# Patent

## Title
Astaxanthine biosynthesis in eukaryotes

## Abstract

The invention relates to a DNA vector comprising (a) a DNA sequence coding for the phytoene desaturase protein that is modified in one position by an amino add exchange providing resistance, and (b) a multiple cloning site into which any DNA sequence can be cloned. The invention also relates to the use of said DNA vector for transforming enkaryotic cells, transformation methods, and transgenic plant cells produced in said manner.

## Background
RELATED APPLICATIONS
This application is a §371 of PCT/EP2006/009881 filed Oct. 12, 2006, which claims priority from German Patent Application No: 10 2005 049 072.7 filed Oct. 13, 2005.
The invention relates to a DNA vector which comprises (a) a DNA sequence which codes for the protein phytoene desaturase which is modified at one position by a resistance-conferring amino acid substitution and (b) a multiple cloning site into which any DNA sequence can be cloned, to its use for the transformation of eukaryotic cells, to transformation methods, and to transgenic plant cells thus prepared.
BACKGROUND OF THE INVENTION
Carotenoids are pigments which are found in all photo-synthetic organisms. They play an important role as components of the photosynthetic reaction center and in mediating protection against photooxidative damage.
The carotenoid biosynthetic pathway proceeds from geranylgeranyl diphosphate (GGDP) to astaxanthin. As the result of the condensation of two molecules of geranylgeranyl pyrophosphate, the phytoene synthase enzyme (PSY) forms the C40 structure phytoene. Starting from phytoene, the phytoene desaturase enzyme (PDS) synthetizes ζ-carotene by eliminating protons and incorporating two double bonds. The intermediate of this synthetic step is phytofluene. ζ-Carotene, in turn, is converted into lycopene in a two-step desaturation reaction, which proceeds via the intermediate neurosporin. The enzyme which is responsible therefor is ζ-carotene desaturase (ZDS). Lycopene is converted into β-carotene via a lycopene cyclase (LCYB). Starting from β-carotene, two further enzymes are involved in the formation of astaxanthin. Firstly, the β-carotene ketolase enzyme (BKT) introduces in each case one keto group at the 4 and the 4′ position. Secondly, in each case one hydroxyl group is attached at the 3 and 3′ positions on the ion ring of the astaxanthin precursor via the carotenoid hydroxylase enzyme (CHY).
Overexpressing a bacterial phytoene synthase from Eriwinia uredovora has made it possible to influence, or stimulate, carotenoid biosynthesis in transgenic tomato plants and thus to increase the amount of carotenoid synthesized by a factor of 2-4 (Fraser, Romer et al. 2002).
However, it is the enzyme phytoene desaturase which plays the central key role in the carotenoid biosynthetic pathway (FIG. 1). The pds genes, which code for phytoene desaturase, have been cloned from cyanobacteria (Chamovitz et al., 1991; Martínez-Férez and Vioque, 1992; Martínez-Férez et al.,. 1994) and higher plants (Bartley et al., 1991; Pecker et al., 1992) and are successfully overexpressed in E. coli. The bleaching herbicide norflurazon is known as a reversible, noncompetitive inhibitor of phytoene desaturase. Mutated forms of phytoene desaturase have been described for the cyanobacterium Synechococcus; they confer resistance to norflurazon to the bacterium. In each case, the resistance-imparting mutations are based on a single amino acid substitution. Within the scope of the mutation studies carried out in Synechococcus, resistances have been found for the following amino acid substitutions: Arg195Pro; Leu320Pro; Val403Gly; Leu437Arg (Linden et al., 1990; Chamovitz et al., 1993). Despite the fact that norflurazon and other such bleaching herbicides have been used for some time for controlling weeds, no resistant naturally occurring plants have been isolated to date.
Among the intermediates and products of the carotenoid biosynthetic pathway, it is in particular the keto carotenoid astaxanthin which is of commercial importance. Astaxanthin has a higher antioxidant activity than other intermediates of the carotenoid biosynthetic pathway. Astaxanthin acts as a quencher of free radicals and active oxygen species (Kobayashi and Sakamoto, 1999), as an enhancer of immune responses (Jyonouchi et al., 1995) and as an anticancer agent (Tanaka et al., 1994, 1995). Owing to its natural effect as potent antioxidant, astaxanthin is also employed as food supplement. It is used as food additive with colorant effect in fish farming.
Some green algae such as, for example, Dunaliella bardawil and Haematococcus pluvialis have the unique ability of accumulating carotenoids under stress conditions. In this context, it is in particular H. pluvialis which is suitable for the natural production of astaxanthin. H. pluvialis is capable of accumulating astaxanthin in amounts of up to 4% of its dry weight. This is why H. pluvialis plays a key role in the commercial production of astaxanthin, since the chemico-synthetic route for the production of astaxanthin is among the most complex which are commercially employed for producing an active ingredient.
The genetic transformation of green algae has been described repeatedly for Chlamydomonas reinhardtii and some Chlorella species (Kindle 1990; Lumbreras et al., 1998; Hawkins and Nakamura, 1999; Kim et al., 2002). Owing to their metabolism, these two algal species are thought to be less interesting for the natural production of carotenoids.
The genetic transformation of H. pluvialis has been described by Teng at al. (2003). The β-galactosidase reporter gene lacZ under the control of the SV40 promoter has been integrated into the algal genome by means of microparticle bombardment. Successfully transformed algal cells must be detected and selected individually with the aid of optical means. Algal cells which are transformed with the lacZ gene have no resistance to toxically, or dominantly, acting selection agents.
Although astaxanthin biosynthesis has been studied in detail during the last 10 years (Lu et al., 1995; Fraser et al., 1998), the production of astaxanthin by H. pluvialis on the large scale of the biotechnology industry remains problematic since, inter alia, cell division is inhibited during astaxanthin biosynthesis (Boussiba and von Vonshak, 1991).
It is an object of the present invention to provide a DNA vector which can be used for suitably genetically modifying the genome of eukaryotic cells, in particular of algae such as H. pluvialis, in order to be able to influence, or increase, the in-vivo synthesis of natural carotenoids and isoprenoids and, in particular, astaxanthin biosynthesis. It is another object of the invention to provide a vector which can be employed as dominantly-selective marker for the transformation in eukaryotic cells, in particular of algae such as H. pluvialis, and which makes it possible to select, in a simple manner, successfully transformed eukaryotic cells, in particular of algae such as H. pluvialis. 
The invention achieves this object by a vector comprising (a) a DNA sequence which codes for the protein phytoene desaturase which has a resistance-conferring amino acid substitution at one position, and (b) a multiple cloning site into which any DNA sequence can be cloned.
In this context, the vectors according to the invention comprise any DNA molecules which can be used as vehicles with the aid of which foreign DNA can be introduced into a cell. They encompass cosmids, phages, viruses, YACs, BACs, more linear DNA molecules and, in particular, circular plasmids.
Within the scope of the invention, the DNA sequence of the enzyme phytoene desaturase (PDS) from H. pluvialis has been isolated from a genomic DNA library and sequenced (SEQ ID NO:1). The corresponding protein sequence of the H. pluvialis phytoene desaturase is shown in SEQ ID NO:2. The subject matter of the invention comprise all nucleic acid sequences which, taking into consideration the degeneration of the genetic code, code for the protein sequence SEQ ID NO:2.
Preferred vectors are DNA vectors in which the DNA sequence (a) which codes for the protein phytoene desaturase, the pds gene, is preferably derived from H. pluvialis and the resistance-conferring amino acid substitution has been introduced by means of directed mutagenesis. Especially preferred vectors are those which code for the protein H. pluvialis phytoene desaturase which has an amino acid substitution from leucine to arginine at position 504 of its amino acid sequence (SEQ ID NO:3).
The amino acid substitution according to the invention from leucine to arginine at position 504 of the H. pluvialis PDS protein corresponds to the Leu437Arg substitution in Synechococcus PCC7942 (FIG. 2). For this purpose, the Leu codon “CTG” is replaced by the Arg codon “CGC” by directed mutagenesis. This mutation imparts, to the mutants, the highest known resistance factor to norflurazon. The resistance which has been conferred imparts, to the mutants according to the invention, a resistance to norflurazon which is up to 70-fold higher in comparison with the wild type. In accordance with the invention, norflurazon concentrations of 0.5-50 μm are used as selection substance.
Further positions in the amino acid sequence of H. pluvialis which are preferred for an amino acid substitution are arginine 264, leucine 388, valin 465 (FIG. 3). In Synechococcus, a resistance-mediating effect is known for the amino acid substitution at homologous positions.
Preferred are vectors which mediate, to transformants, a resistance to herbicides, in particular bleaching herbicides, and especially preferably norflurazon.
Within the scope of the invention, multiple cloning site (MCS), also referred to as polylinker, is understood as meaning a region in a nucleic acid sequence which has a large number of different utilizable cleavage sites for restriction endonucleases without the function of other elements of the nucleic acid sequence being adversely affected when a restriction hydrolysis takes place. Preferably, the DNA molecule is hydrolyzed only at one position by restriction endonucleases, whose cleavage sites are defined in the MCS. Examples of conventional MCSs are well known to the skilled worker from commercially available vectors and plasmids. Within the meaning of the present invention, MCS is furthermore understood as meaning any restriction cleavage site within a vector which can be used in order to clone in, into the vector sequence, any DNA sequences in a directed or undirected fashion without in the process adversely affecting other functional elements according to the invention of the vector.
An embodiment, of an MCS, which is preferred in accordance with the invention is shown in SEQ ID NO:4. In a preferred embodiment, the DNA vector according to the invention has the sequence SEQ ID NO:5 (see also FIG. 4).
The present invention preferably relates to those DNA vectors which comprise, as any DNA sequence to be cloned in, a coding sequence in the multiple cloning site.
Coding sequence is understood as meaning any DNA sequence which codes for a complete active protein or a protein fragment which has biological activity.
Especially preferred are coding sequences of plant origin. Especially preferred are furthermore coding sequences which comprise at least one promoter sequence. In this context, preferred promoters are those which make possible a constitutive transcription or expression of the coding sequences which are under their control. A preferred promoter is the β-tubulin promoter. Particularly preferred are the promoter sequences which are selected from the group consisting of H. pluvialis promoters of the actin gene (SEQ ID NO:6) and of the Rubisco gene (SEQ ID NO:7).
The invention furthermore relates to DNA vectors in which the coding sequence comprises a functional gene to be expressed, in addition to at least one promoter gene. Especially preferred in this context are DNA vectors which comprise a coding sequence which is selected from the group consisting of carotenoid biosynthesis genes, astaxanthin biosynthesis genes and isoprenoid biosynthesis genes. Particularly preferred are gene sequences of β-carotene ketolase, carotenoid hydroxylase, ζ-carotene desaturase, phytoene synthase, leucopene cyclase, deoxyxylulose synthase and 1-deoxy-xylose 5-phosphate reductoisomerase (Berthold et al., 2002, Hallmann and Sumper, 1996, Mahmoud and Croteau, 2001).
The invention furthermore relates -to the use of the vector according to the invention for transforming eukaryotic cells, in particular single-celled plant cells. It is especially preferred to use the vector according to the invention for the transformation of algal cells, especially. H. pluvialis cells. Within the scope of the invention, transformation is understood as meaning the introduction of foreign DNA into an organism.
The use of the vector according to the invention as selective marker for transformation purposes is also a subject matter of the invention. Especially preferred in this context is the use as dominant selective marker.
A selective marker within the meaning of the present invention mediates, to a transformed organism, a property by means of which this organism can be distinguished readily from untransformed organisms of the same species. A dominant-selective marker is understood as meaning a marker which mediates a property by means of which selection pressure can be exerted within the species so that, in a population of transformed and untransformed organisms of the same species, only the transformed organisms are viable. By adding norflurazon to growth media it is possible, for example, to select H. pluvialis cells which have been successfully transformed with the vector according to the invention of SEQ ID NO:4 from untransformed cells which are not viable under these conditions. The selection of the transformants is accomplished in accordance with the invention with norflurazon concentrations of 0.5-50 μm. The selection can be accomplished in liquid culture or by adding the herbicide to nutrient media plates.
Using mutated PDS as the first dominant selective marker for the transformation of eukaryotes, in particular algae such as H. pluvialis, the present invention provides an important contribution to the biotechnological utilization of eukaryotes, in particular algae such as H. pluviadis. For example, the vector according to the invention can be used with or without insertion of a coding sequence into the MCS in order to influence, or to modify, carotenoid biosynthesis in transformants.
The invention furthermore relates to a method of transforming eukaryotic cells using a vector according to the invention. Such transformation methods are known to the skilled worker. They comprise, for example, the use of PEG, glass beads, electroporation and micro-particle bombardment. Especially preferred in accordance with the invention is a method in which the transformation is carried out by means of particle bombardment. A preferred embodiment in this context is the particle bombardment with tungsten or gold particles 0.4 to 1.7 μm in size which have previously been coated with vector DNA according to the invention, carried out at a pressure of from 500 to 2500 psi and in vacuo. After the transformation, the cells are preferably regenerated in OHA liquid medium (2.42 g Tris-acetate pH 6.8) with shaking overnight in the dark. The cells are then plated on OHA plates under selection pressure with 0.7% strength OHA agarose. Transformants can be observed after 3-4 weeks under light-dark-intervals of equally long intervals of light (15-25 μE*m−2*s−1) and dark (in each case 6-12 h). The transformation efficacy is approximately 1*10−4 to 10*10−8 cells/μg DNA, preferably 1*10−6 cells/μg DNA.
The invention additionally comprises a transgenic plant cell and its progeny which, after transformation with the DNA vector according to the invention, is characterized in that it features an incorporation of the introduced DNA into the nuclear genome. Preferred in this context are transgenic plant cells and their progeny which display a single or multiple incorporation of the introduced DNA into the nuclear genome. Particularly preferred are transgenic plant cells and their progeny in which the introduced gene is expressed constitutively.

    SEQ ID NO:1: Nucleic acid sequence of the pds gene which codes for the protein H. pluvialis phytoene desaturase
    SEQ ID NO:2: Protein sequence of the H. pluvialis phytoene desaturase
    SEQ ID NO:3: Protein sequence of the phytoene desaturase with Leu504 Arg amino acid substitution
    SEQ ID NO:4: Nucleic acid sequence of the MCS of the vector Plat-pdsMod4.1 according to the invention
    SEQ ID NO:5: Nucleic acid sequence of the preferred DNA vector Plat-pdsMOD4.1
    SEQ ID NO:6: Nucleic acid sequence comprising the actin promoter (SmaI fragment). The nucleic acid sequence comprises coding regions with introns, exons and the promoter sequence, which is indicated (−).
    SEQ ID NO:7: Nucleic acid sequence comprising the rbsc promoter (Rubisco small subunit) (Pstl fragment). The nucleic acid sequence comprises coding regions with introns, exons and the promoter sequence, which is indicated.





FIG. 1: Carotenoid biosynthetic pathway of geranylgeranyl diphosphate to astaxanthin
FIG. 2: Comparison of phytoene desaturase from Synechococcus and H. pluvialis with known mutations and resistance factors (RF) to norflurazon
FIG. 3: Amino acid sequence alignment of the PDS proteins from H. pluvialis and Synechococcus. Positions preferred for an amino acid substitution imparting resistance to norflurazon are indicated.
FIG. 4: Plasmid mark of the vector Plat-pdsMod4.1; MCS (multiple cloning site), on (origin of replication)
FIG. 5: Southern blot of genomic DNA after digestion with XbaI and XhoI, and detection via phytoene desaturase probe. Marker (M) in kilobases (kb)
FIG. 6: (A) Northern blot of three WT strains put under continuous high light stress (120 μE*m−2*s−1) (WT 0, 18 and 36 hours) and the transformants P1-P13. (B) Western blot with antibodies against the phytoene desaturase protein. The band migrates at the level of the calculated 55 kDa. Phytoene desaturase mRNA (pds), carotenoid hydroxylase mRNA (hyd), phytoene desaturase protein (PDS).
FIG. 7: Representation of the nonphotochemical quenching (NPQ). NPQ=(Fom-F′m)/F′m Fom is the maximum fluorescence of dark-adapted organisms after a saturating light pulse. F′m maximum fluorescence after saturating light pulses in defined intervals at 20 s.
FIG. 8: Representation of the accumulation of the keto carotenoid astaxanthin in the WT H. pluvialis and two transformants (P3, P13) without added herbicide under continuous high light (175 μEm−2 s−1). Dry weight (DW), time in hours (h).
FIG. 9: Transformation vector for the constitutive expression of the hydroxylase gene (hyd) under the control of the promoter of the small Rubisco subunit (rbcS2)



EXAMPLES
Preparation of the Transformation Vector for the Transformation of H. pluvialis 
A portion of the phytoene desaturase 6.1 kilobases in size was isolated from a genomic DNA library of H. pluvialis and subcloned into the multiple cloning site (MCS) of the pBluescriptSK vector (stratagene) via the restriction cleavage sites XbaI and XhoI. A nucleotide substitution from CTG to CGC was carried out via directed mutagenesis with the primers PDS-Qmut-plu2 CCA AGC AGA AGT ACC GCG CCT CCA TGG AGG G (SEQ ID NO: 8)and PDS-Qmut-min2 CCC TCC ATG GAG GCG CGG TAC TTC TGC TTG G (SEQ ID NO: 9); the plasmid was named pPDS-Q2. This nucleotide substitution led to an amino acid substitution from leucine to arginine in codon 504 (FIG. 2), and confers to mutants a resistance to norflurazon.
Starting from the plasmid pPDS-Q2, the terminal restriction cleavage sites XbaI and XhoI were eliminated by PCR with the specific primers, and two new EcoRV cleavage sites were introduced. Via these restriction cleavage sites, the mutated phytoene desaturase gene was subcloned into an NaeI cleavage site of a pBluescriptSK vector. The multiple cloning site (SEQ ID NO:4) of the vector is thereby free for other cloning steps. The transformation platform thus prepared was named Plat-pdsMod4.1 (FIG. 4).
Transformation of H. pluvialis with the Transformation Vector Plat-pdsMod4.1
H. pluvialis cells were grown for 4 days in liquid medium under standard conditions (light-dark-rhythm of 12 hours light (20 μE*m−2*s−1) and 12 hours dark) up to a cell density of 3.5*105 cells/ml (Kobayashi, Kakizono et al., 1991). The cells were centrifuged for 5 minutes at 16° C. and 4000×g, resuspended and in each case 1*108 cells were plated onto nylon filters (Roche). The filters were transferred to OHM medium plates (Fabregas, Dominguez et al., 2000) and dried until transformation was performed. The tungsten particles which were 0.4-1.7 μm in size were coated with 2 μg of Plat-pdsMod4.1 vector DNA following the protocol of Klein and its coworkers (Klein, Wolf et al., 1987). The particle gun PDS-1000/He from Bio-Rad was used for the transformation at a pressure of 1350 psi and a vacuum of 25 mmHg. Otherwise, the standard settings were retained.
After the transformation, the cells were regenerated on the nylon filter in OHA liquid medium (2.42 g Tris-acetate PH 6.8) overnight with gentle shaking in the dark. Thereafter, the cells were briefly centrifuged and plated with 0.7% OHA top agarose onto 10 to 20 OHA plates (5 μm norflurazon). The first transformants were observed after a growth phase of three to four weeks under a light-dark rhythm of 12 hours light (20 μE*m−2*s−1) and 12 hours dark. The transformation efficacy is approximately 1*10−8 cells/μg DNA.
Molecular Analyses of the Cells Transformed with the Vector Plat-pdsMod4.1
The positive transformants were inoculated repeatedly onto OHA plates with a norflurazon concentration of 0.7 μM. At these concentrations, the growth of the WT cells is greatly inhibited. The transformants were grown in liquid medium with a norflurazon concentration of 3 μM in order to subject the transformants to molecular analysis via Southern, Northern and Western blot analyses.
Southern Blot, Verification of the Integration of the Transformation Plasmid in the Genome of H. pluvialis 
After four days' growth under standard conditions at a norflurazon concentration of 3 μM in liquid medium, the cells were isolated by centrifugation, and the genomic DNA was isolated from the various transformants and several WT controls. The genomic DNA obtained was digested with the restriction enzymes XbaI and XhoI and separated on an 0.8% strength agarose gel. The DNA was blotted by standard methods and hybridized with a probe for PDS (FIG. 5).
The Southern blot clearly reveals the endogenous phytoene desaturase at approximately 5.9 kb in all transformants and in WT. The additionally integrated mutated phytoene desaturases migrate in all cases further up than the endogenous phytoene desaturase. It is known from other organisms such as, for example, the fungus Neurospora crassa that vectors frequently integrate into the genome in the form of tandem repeats (Cogoni and Macino 1997). This phenomenon explains the very pronounced bands in transformants P6, 7, 11 and P13. the genomic DNA was additionally also hybridized with a probe for the ampicillin resistance cassette of the transformation vector Plat-pdsMod4.1. Under these conditions, several pronounced bands were visible in all cases, with the exception of the transformant P3.
Northern and Western Blot Analyses of the Transformants P1-P13
After four days' growth under standard conditions at a norflurazon concentration of 3 μM in liquid medium, the cells were isolated by centrifugation, and the RNA was isolated from the various transformants and several WT controls. For the further analysis of the transformants' transcription patterns, the RNA samples were separated on a 1% denaturing agarose gel, blotted, and hybridized with a probe for the phytoene desaturase mRNA (pds) and for the carotenoid hydroxylase mRNA (hyd) (FIG. 6A). The hydroxylase acts as internal standard in order to allow statements on the transformants' stress status. Hydroxylase is induced under stress conditions such as stress caused by light or salt and is otherwise below the detection limit (Steinbrenner and Linden 2001; Steinbrenner and Linden 2003).
To obtain protein samples, the cells were likewise harvested after four days' growth under standard conditions and separated on a 12.5% strength SDS polyacrylamide gel. The phytoene desaturase protein was detected with a specific antibody (Grunewald, Eckert et al., 2000).
The WT cells which have been placed under continuous high light conditions (120 μE*m−2*s−1) show, for phytoene desaturase (pds) a basal expression of the pds gene at the point in time of induction (0 hours). This signal increases in the course of 18 hours. The carotenoid hydroxylase's behavior under these induction conditions is adequate, the transcription is somewhat delayed.
The expression patterns of the phytoene desaturase mRNA of the transformants P1, P2, P5 and P6 are comparable with the basal expression of the WT at 0 hours under standard conditions. Indeed, the expression levels for the transformants P8, P9 and P10 are somewhat below the level of the basal expression of the WT at 0 hours. In two further independent Northern blot analyses, the expression of the transformants P8, P9 and P10 corresponded to the basal expression of the WT at 0 hours. Here, the transformants P3, P4, P7, P11, P12 and P13 show an elevated transcription level which, in comparison with the continuous high light control is between 0 hour and 18 hours; this was also observed in two further independent Northern blot analyses. In this case, the increase cannot be attributed to induction as a result of stress, but to the multiple incorporation of the transformation vector Plat-pdsMod4.1 in the genome of H. pluvialis. The expression of carotenoid hydroxylase as stress control shows no increased transcription level under these standard conditions.
In the Western blot with an antibody against PDS, the protein amount increases uniformly up to the 36-hour-value (FIG. 6B).
The protein quantities of all transformants are slightly above the 0 hour value of the WT control. The protein quantities of transformants P3, P5, P7, P11 and P13 are again increased over the other levels and comparable with the protein quantity of the WT 18-hour-value under continuous high light conditions. The higher transcription levels of phytoene desaturase can also be observed at the protein level in transformants P3, P7, P11 and P13.
Physiological Studies of the Plat-pdsMod4.1 trans-Formants
Determination of the Nonphotochemical Quenchings (NPQ) of the Plat-pdsMod4.1 Transformants
Some higher plants with modified carotenoid biosynthesis have been studied in greater detail in the past. Thus, tobacco has been transformed with the phytoene desaturase gene crtl from Erwinia uredovora (Misawa, Yamano et al., 1993). In one of the resulting transformants, named ET4-208, a higher resistance to norflurazon was observed. Later, a modified carotenoid composition in the transgenic crtl plants in comparison with untransformed controls has also been detected (Misawa, Masamoto et al., 1994).
A possibility of studying modifications in the carotenoid composition in transgenic plants is measuring the chlorophyll fluorescence. Firstly, this is a very rapid method of screening photosynthetic mutants; secondly, a modification of the xanthophyll contents is reflected in the chlorophyll fluorescence (Niyogi, Bjorkman et al., 1997).
The various transformants were grown for three to four weeks on OHA plates without norflurazon under standard conditions. Prior to the measurements, the plates were dark-adapted for 24 hours. Using a PAM fluorimeter (Walz, Germany), the chlorophyll fluorescence of all transformants was measured, and the NPQ was calculated via the following formula: NPQ=(Fom-F′m)/F′m 
Fom is the maximum fluorescence of the dark-adapted organisms after a saturating light pulse. F′m is the maximum fluorescence after saturating light pulses in defined intervals; in our case, the intervals were 20 s. A good review article on this subject was written by Kate Maxwell and Giles N. Johnson (Maxwell and Johnson 2000).
All of the transformants measured, with the exception of P3 and P13, showed fluorescence curves and NPQ curves like the wild type (WT). The NPQ of transformants P3 and P13 is approximately 50% higher over the entire duration of the measurement than in the case of the WT (FIG. 7).
Comparison of the Distribution of the Pigmented Carotenoids in the WT H. pluvialis and of Three Transformants without Added Herbicide
The transformants were grown for four days under standard conditions in liquid medium without added herbicide, and the cells were isolated by centrifugation and the pellets were freeze-dried. The dry weights of the pellets were determined and the cells were comminuted in a mortar with addition of methanol. The chlorophylls were measured photometrically and later hydrolyzed by addition of 6% strength KOH. The carotenoids were extracted in petroleum ether/ether (b.p. 35° C.-60° C.) (9:1 v/v), the mixtures were evaporated and the residues were taken up in 100% strength acetone. The samples were then separated via an HPLC column.







TABLE 1







HPLC analysis of the total pigment extract of


H. pluvialis WT and the phytoene desaturase transformants


P1, P3 and P13. Percentage distribution of the


carotenoids of the total carotenoid.















Transfor-



H. pluvialis
Transformant
Transformant
mant



WT
P1
P3
P13















Neoxanthin
9.6
9.6
12.9
8.9


Violaxanthin
8.6
8.3
11.7
11.23


Lutein
65.3
62.9
65.8
64


β-Carotene
16.6
19
9.6
15.7









As can readily be seen from table 1, the percentage distribution of the individual carotenoids in the WT and transformant P1 is very similar. In comparison, the carotenoid compositions in transformants P3 and P13 have changed drastically. In P3, the xanthophylls are increased by approximately 6% and the β-carotene content is reduced by this value in comparison with the WT. In transformant P13, the violaxanthin content is increased by approximately 3%. Zeaxanthin, which is directly involved in the NPQ process, cannot be measured under standard conditions. This would require HPLC measurements of cells which have been subjected to high light stress. However, the increase in the violaxanthin contents of approximately 3% of transformants P3 and P13 still permits a direct coupling with the increased NPQ of these transformants since the violaxanthin pool under high light conditions is converted into zeaxanthin.
Carotenoid Composition of the H. pluvialis Transformants P3, P13 and WT Under Low Light Conditions
It is known that the heterologous expression of the pds gene from Erwinia uredovora in tobacco modifies the carotenoid composition in the leaf (Misawa et al., (1994)). The analysis of norflurazon-resistant Synechococcus mutants confirmed that it is the phytoene desaturation in carotenoid biosynthesis which is the rate-limiting step in cyano bacteria (Chamovitz et al., (1993)). To confirm these results, the P3, P13 and WT H. pluvialis transformants were grown under low light conditions, and all carotenoids were extracted. None of the transformants studied showed altered carotenoid quantities in comparison with the dry weight. A subsequent HPLC analysis of the carotenoid composition in transformants P3, P13 and WT revealed, for transformant P3, a slightly increased amount of xanthophylls violaxanthin, whereas the amounts of lutein and neoxanthin were slightly reduced.
Comparison of the Accumulation of the Keto Carotenoid Astaxanthin in the WT H. pluvialis and Transformants P3 and P13 without Added Herbicide Under Continuous High Light
To carry out the high light experiments, the H. pluvialis cells were grown for 4 days under standard conditions. After this period, the light intensity was increased from 20 μEm−2 s−1 to 175 μEm−2 s−1 and the light/dark rhythm to 24 hours continuous light. At certain times after the induction (0, 6, 12, 24, 48, 72 hours), samples were taken and centrifuged.
Carotenoids were extracted as described by Boussiba et al., (1992). In this manner, the dry weight (DW) of the freeze-dried cells was determined, and the material was again comminuted in a mortar with addition of seesand, 30% methanol and 5% KOH. After hydrolysis of the chorophylls for 10 minutes at 70° C., the samples were centrifuged for 5 minutes at 4000 rpm, and the chlorophyll-containing supernatant was discarded. The pellet was taken up in 100% DMSO (dimethyl sulfoXide) and heated for 10 minutes at 70° C. This extraction step was repeated until the pellet had lost its reddish color. Astaxanthin and its esters have their main absorption maximum at 492 nm. However, a measurement of this wavelength was not carried out since the total carotenoids (mainly β-carotene, lutein and violaxan-thin) too showed absorption in this wavelength range. With the aid of an astaxanthin standard (Sigma, in 100% DMSO), a calculation factor was thus determined which permitted the determination of the astaxanthin content in a wavelength range (550 nm) in which the total carotenoids no longer absorbed. Thus, the absorption of the astaxanthin standard was only measured at a wave-length of 492 nm (A492) and subsequently at a wavelength of 550 nm, (A550). The absorption values were measured at 550 nm and then multiplied by the quotient A492/A550=3.2, and the astaxanthin content was calculated with an astaxanthin absorption coefficient (E1%/1 cm=2220) using the formula of Davies (1976).
Six hours after changing the light conditions, no astaxanthin was detectable as yet (FIG. 8). After 8 hours' exposure to high light, the transformant P3 showed a visibly red phenotype, while the WT and the other transformants remained green. 12 hours after induction, the amount of astaxanthin formed in the WT and in transformant P13 amounted to 1.47 mg/g DW and 1.32 mg/g DW; in contrast, the value which the transformant P3 had reached was 2.37 mg/g DW. After 24 hours, the astaxanthin quantity measured, of the WT and of the transformant P13, was 3.48 mg/g DW and 3.79 mg/g DW, respectively. At this point in time, transformant P3 showed an approximately 40% increase with an astaxanthin quantity of 6.22 mg/g DW in relation to the wild type. After 48 hours under continuous high light, the WT and the P13 transformant showed values of 8.6±1.4 mg/g DW and 8.4±1.6 mg/g DW, respectively. An astaxanthin accumulation which was increased by 26% in comparison with the WT and P3 was determined for the transformant P3 after 48 hours (11.4±0.9 mg/g DW). 72 hours after continuous light stress, the WT and the P13 transformant revealed an amount of 10.5 mg/g DW and 10.1 mg/g DW, respectively, with the transformant P3, with a value of 12.1 mg/g DW, showing an astaxanthin accumulation which was 14% higher.
Thus, the transformant P3 differed greatly in comparison with the transformant P13 and the WT with regard to its accumulation of astaxanthin under continuous high light. During the continuous-light experiment, P3 showed an increase in the astaxanthin quantities by approximately 40% after 24 hours, in comparison with the WT. This difference drops to 26% after 48 hours and reaches a value of 14% after 72 hours.
The results demonstrate that the phytoene desaturation step in H. pluvialis is rate-limiting under high light conditions, but not under low light conditions.
Increasing Astaxanthin Biosynthesis by using β-carotene Ketolase Under the Control of Constitutive Promoters
Vector Construction and Transformation
Using the transformation platform Plat-pdsMod4.1, transformation plasmids with promoters of the actin gene or of ribulose bisphosphate carboxylase followed by a cloning site for various restriction endonucleases were prepared; both promoters were isolated from a genomic DNA library. These platforms furthermore comprise the 3′-untranslated region of the respective genes, which also act as polyadenylation signal for introduced cDNAs. The cDNAs of carotenoid hydroxylase and of β-carotene ketolase were amplified via PCR and provided with terminal restriction cleavage sites. This made it possible to introduce these cDNAs in-frame into the transformation platform. The constructs in question were also prepared for genomic DNA of the two carotenoid biosynthesis genes. FIG. 9 shows a transformation vector for the constitutive expression of the hydroxylase gene under the control of the small Rubisco subunit promoter.
Three constructs under the control of an actin promoter with different translation starts, and in each case one construct under the control of the Rubisco promoter, were cloned, and transformed into H. pluvialis, for the cDNAs and the genomic hydroxylase sequences. The selection pressure was lowered by reducing the norflurazon concentration from 5 μM to 3 μM. This was intended also to obtain transformants which, on the one hand, have a lower resistance to norflurazon, but whose chances of growth, on the other hand, were reduced as the result of a shift of the metabolic equilibrium toward zeaxanthin or canthaxanthin. A shift of this equilibrium might mean, for example, a reduced formation of lutein, which plays an important role in the association of antenna complexes in the thylakoid membrane. The formation of the chlorophylls, or else the formation of tocopherols, might also be affected by such a shift, since those substances also diverge from the very early isoprenoid biosynthesis.
Selection of Transformants
Following transformation, the colonies obtained were transferred to other medium plates comprising norflurazon. Thereafter, 15 transformants of each construct were transferred into liquid medium under very low selective pressure.
After a further growth phase, where none of the transferred transformants showed a pronounced carotenoid accumulation of canthaxanthin or zeoxanthin, the cells were placed under high light (120 μE*m−2*s−1) for 24 hours.
The transformants which have been transformed with the hydfoxylase constructs of genomic DNA under the control of the Rubisco promoter (Plat-rbcS2gHyd) and of the second actin promoter (Plat-ActPII-gHyd) (second translation start with an intron) showed no reddish-brown colored cells in comparison with the transformants of the other hydroxylase constructs.
In the case of the ketolase construct transformants, those which had been transformed with the cDNA and the ketolase gene under the control of the second actin promoter (Plat-ActPIIgBkt and Plat-ActPIIcBkt) (second translation start with an intron) were subjected to further studies.
Southern Blot and Northern Blot Analyses of the Hydroxylase Constructs
10 among the in each case 15 pre-screened transformants were selected and grown on for molecular-biological studies. After the genomic DNA of the individual transformants had been isolated, they were subjected to restriction digestion and separated on an agarose gel, blotted and hybridized with a probe for hydroxylase. Among the 10 transformants which were under the control of the second actin promoter (Plat-ActPII-gHyd), two transformants unambiguously revealed additional hydroxylase copies in the genome. Among 10 transformants under the control of the Rubisco promoter (Plat-rbcS2gHyd), 6 were positive with additional copies in the genome. Reducing the norflurazon concentration on the selection plates in order to favor growth-disadvantaged transformants clearly has a negative effect on the selection.
The RNA of the remaining transformants was extracted and separated on a denaturing agarose gel, blotted and hybridized with a probe for the hydroxylase mRNA. Two of the transformants showed an increased transcript level of hydroxylase mRNA. Control experiments with other probes for carotenoid biosynthesis genes β-lycopene cyclase and phytoene synthase also revealed an increased transcript level in these two transformants.
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## Claims

1. An isolated expression vector which comprises:
(i) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2 with one of the following amino acid modifications: a modification at amino acid 504, changing leucine to arginine; a modification at amino acid 264, changing arginine to proline; a modification at amino acid 388, changing leucine to proline; a modification at amino acid 465, changing valine to glycine, wherein said nucleic acid molecule encodes a modified phytoene desaturase, which confers disease resistance, and (ii) a multiple cloning site which permits cloning of an additional nucleic acid molecule into said vector.

2. The expression vector of claim 1, further comprising a nucleic acid molecule which confers resistance to herbicides when said vector is inserted into a transformant.

3. The expression vector of claim 2, wherein said herbicide is a bleaching herbicide.

4. The expression vector of claim 3, wherein said bleaching hebicide is norflurazin.

5. The expression vector of claim 1, further comprising a nucleic acid molecule which encodes a protein at said multiple cloning site.

6. The expression vector of claim 5, wherein said nucleic acid molecule is identical to a nucleic acid molecule found in a vegetable.

7. The expression vector of claim 5, further comprising a promoter sequence.

8. The expression vector of claim 5, wherein said promoter sequence a Haematococcus act gene promoter, a Haematococcus Rubisco gene promoter, or a β-tubulin promoter.

9. The expression vector of claim 7, wherein said nucleic acid molecule which encodes a protein is a carotenoid biosynthesis gene, an astaxanthin biosynthesis gene, or an isoprenoid biosynthesis gene.

10. A method for transforming a eukaryotic cell comprising contacting said eukaryotic cell with the expression vector of claim 1 under conditions favoring transformation thereby.

11. The method of claim 10, wherein said eukaryotic cell is a single cell plant.

12. The method of claim 11, wherein said single cell plant is an algae.

13. The method of claim 12, wherein said algae is a H. pluviales.

14. A method for determining if a cell has been successfully transformed comprising contacting said cell with the expression vector of claim 1 and determining resistance to a herbicide as a determination of successful transformation.

15. The method of claim 10, comprising transforming said cell via particle bombardment.

16. The method of claim 15, comprising carrying out said particle bombardment with tungsten particles of from 0.4 to 1.7 μm in diameter, a pressure of from 500 to 2500 pounds per square inch, in vacuo.

17. A transgenic plant cell comprising the expression vector of claim 1 in its nuclear genome.

18. The transgenic plant cell of claim 17, comprising a plurality of copies of said expression vector in its genome.