.schoedl.de/ index.html). Microalgae codon usage was calculated from 602 genes and 26 genes in C. reinhardtii and P. tricornutum, respectively.

Codon usage Bias in codon usage is an important limitation for the expression of heterologous genes in microalgae. Biased usage means that a codon frequently used in certain
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understood. Several elements involved in the silencing of transgenes in Chlamydomonas have recently been isolated [56]. Transcriptional gene silencing can occur through an altered chromatin structure that is associated with cytosine methylation of the promoter regions. However, single-copy transgenes can be transcriptionally silenced without detectable cytosine methylation of introduced DNA [57,58]. Post-transcriptional transgene silencing can take place by an RNA-mediated process called RNA interference (RNAi). RNAi forms RNA-induced silencing complexes that promote RNA degradation [59]. It is worth noting that mechanistic connections between epigenetic transcriptional silencing and DNA double-strand-break repair have recently been proposed [58]. This disadvantage for gene expression can result in the useful technique of RNAi [12]. The low efciency of homologous nuclear recombination in microalgae that prevents the direct isolation of desired mutants can be overcome by an antisense mRNA strategy or by RNAi, with varying degrees of success [60] (http://www.biology.duke.edu/ chlamy/methods/antisense.html). The best results were obtained from fusion of a 50 genomic DNA fragment of the gene with the corresponding cDNA in an antisense orientation. This construct is predicted to form a doublestranded RNA with a hairpin that induces post-transcriptional gene silencing [61,57]. Concluding remarks Initial difculties associated with the expression of foreign genes in microalgae have stimulated both a search for creative solutions, such as the synthesis of Gfp or Luc adapted to Chlamydomonas codon usage, and a signicant increase in the availability of promoters and selectable marker genes. Nevertheless, the number of species that have been successfully genetically modied remains extremely low. More work is needed to transform new species of microalgae, especially those that have commercial value. Silencing of transgenes remains the main limitation for stable expression of foreign genes, although this problem is not unique to microalgae, being observed also in plants, animals and fungi. A better understanding of the mechanisms that control the regulation of gene expression in eukaryotes is therefore needed. The use of transgenic microalgae as efcient cell-factories for the production of recombinant vaccines, mammalian antibodies and added-value compounds, such as carotenoids, polyunsaturated fatty acids, hydrogen or bio-fuel, is clearly the immediate challenge for this technology.
Acknowledgements
We acknowledge nancial support from the Spanish Ministry of Science and Technology (BMC2002-03325 and the Ramon y Cajal research programme).

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