TITLE: Decreasing RUBISCO content of algae and cyanobacteria cultivated in high carbon dioxide

INVENTORS: EISENSTADT, Doron; UFAZ, Shai; SCHATZ, Daniella and GRESSEL, Jonathan

ASSIGNEE: TransAlgae (Israel) Ltd.

PRIORITY: This application claims priority of the U.S. Provisional applications number 61/191,169 filed on September 5 ^th 2008 and 61/191,453 filed on September 9 ^th 2008.

SEQUENCE LISTING: This application contains sequence data provided on a computer readable diskette and as a paper version. The paper version of the sequence data is identical to the data provided on the diskette

FIELD OF THE INVENTION This invention relates to the field of genetically engineering algae and cyanobacteria to grow more efficiently on industrial waste emissions of carbon dioxide and is applicable for use with algae and cyanobacteria cultured in closed bioreactors and covered or open ponds for producing high value products, as well as biofuels. It builds on integrating principles of genetic engineering, photosynthetic physiology and biochemistry, chemical engineering of bioreactors, and waste emission engineering.

BACKGROUND OF THE INVENTION

A major breakthrough in the large scale cultivation of algae and cyanobacteria to produce commercially useful products was the discovery that many such species could be cultivated with flue gas (up to 80% CO ₂ ) or even pure CO ₂ whereas most other organisms (plants and animals) are "biochemically anaesthetized" at CO ₂ levels of 5% or higher, slowing all metabolism. This opened the way for cultivating such organisms on CO ₂ emissions to the environment (Murakami and Ikenouchi 1997, Negoro, et al. 1993).

Cyanobacteria have already begun to be genetically engineered to utilize these elevated CO ₂ levels by over expressing genes encoding rate limiting enzymes of the

"dark reactions" (CO ₂ assimilating reactions that utilize NADPH and ATP from the light reactions) of photosynthesis. Thus, for example, engineering genes from rice encoding for cytosolic fructose- 1,6-bisphosphate aldolase and spinach triose phosphate isomerase in cells of a cyanobacterium doubled their activities and greatly increased photosynthetic efficiency and biomass yields (Kang et al. 2005, Ma et al.

2007).

However, one can only over-express enzymes to a limited extent, as the cell does not have unlimited capacity. Therefore, the existing methods have only a limited capacity and there is a need to improve these methods to enhance the use of elevated carbon dioxide concentration in algal cultures.

SUMMARY OF THE INVENTION

The instant invention provides a solution to this existing problem by reducing the levels of other enzymes to "make room" for the over expressed, rate limiting enzymes such as fructose- 1,6-bisphosphate aldolase (ALD), chloroplast triosephosphate isomerase (TPI) or acetyl CoA carboxylase (ACCase) that enhance sink capacity to utilize fixed carbon dioxide.

Ribulose-l,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the key photosynthetic enzyme that catalyzes the first step of CO ₂ fixation. The chloroplast localized holoenzyme of plants and algae in Sub-kingdom Viridaeplantae, Phylum Chlorophyta (heretofore referred to as green algae) contain eight nuclear genome encoded small subunits and eight chloroplast genome encoded large subunits. In red lineage algae (Sub-Kingdom Chromobiota, Phylum Haptophyta, Heterokonotophyta, Bacillariophyta and others (Table 1), all sub-units are encoded in the chloroplast. Table 1 Phytogeny of some of algae used

Genus Family Order Phylum Sub- Kingdom

Chlamydomonas Chlamydomonadaceae Volvocales Chlorophyta Viridaeplantae

Nannochlorϊs Coccomyxaceae Chlorococcales Chlorophyta Viridaeplantae

Tetraselmis Chlorodendraceae Chlorodendrales Chlorophyta Viridaeplantae

Phaeodactylum Phaeodactylaceae Naviculars Bacillariophyta Chromobiota

Nannochloropsis Monodopsidaceae Eustigmatales Heterokontophyta Chromobiota

Pavlova Pavlovaceae Pavlovales Haptophyta Chromobiota

Isochrysis Isochrysidaceae Isochrysidales Haptophyta Chromobiota

Phylogeny according to: http://www.algaebase.org/browse/taxonomy/

Note: Many genes that in higher plants and Chlorophyta are encoded in the nucleus are encoded on the chloroplast genome (plastome) of Chromobiota, red lineage algae (Grzebyk, et al. (2003).

The present consensus is that even more RUBISCO is needed for efficient photosynthesis, as demonstrated in the recent suggestion that elevated RUBISCO would enhance the rate of photosynthesis in algae commercially cultivated for biofuels (Huntley and Redalje 2007). Counter-intuitively, the inventors of this disclosure chose to "make room" for other enzymes by reducing concentration of the RUBISCO enzyme, typically considered to be rate limiting for CO ₂ assimilation in photosynthetic cells; RUBISCO can comprise up to 70% of the soluble protein in plant cells.

Photosynthesis produces the sugars needed for the biosynthesis of the specific primary and secondary metabolites of interest in each case (biofuels, pigments, enzymes, pharmaceuticals, starch, etc.). RUBISCO is the first enzyme in the dark reactions of photosynthesis, "fixing" carbon dioxide onto an organic molecule. The later reactions use NADPH and ATP generated by the light reactions to reduce the fixed CO ₂ to carbohydrates. RUBISCO was the best enzyme evolution could produce pre-antiquity, which was of little matter early in the evolution of earth, e.g. in the Archean era 3.5 billion years ago when CO ₂ concentrations in the atmosphere were thought to be at least 100 times more than at present (Fig. 1.4 in Falkowski and Raven, 1997), and the very low affinity for CO ₂ to RUBISCO was of little consequence. As oxygenous photosynthesis began to remove CO ₂ from the atmosphere and elevate atmospheric O ₂ , the levels of RUBISCO became limiting, and evolution gradually increased the levels of RUBISCO in photosynthetic organisms to the present high levels. Presently, high concentrations of CO ₂ can be inexpensively provided to algae and cyanobacteria in culture from industrial combustion of fuels. This will lead to much of the RUBISCO being superfluous, and decreasing its content "makes way" for over-expressing other enzymes needed for photosynthesis as well as releasing resources for more soluble products. This would be one way of sequestering large amounts of carbon dioxide, presently imperative due to the purported relationships between elevated global carbon dioxide levels and global warming. Even though the small subunit of RUBISCO is encoded by a family of genes, the gene products are interchangeable, with considerable consensus among them.

According to this disclosure, reducing RUBISCO in green algae can be done by using either antisense or RNAi technology, both targeting the consensus sequences of the small nuclear encoded subunit, which, in many cases, has been shown to control the biosynthesis of both subunits. Obviously, total suppression is undesirable, as RUBISCO is needed for CO ₂ fixation. The level of suppression that is advantageous is a function of: a. experimental determination for each pond or bioreactor condition and concentration of CO ₂ introduced into the system; and b. the availability of the CO ₂ to the organisms in the medium. Reducing RUBISCO in red lineage algae according to this disclosure, can be done by knockout of one or both RUBISCO subunits in the chloroplast by homologous recombination and transformation of the same RUBISCO gene under a mutated promoter, which will decrease the RUBISCO expression level.

Many algae and especially cyanobacteria have atypical (for higher organisms) G:C contents and consequently atypical codon usages and DNA sequences. While there is a high consensus in amino acid sequences in RUBISCO subunits, it is far less at the nucleotide level, requiring sequencing of the genes encoding RUBISCO subunits from each target organism before embarking on generating RNAi or antisense constructs and engineering them into the cell thus reducing RUBISCO according to the codon usage of the target organism.

One drawback to genetically engineering algae and cyanobacteria for large scale cultivation is the risk of inadvertent "spills" into the environment. It is highly unlikely (i.e. as near to impossible as a scientist can evaluate) that organisms optimized to live and grow in an atmosphere of >5% CO ₂ , yet having a lower than normal RUBISCO content can survive for long in nature where the CO ₂ concentration is <0.5%, as the engineered organism has lost most of its ability to scavenge CO ₂ from the environment. Thus, this risk from inadvertent transgenic release is negated, whether the organism is engineered just for lowered RUBISCO, or engineered with lowered RUBISCO and elevated other enzymes, whether photosynthesis related, or related to other properties.

The present invention relates to the use of algae or cyanobacteria cultivated in ponds or bioreactors that have had their RUBISCO contents transgenically lowered by antisense or RNAi or other transgenic technologies giving rise to similarly lowered RUBISCO contents. These novel organisms are especially adapted to thrive in cultivation with high CO ₂ levels in the medium, allowing for the over-expression of other, rate limiting enzymes of photosynthesis as well as enzymes encoding for other desirable traits. Thus, these organisms are platforms for further engineering.

In one embodiment the small subunit of RUBISCO is subjected to RNAi suppression and in another it is subjected to antisense. In yet another embodiment both large and small subunits are suppressed by chloroplast transformation of the rbcLS gene cluster under the control of a mutated promoter replacing the endogenous promoter and genes.

In other embodiments DNA encoding other traits is either engineered in tandem with the RUBISCO suppression simultaneously (co-transformation) or subsequent to the engineering of partial RUBISCO suppression. In such embodiments algae or cyanobacteria with reduced RUBISCO levels are used as a platform for further engineering of other desired traits, with greater efficiency of organism activity. These include genes encoding enzymes such as fructose- 1,6-bisphosphate aldolase (ALD), chloroplast triosephosphate isomerase (TPI) or acetyl CoA carboxylase (ACCase) that enhance sink capacity to utilize fixed carbon dioxide. Conversely, other transgenic traits may be in the algae or cyanobacteria prior to transformation for partial RUBISCO suppression. According to one embodiment the alga or cyanobacterium transformed to express reduced RUBISCO content is also transformed to express pharmaceutical or industrial proteins, such as human insulin AAN39451 or AY138590 or thermostable phytase, such as disclosed in US patent 6720174. Other desired proteins to be expressed in transgenic algae or cyanobacteria with reduced RUBISCO content are storage proteins, such as 15kDZein or BHL8. The transgenic algae expressing reduced RUBISCO content may also be transformed to express altered oil or lipid contents.

A SHORT DESCRIPTION OF THE DRAWINGS Figure 1. Map of the plasmid pSI-PDS-rbcS RNAi containing the cassette designed to induce RNAi of C. reinhardtii RUBISCO small subunit An inverted repeat of the first 234 bp of RbcS2 coding region encoding the RUBISCO small subunit is cloned downstream to the pds gene conferring resistance to the herbicide flurochloridone. The transgene is under the control of the HSP70-RbcS2 promoter and RbcS2 terminator (taken from pSI103, Sizova et al, 2001).

Figure 2. Map of the plasmid pSI-rbcS-AS containing the cassette designed to induce antisense of C. reinhardtii RUBISCO small subunit and the psaD-Ble cassette conferring resistance to the antibiotic Zeocin. The coding region of RbcS2 from C. reinhardtii encoding the RUBISCO small subunit is cloned in antisense orientation downstream to the HSP70-rbcS promoter.

DETAILED DESCRIPTION OF THE INVENTION

Algae and cyanobacteria with biotechnological utility are chosen from among the following, non-exclusive list of organisms. List of species:

Pavlova lutheri, Isochrysis CS-J 77, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina CS- 190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp.,Chlamydomonas reinhardtii as representatives of all algae species. The phylogeny of the algae is summarized in Table 1. Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongaues BP-J are used as representatives of all cyanobactrial species. Algae and cyanobacteria with partially suppressed RUBISCO are achieved by standard molecular biological procedures, as outlined in numerous texts and papers. First, consensus sequences of the large and small subunits of RUBlSCO are used to "fish out" the respective genes by low stringency PCR using a consensus sequences chosen to have the least number of nucleotide variants. Standard software is used to design degenerate primers according to these consensus sequences. After fishing out fragments of the genes, larger segments of the genes are obtained using the RACE (rapid amplification of cDNA ends) technique. The resulting sequences are used to design anti-sense and RNAi constructs that are then inserted into respective cassettes and transformed into the algae and cyanobacteria using techniques readily available to those skilled in the art. Different cassettes are used having different promoters such that a large variety of expression levels are achieved, so that RUBISCO will be reduced by varying amounts. A large number of transformation events were generated for each algal species, and the best transformants chosen as described below.

The growth rates of the transformants are measured under conditions of various levels of high CO ₂ (1%; 5%; 14%; 100%) and those that appear best are rechecked in mini bioreactors and pilot scale ponds to ascertain which have the best yield, under a variety of environmental conditions and CO ₂ concentrations.

The best transformants of each organism can then be used as platforms for inserting other genes into the algae or cyanobacteria to optimize the production of valuable compounds. The algae come from a large taxonomical cross section of species (Table 1)

The general approach for green algae is as follows:

1. Cloning of the algae RUBISCO small subunit (rbcS) cDNA in antisense (AS) orientation under the control of a constitutive promoter such as the rbcS promoter and 3 'rbcS terminator, downstream to a selectable marker. The selectable marker can be Sh ble, which confers resistance to the antibiotic Zeocine, the pds gene, which confers resistance to fluridone and flurochloridone. 2. Generation of an RNAi cassette (as described in detail in Schroda, 2006) of the algae rbcS gene comprising a 300 bp cDNA/cDNA inverted repeat under the control of a constitutive promoter downstream to a selectable marker described above. The general approach for red lineage marine algae species (Sub-kingdom Chromobiota, Table 1), is to replace the chloroplast RUBISCO small or large subunit with a DNA construct containing the same RUBISCO subunit gene controlled by a mutated promoter, use antisense or with a chloroplast expression vector, and directly transform the chloroplasts, as has been done with Chlamydomonas (Franklin and Mayfield, 2004)

The general approach for cyanobacteria is as follows:

Cloning of the RUBISCO small subunit (rbcS) or large subunit (rbcL) gene from a cyanobacteria species under the control of mutated promoter and replacing the respective endogenous gene with the cloned cassette using homologous recombination, as described in Clerico et al. (2007).

The methodology used in the various steps of enabling the invention is described here below: Nucleic Acid Extraction Genomic DNA is isolated using either the

Stratagene (La Jolla, CA, USA ) DNA purification kit or a combination of the QIAGEN (Valencia, CA, USA ) DNeasy plant mini kit and phenol chloroform extraction method (Davies et al. 1992). Total RNA is isolated using either the QIAGENS Plant RNeasy Kit or the Trizol Reagent (Invitrogen, Carlsbad, CA, USA ). RACE analysis The full length RbcS small and large subunits from algae or cyanobacteria with unknown genomic sequences are determined by 3' and 5' RACE and nested PCR using the First Choice RLM-RACE Kit (Ambion, Austin, TX, USA), as described by Liu and Gorovsky (1993). Transformation of plasmid DNA Transformation of Chlamydomonas Algae cells in 0.4 mL of growth medium containing 5% PEG (polyethylene glycol MW6000) were transformed with the plasmid from examples 1 and 2 by the glass bead vortex method (Kindle, 1990). The transformation mixture was then transferred to 50 mL of non-selective growth medium for recovery and incubated for at least 18 h at 25°C in the light. Cells were collected by centrifugation and plated at a density of 10 ⁸ cells per Petri dish. Transformants were grown on fresh TAP or SGII agar plates containing a selection agent for 7-10 days in 25°C Transformation of marine algae I. Electroporation

Fresh algal cultures are grown to mid exponential phase in artificial sea water (ASW)+f72 media. Cells are then harvested and washed twice with fresh media. After resuspending the cells in 1/50 of the original volume, protoplasts are prepared by adding an equal volume of 4% hemicellulase (Sigma) and 2% Driselase (Sigma) in ASW and are incubated at 37 ⁰ C for 4 hours. Protoplast formation is tested by Calcofluor white (Fluka) staining. Protoplasts are washed twice with ASW containing 0.6M D-mannitol (Sigma) and 0.6M D-sorbitol (Sigma) and resuspended in the same media, after which DNA is added (lOμg linear DNA for each lOOμl protoplasts). Protoplasts are transferred to cold electroporation cuvettes and incubated on ice for 7 minutes, then pulsed in a BTX

ECM830 (Harvard Apparatus, Holliston, MA, USA) electroporation apparatus. A variety of pulses is usually applied, ranging from 1000 tol500 volts, 10-20ms each pulse. Each cuvette is pulsed 5-10 times. Immediately after pulsing the cuvettes are placed on ice for 5 minutes and then the protoplasts are added to 250μl of fresh growth media (without selection). After incubating the protoplasts for 24 hours in low light, 25°C the cells are plated onto selective solid media and incubated under normal growth conditions until single colonies appear. II. Microporation

A fresh algal culture is grown to mid exponential phase in ASW+f/2 media. A 10 mL sample of the culture is harvested, washed twice with Dulbecco's phosphate buffered saline (DPBS, Gibco) and resuspended in 250μl of buffer R (supplied by Digital Bio, Seoul, Korea, the producer of the microporation apparatus and kit). After adding 8μg linear DNA to every lOOμl cells, the cells are pulsed. A variety of pulses is usually needed, depending on the type of cells, ranging from 700 to 1700 volts, 10-40ms pulse length; each sample is pulsed 1-5 times.

Immediately after pulsing the cells are transferred to 200μl fresh growth media (without selection). After incubating for 24 hours in low light, 25 ⁰ C, the cells are plated onto selective solid media and incubated under normal growth conditions until single colonies appear. III. Particle bombardment

A fresh algal culture is grown to mid exponential phase in ASW+f/2 media. 24 hours prior to bombardment cells are harvested, washed twice with fresh

ASW+f/2 and re-suspended in 1/10 of the original cell volume in ASW+f72. 0.5 mL of each cell suspension is spotted onto the center of a 55mm Petri dish containing 1.5% agar solidified ASW+f/2 media. Plates are left to dry under normal growth conditions. Bombardment is carried out using a BioRad

PDS 1000/He system according to the manufacturer's (BioRad) instructions, using MlO tungsten powder for cells larger than 2 microns in diameter, and tungsten powder comprised of particles smaller than 0.6 microns (FW06, Canada

Fujian Jinxin Powder Metallurgy Co., Markham, ON, Canada) for smaller cells.

The tungsten is coated with linear DNA. 1 100 or 1350psi rupture discs are used.

All disposables are supplied by BioRad. After bombardment the plates are incubated under normal growth conditions for 24 hours followed by transferring the cells onto selective solid media and incubated under normal growth conditions until single colonies appear. For chloroplast transformation, this method is carried out in the same way, but the resulting transformants are screened for the presence of the transgene in the chloroplast.

Transformation of cyanobacteria For transformation to Synechococcus PCC7002, cells are cultured in 100 mL of BG-I l + Turks Island Salts liquid medium

(http://www.crbip.pasteur.fr/fiches/fichemedium.jsp?id=54 8 ) at 28°C under white fluorescent light and cultured to mid exponential growth phase. To 1.0 mL of cell suspension containing 2xlO ⁸ cells, 0.5-1.0 μg of donor DNA (in 10 mM Tris/1 mM EDTA, pH 8.0) is added, and the mixture is incubated in the dark at 26°C overnight. After incubation for a further 6 h in the light, the transformants are selected on BG-11 + Turks Island Salts 1.5% agar plates containing a selection agent until single colonies appear.

There is no prior art known to us of previously transforming the following species: Pavlova lutheri, Isochrysis CS-177, Nannochloropsis oculata CS-J 79, Nannochloropsis like CS-246, Nannochloropsis salina CS- 190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp. nor has microporation been used previously for transforming algae cyanobacteria or higher plants. RNA extraction, cDNA synthesis and quantitative RT-PCR analysis Total RNA is isolated using either the QIAGENS Plant RNeasy Kit or the Trizol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA is synthesized using 3 μg total RNA as a template using SuperscriptII kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Real-time quantitative PCR reactions are preformed in an optical 96-well plate using the ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Scoresby, Victoria, Australia) and SYBR Green I for monitoring dsDNA synthesis. For all PCR reactions the following standard thermal profile is used: 50 ⁰ C for 2 min; 95°C for 15 min; 40 cycles of 95°C for 15 sec and 6O ⁰ C for 1 min. In order to compare data from different cDNA samples, C _τ (threshold cycle) values for all genes are normalized to the CT values of Ubiquitin, or 16S rDNA for algae and cyanobacteria, respectively, which are used as internal references in all experiments. All primers are designed using the Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA). The sequences of sense and antisense designed primers correspond to two consecutive exons of the studied genes, excluding any genomic DNA amplification. The real-time PCR data is analyzed using the comparative CT-method with appropriate validation experiments performed beforehand (Applied Biosystems, User Bulletin #2, http://home.appliedbiosystems.comΛ. All experiments are repeated at least three times with cDNA templates prepared from three independent colonies of algae or cyanobacteria and every reaction is set up in triplicates.

Protein extraction 1 to 10 mL cells at 5xlO ⁶ cell/mL are harvested and resuspended in 500μl extraction buffer (5OmM Tris pH=7.0; ImM EDTA; 10OmM NaCl; 0.5% NP-40; and protease inhibitor (Sigma cat# P9599). Then 100 μl of glass beads (425-600 μm, Sigma) are added and cells are broken in a bead beater (MP FastPrep-24, MP Biomedicals, Solon, OH, USA) for 20 sec. The tube content is centrifuged for 15 min, 13000xg, at 4°C. The supernatant is removed to new vial.

Protein separation by PAGE and western analysis Extracted proteins are separated on a 4-20% gradient SDS-PAGE (Gene Bio-Application Ltd., Kfar Hanagid, Israel, at 160V for 1 hr. They were then either stained by Coomassie (Sigma) or blotted onto PVDF (Millipore, Billerica, MA, USA) membranes for 1 h at 100 volts in the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). The proteins are detected with the RbcL RUBISCO large subunit, form I and form II antibody (Agrisera, Vannas, Sweden) diluted to a ratio of 1 :10000 in antibody incubation buffer (5% skim milk, Difco). An alkaline phosphatase conjugated anti- rabbit antibody (Millipore, Billerica, MA, USA), at 1:10000 dilution in the same buffer was used as a secondary antibody. Detection was carried out using the standard alkaline phosphatase detection procedure (Blake et al., 1984).

Physiological Assessment To assess physiological properties of genetically modified algae compared with their relevant wild type strains and other algal candidates we perform a set of procedures that enable us to evaluate each strain. Initially, each genetically modified strain is checked for the modified trait, (reduced RUBISCO content). A screening process is established where colonies of transgenic algae or cyanobacteria are allowed to grow on solid media supplemented with selection reagent (an antibiotic or herbicide) to check if the desired trait has been established. Next, the fastest growing colonies are picked and transferred to liquid medium for further physiological evaluation. This includes:

1. Growth rate

2. Photosynthetic activity at ambient and high carbon dioxide concentrations

3. Respiration activity

4. Tolerance to a-biotic parameters 5. Lipid content

6. Protein content

An overall report is generated for each strain that is used to estimate the feasibility of using the strain.

Growth Rate Growth rates are measured using one or more of the following techniques:

• Direct cell count

• Optical density at a relevant wavelength (e.g. 730nm)

• Pigment/Chlorophyll concentration (where this method is applicable)

• Percentage of packed volume Photosynthetic Activity One of the important parameters indicating the welfare of a photoautotrophic culture is its photosynthetic capability. To measure this, one or more of several methodologies are applied:

• Oxygen evolution - using Clark Type electrodes. • Variable fluorescence - using PAM (Pulse Amplitude Modulated fluorometry) Oxygen consumption in darkness is also evaluated in order to estimate net photosynthetic potential of the algal culture. As part of the photosynthetic evaluation several abiotic parameters that potentially influence the physiological state of a culture are followed.

• Light intensity tolerance (at a given cell density) is evaluated. P/I (photosynthesis vs. irradiance) curves are used to determine optimal light intensity per cell.

• Performance at different CO ₂ levels (e.g. ambient; 1%; 5%; 14%; 100%). This is coupled with pH tolerance.

• Temperature tolerance. Each culture is tested at optimal temp. In addition, temperatures are raised temperatures to the highest points possible without inhibiting other culture activities.

Growth conditions Cells of eukaryotic marine cultures and transformants thereof are grown on artificial seawater medium (Goyet, 1989) supplemented with f/2 (Guillard, 1962). Marine cultures are grown at 18-20 ⁰ C with a 16/8 h light/dark period. Fresh water cultures (e.g. the diploid wild-type Chlamydomonas reinhardtiϊ) and transformants thereof are grown photoautotrophically on liquid medium, using mineral medium as previously described (Harris, 1989), with the addition of 5mM NaHCO ₃ ^" , with continuous shaking and illumination at 22°C. Marine cyanobactertial cultures and transformants thereof are grown in BG 11 medium BGl 1 (Stanier et al., 1971) supplemented with Turks Island Salts and with 20 mM HEPES-NaOH buffer pH 7.8 (http://www.crbip.pasteur.fr/fiches/fichemedium.jsp?id=548). Cyanobacterial cultures are grown at 25°C where relevant under continuous white light, with constant CO ₂ -air bubbling.

Growth Rate Estimation Cells are harvested in the logarithmic growth phase and re-suspended in fresh growth media. Cultures are brought to a cell density corresponding to ~3 μg/mL chlorophyll a. Light intensity is optimized for each culture and temperature is maintained at growth temperature ±1°C. Where required, cells are concentrated by centrifugation (300Og, 5 min) and re-suspended in fresh media. A time-series sampling procedure is followed where a subsample of each culture is collected and the number of cells per mL is estimated. As well as direct counting, optical density at different wavelengths, percentage of packed volume and chlorophyll concentrations are also measured.

Photosynthetic Activity: Oxygen evolution Measurements of O ₂ concentrations are performed using a Clark type O ₂ electrode (Pasco Scientific, Roseville, CA, USA). Twenty mL of cell suspension containing 15 μg chlorophyll/mL are placed in the O ₂ electrode chamber, at relevant temperature. Cells are exposed to various light intensities and regimes (e.g. flashing light). Incubations in darkness are performed in these air-tight vessels to follow oxygen consumption in the dark.

Fluorescence measurements Electron transfer activity of photosystem II is measured by pulse modulated fluorescence (PAM) kinetics using PAM-101 (WaIz,

Effertlich, Germany). Light intensity (measured at the surface of the chamber) of the modulated measuring beam (at 1.6 kHz frequency) is 0.1 μmol photons m ^~2 s ^"1 . White actinic light is delivered at 50-1500 μmol photons m ^"2 s ^"1 as required in different experiments and is used to assess steady state fluorescence (F _s ). Maximum fluorescence (F _m ) is measured with saturating white light pulses of 4000 μmol photons m ^"2 s ^"1 for 1 s.

Additional experiments

• Light intensity tolerance (at a given cell density) is evaluated. P/I (photosynthesis vs. irradiance) curves are used to determine optimal light intensity per cell. Four mL of cell suspension containing 15 μg chlorophyll/mL are placed in the O ₂ electrode chamber, at relevant temperatures and various light intensities. Oxygen evolution rates are measured at each light intensity.

• Performance at different CO ₂ levels (e.g. ambient; 1%; 5%; 14%; 100%). Growth rate estimations and photosynthetic activity (methodology described above) are evaluated when cultures are maintained at different CO ₂ levels.

• Temperature tolerance. Each culture is tested at optimal temp. In addition, we attempt to raise temperatures to the highest point possible without inhibiting other culture activities.

The invention is now described by means of various non limiting examples using the above methods: EXAMPLE 1: Generation of C. reinhardtii expressing RNAi of RbcS2B gene under the control of the HSP70-rbcS2 promoter

For generation of RNAi of rbcS2 (ACCESSION NO: X04472), a 774 bp fragment (SEQ ID NO:1) corresponding to forward and reverse orientation of nucleotides 1 to 234 of rbcS2 gene separated by 246 bp spacer region comprised from the 3 ^rd intron of the rbcS2 gene (REGION: 1947..2184), was custom synthesized by DNA2.0 Inc, (Menlo Park, CA, USA). The 774 bp region (SEQ OD NO:1) was then cloned into BamHI restriction site in plasmid pSI-PDS downstream to the pds gene, generating the plasmid pSI-PDS rbcS RNAi (Figure 1). The plasmid was transformed to C. reinhardtii CW 15 strain (CC-400) and transfromants were selected on SGII medium supplemented with 3x10 ⁷ M flurochloridone (FCD). FCD resistant colonies were transferred to liquid media for DNA and protein extraction. Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp are transformed with the above cassette using to the transformation methods described above. Total proteins are separated on 4-20% gradient SDS-PAGE (Geba, Israel) and stained with Coomassie blue or transferred to PVDF membranes (Millipore, Billerica, MA, USA) for western blot analysis using the anti RbcL RUBISCO large subunit, form I and form II antibody (Agrisera, Vannas, Sweden) .

Colonies with reduced RUBISCO levels are further analyzed as described in examples 5 to 7.

EXAMPLE 2. Generation of C. reinhardtii expressing the rbcS2 gene in antisense orientation under the control of the HSP70-rbcS2 promoter For the generation of plasmids containing the C. reinhardtii rbcS2 gene in antisense orientation under the control of the HSP70-rbcS2 promoter (Figure 2), the 579 bp fragment of the C. reinhardtii rbcS2 gene was PCR amplified with primers BstBI- rbcS2B: GCTTCGAATCAACGAGCGCCTCCATTTAC (SEQ ID NO:2), and Xhol- rbcS2 AS GCCTCGAGATGGCCGCCGTCATTGCCAA (SEQ ID NO:3) containing the BstBI and Xhol sites at their 5' and 3' regions, respectively, and was cloned into pGEM-T vector (Promega, Madison, WI, USA). The BstBI-XhoI fragment was then introduced into the BstBI/XhoI sites of plasmid pSI-PDS rbcS RNAi, replacing the pds-rbcS RNAi cassette (Example 1). A psaD-Ble fragment (comprising the BIe selectable marker (SEQ ID NO: 4 ) under the control of the psaD promoter (SEQ ID NO:5), excised from pGenD-Ble) was further ligated into the plasmid using Notl restriction site. The resulting pSI-rbcS-AS plasmid was then transformed to C reinhardtii CW 15 (CC-400) and transformants were selected on TAP medium supplemented with 5 μg/mL Zeocin. Approximately 100 Zeocin resistant colonies were transferred to liquid media for protein extraction and rbcS level analysis.

Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp are transformed with the above cassette using to the transformation methods described above. Colonies with reduced RUBISCO levels are further analyzed as described in examples 5 to 7.

EXAMPLE 3. Generation of cyanobacterial transformants with reduced rubisco expression levels

In order to reduce expression level of rbcL in the cyanobacterium Synechococcus PCC7002, the native rbcL promoter is replaced with a mutated one. The rbcL region (SEQ ID NO: 6 ) is synthesized with random mutations in the promoter region (nucleotides 1165-1638 in SEQ ID NO: 6) and a spectinomycin resistance cassette upstream of the promoter. Resulting fragments are then cloned into pGEM-T (Promega, Madison, WI, USA) to create a library of plasmids containing a myriad of mutated promoters. The resulting library is transformed into Synechococcus PCC7002, and following homologous recombination (that occurs naturally in cyanobacteria) clones are screened for transformants with reduced RUBISCO content.

EXAMPLE 4. Chloroplast transformation of red lineage algae

To reduce rbcS expression level of red lineage marine algae, the sequence of the algae chloroplast DNA is obtained using 454 sequencing (CD Genomics, Shirley, NY, USA). Then, a DNA fragment containing the rbcS gene and its flanking regions is obtained by PCR on DNA isolated from the marine algae. The rbcS coding sequence is then cloned under a mutated rbcL promoter and rbcL terminator together with a spectinomycin resistance gene cassette comprising rbcL promoter, bacterial AAD gene (SEQ ID NO:7) and rbcL terminator as described in Takahashi, (1991). This construct is then transformed to the algae chloroplast DNA using particle bombardment as described in the methods part, and according to Spectinomycin resistant colonies are then selected and analyzed using PCR on genomic DNA to confirm the homologous recombination. Positive colonies are then selected for further analysis as described in Examples 5-7.

EXAMPLE 5. Demonstration that transformed algae and cyanobacteria have optimal photosynthesis at elevated CO ₂ Cultures of reduced RUBISCO-content transformants of algae and cyanobacteria are compared to those of their respective wild type. While the latter reveal maximal photosynthesis rates at concentrations of 0.03- 1% CO ₂ , transformed algal and cyanobacterial cells exhibit maximal photosynthesis rates at CO ₂ concentrations above 4%. The increased CO ₂ concentrations compensated for reduced RUBISCO contents.

EXAMPLE 6. Demonstration that transformed algal and cyanobacterial strains cannot compete with wild type cultures at ambient CO ₂ concentrations

The algal and cyanobacterial transformants described above function best under bioreactor and/or pond conditions at high CO ₂ concentrations. An additional benefit arising from this condition is that these strains cannot cope with natural occurring conditions such as ambient CO ₂ concentration. Being currently at 0.03% in the atmosphere, CO ₂ becomes a major limiting factor for the transformants cultured with ambient carbon dioxide levels. In order to demonstrate such growth limitation reduced-RUBISCO-content transformants are co-cultured with wild-type cells at ambient CO ₂ concentrations. A time-sequence sampling protocol is followed and cells are collected from the growth vessels. Cells are then transferred to plates for colony isolation, then replicas are made of each colony. One plate contains normal growth media while its duplicate contained a selection factor (e.g. antibiotics / herbicides). This enables the differentiation between wild-type cells and transformants, allowing following wild-type cells outcompeting reduced RUBISCO content transformants co-cultivated under ambient carbon dioxide.

EXAMPLE 7. Demonstration that transformed algal and cyanobacterial strains have increased levels of photosynthesis when sink-enhancing genes are transformed into these strains

Reduced-RUBISCO-content transgenic algae and cyanobacteria are further transformed with sink-enhancing genes. As was previously demonstrated by Miyagawa et al., (2001), overexpression of cyanobacteria fructose-1,6- /sedoheptulose-l,7-bisphosphatase (FBP/SBPase) (SEQ ID NO:8) in tobacco enhances photosynthesis and growth, is used. The reduced-RUBISCO-content x FBP/SBPase transformants are compared with the reduced-RUBISCO-content transformants alone under conditions of 14% CO ₂ . Oxygen evolution is followed as an indication for photoautotrophic assimilation, and higher oxygen production rates are observed with the reduced-RUBISCO-content x FBP/SBPase transformants. This implies higher Ci assimilation rates, and therefore suggests enhanced energy harvesting even at when RUBISCO levels are reduced.

Growth rate estimation

Cultures of Reduced-RUBISCO-Content (RRQ transformants of green algae (Tetraselmis suecica, Tetraselmis chuii, Nannochloris sp. and Chlamydomonas reinhardtii) are compared to those of their respective wild type. Wild type cells reveal a saturation-curve-like pattern where growth rates increase with increasing CO ₂ concentrations. At ambient CO ₂ concentrations, reduced-RUBISCO-content cells exhibit reduced growth rates. Their doubling times are reduced (at ambient CO ₂ ) but increased with increasing CO ₂ concentrations.

Photosynthetic Activity: Oxygen evolution and PAM Fluorescence

Again, cultures of Reduced-RUBISCO-Content transformants of green algae (Tetraselmis suecica, Tetraselmis chuii ,Nannochloris sp and Chlamydomonas reinhardtii) are compared to those of their respective wild type. Wild-type cells reveal a typical P/I saturation curve. In contrast, reduced-RUBISCO-content cells exhibit a slight decrease in optimal light intensity, i.e. saturation and inhibition occurs at lower light intensities. When CO ₂ levels are raised to 14% or more, P/I curves of reduced-RUBISCO-content cells return to normal parameters. The increase OfCO ₂ concentrations compensates for the reduced RUBISCO content.

Lipid and Protein contents Finally, we test reduced-RUBISCO-content transformants for lipid and protein content, and compare them to those of wild type cells. Lipid and protein content are lower in the transformants than in wild type cells at ambient CO ₂ concentrations. However, when CO ₂ levels are increased to 14% or more, lipid and protein contents exceed those of wild type cells.

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