# Patent

## Title
Genetically engineered herbicide resistance for maintaining axenic cultures

## Abstract

This disclosure provides herbicide resistant algae and cyanobacteria. This disclosure also provides a method to cultivate algae and cyanobacteria in axenic cultures without contaminating species. Moreover, this disclosure provides transgenic algal and cyanobacterial cells that are capable of high production in high light intensities as typically applied in cultivation. Furthermore, a novel transformation method is provided for algal cells.

## Background
PRIORITY
This application claims priority of U.S. Provisional Patent No. 61/191,167 filed on Sep. 5, 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 cultivation of algae and cyanobacteria. More specifically this invention relates to methods to maintain axenic cultures. The invention relates to management of algal culture by means of genetic modification
BACKGROUND OF THE INVENTION
A major problem in the commercial cultivation of algae and cyanobacteria in axenic culture in bioreactors or open or closed ponds is that they can become contaminated by other, highly competitive but unwanted species of algae and cyanobacteria, fungi and bacteria, as well as by rotifers and other zooplankton that devour the desired species in the cultures. (Sheehan et al. 2004). Fluridone is the only USEPA-approved systemic herbicide that is commonly used for control of aquatic weeds (but not algae) in large bodies of water. It is a noncompetitive inhibitor of the enzyme phytoene desaturase (PDS), which is one of the first dedicated enzymes of the plant carotenoid biosynthesis pathway. Under high light intensities, carotenoids stabilize the photosynthetic apparatus by quenching the excess excitation energy; therefore, inhibition of phytoene desaturase decreases colored carotenoid concentration and causes photo-bleaching of green tissues (Böger and Sandmann 1998).
The pds gene was cloned from the herbicide-susceptible as well as from the recently-evolved, herbicide-resistant biotypes of the water weed hydrilla [Hydrilla verticillata (Lf) Royle]. Three separate and independent single-point mutations of the codon 304 encoding for Arg (Arg304) in pds were identified in the resistance biotypes (Michel et. al., 2004; Michel et. al., 2004 Patent application WO/2004/007691). The codon usage for Arg304 in the wild-type Hydrilla is CGT and single-point mutations yielding either Ser (AGT), Cys (TGT), or His (CAT) substitutions were identified in the fluridone resistance biotypes of Hydrilla. The resistant biotypes had biomass and-β carotene accumulations of up to 72% and 77% of the content in untreated plants, respectively, while in the susceptible population, fluridone strongly inhibited biomass accumulation and β-carotene accumulation, showing only 10% of the levels found in untreated plants (Michel, et al. 2004). Many fungi and bacteria that synthesize carotenoids as a photoprotectant are sensitive to PDS inhibitors.
Protoporphyrinogen oxidase (PPO; protox) is the last common enzyme in the tetrapyrrole biosynthetic pathway that produces heme and chlorophyll (Beale & Weinstein, 1990). In plants chlorophyll biosynthesis takes place exclusively in plastids, whereas heme is produced in both plastids and mitochondria. In both organelles, PPO converts protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX). Two different nuclear genes, PPX1 and PPX2, encode plastid and mitochondrial PPO isozymes, respectively. When susceptible plants are treated with PPO inhibitors, the substrate of PPO, protogen IX, accumulates and is exported from the organelles into the cytoplasm where herbicide-insensitive peroxidase-like enzymes in the plasma membrane convert it to proto IX. Proto IX accumulates in the cytoplasm and, in the presence of light, induces the formation of singlet oxygen that is damaging to cell membranes.
Herbicides that act by inhibiting protoporphyrinogen oxidase are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites (Lermontova et. al., 1997; Watanabe et. al., 2001). Evaluation of a PPO inhibitor-resistant A. tuberculatus biotype revealed that resistance was an incompletely dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare (Patzoldt et. al., 2006; Gressel and Levy 2006; Tranel et al., 2007).
Even if fluridone/flurochloridone and protox-inhibiting herbicides are known, their use in algal or cyanobacterial culture has not been possible because the cultured photosynthetic algae or cyanobacteria would also be killed. Moreover, there is an unsolved problem of contamination of alga culture ponds and bioreactors with unwanted species such as rotifers and other zooplankton, which are not controlled by phytoene desaturase or protox-inhibiting algae. This disclosure provides solution to each of these unsolved contamination problems.
SUMMARY OF THE INVENTION
To overcome these problems this invention provides algae and cyanobacteria that are genetically-engineered with herbicide resistance genes and cultured under elevated carbon dioxide conditions to allow control of specific algae and cyanobacteria species as well as zooplankton. Application of small amounts of these herbicides singly or as a cocktail to the cultured ponds will prevent the growth of unwanted algae, cyanobacteria, fungi and bacteria and their establishment in the ponds, and cultivation at elevated carbon dioxide levels will control the rotifers and other zooplankton
Accordingly, the present invention relates to transgenic algae and cyanobacteria that are capable of being cultivated in ponds where wild type cells and unwanted species can easily be controlled.
The present invention also relates transgenic algae and cyanobacteria that are capable of high production in artificial conditions, but which have impaired capability to compete in natural environments.
The present invention further relates to herbicide resistance genes that are either co-transformed with other genes such as genes encoding modified protein and starch synthesis, reduced Rubisco, reduced antenna size or modified lipid biosynthesis or the genes are constructed together on the same plasmid and are transformed to the desired algae and cyanobacteria. New genes can be also introduced in the background of transgenic algae that were transformed with the mentioned genes.
The present invention also relates to a novel transformation method for algae and cyanobacteria.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Schematic illustration of the constructs of the synthetic pds gene (SEQ ID NO:1) under various promoters in various expression vectors as used in this disclosure.
FIG. 2. Chlamydomonas cells were transformed with the synthetic pds (SEQ ID NO:1) gene harboring the histidine mutation (right panel) as well as control cells that were not transformed with DNA (left panel) and plated on SGII medium containing 10−7M flurochloridone. Growth of transformants was visible within two weeks. 100 of the clones were picked for further studies.
FIG. 3. Nannochloropsis cells were transformed with the pSI103-PDS construct (right panel) as well as control cells which were not transformed with DNA (left panel) plated on artificial sea water (ASW)+1 μM flurochloridone. The resistant material is also cross resistant to the phytoene desaturase inhibitors fluridone, picolinafen, and norflurazon.
FIG. 4. PCR analysis to the Chlamydomonas bulk transformants with pSI103-PDS showing the insertion of the pds gene into the transformants (denoted by arrow). The numbers represent the following: wild type (1), transformed 1 (2), transformed 2 (3), positive control (the transformed plasmid) (4), no template DNA (5), molecular weight markers (6). More explanations are provided in the examples below.
FIG. 5. PCR analysis of the Nannochloropsis oculata cells that were transformed with pSI103-PDS showing the insertion of the pds gene into the transformants (denoted by arrow).
A. By electroporation: The numbers represent the following: wild type (1), transformed 1 (2), transformed 3 (2), positive control (the transformed plasmid) (4), no template DNA (5), and molecular weight marker (6).
B. By microporation, showing the insertion of the pds gene into the transformed cells. Pos—positive control; NTC—no template control; WT—wild type-DNA—colonies that were not transformed with DNA and grew on the selection plates; 1-19—transformed colonies.
FIG. 6: Dose response for the Chlamydomonas pds transformants colonies. Wild type and pds transformed colonies (pds I35, pds II34, pds II 22, pds I44, pds I38, pds II28, pds II14, pds II5, pds I40) were inoculated at OD750=0.1 and flurochloridone was applied at the concentrations of 0.1, 0.3 and 0.5 μM. Cultures were grown for a week before picture was taken.
FIG. 7: Mixed culture of the alga Chlamydomonas reinhardtii and the cyanobacterium Synechococcus PCC 7942 as visualized under the microscope.
FIGS. 8A and B: Contamination test of Chlamydomonas pds transformant II5 versus the cyanobacterium Synechococcus PCC 7942. Chlamydomonas transformants and the cyanobacterium Synechococcus PCC7942 were inoculated alone and in mixed culture in a ratio of 10:1 with and without flurochloridone. Aliquots were removed daily and counted under the microscope. 8A represents the Chlamydomonas pds transformants counts and 8B represents the cyanobacterium Synechococcus PCC7942 counts.
FIG. 9. Oxygen evolution curves of Chlamydomonas reinhardtii (WT) and pds (pdsI38, pds I44) transformants under 1000 μmol photons m−2 s−1. Dark incubation for approximately 12 min was applied, followed by  ̃8 min of illumination. Rates of dark oxygen consumption and net oxygen evolution were calculated and compared.
FIG. 10 Enhanced photosynthesis of Chlamydomonas reinhardtii pds transformants (pdsII5; pds I44; pds II22; pds I38; pds I40) versus wild-type (WT). Incubations lasted approximately 10 minutes. A and B represent two different experiments conducted with the same strains.
FIG. 11 A Chemical structure of butafenacil. B. Butafenacil dose response on wild type algae and cyanobacteria. Strains were cultured at OD648=0.1 in 24 well plate and butafencil was applied at concentrations of 10−5,6,7M. After a week OD648 of the cultures was measured.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides genetically modified algae and cyanobacteria that are resistant to one or more herbicides, whereby the transgenic strains can be cultivated in axenic monoculture by controlling unwanted species with herbicide applications. The present invention further provides a method to limit the establishment of the transgenic strains in natural ecosystems through their impaired photosynthetic activity in naturally occurring light conditions.
Moreover, the present invention provides a method to cultivate the transgenic algae and/or cyanobacteria in monoculture by controlling unwanted species with herbicides and prevent growth of zooplankton by use of high carbon dioxide concentrations.
In one embodiment the Hydrilla pds-gene (Michel et. al., 2004) was synthesized de novo according to the appropriate codon usage of the desired algae/cyanobacteria. The de novo synthesized herbicide resistant pds gene is then cloned for algae under the control of rbcS2 and or fcpA/35S/ubiquitin/tubulin promoters and 3′rbcS2/fcpA/fcpB terminators, in the plasmids pSI103 and or pPHAT1 (Sizova et. al 2001; Lioudmila, et. al 2000) and for cyanobacteria under the constitutive promoter of the rbcLS operon (Deng and Coleman 1999) in the plasmid pCB4 as well as into various expression vectors, allowing various levels of expressions driven by different promoters, including constitutive, inducible and log phase temporal promoters. The pds transformants are selected for the highest levels of the appropriate herbicide resistances with the least effects on growth in special situations.
In another embodiment the Amaranthus tuberculatus ppo-gene (Patzoldt et. al., 2006) was synthesized de novo according to the appropriate codon usage of the desired algae/cyanobacteria. The de novo synthesized herbicide resistant ppo gene is then cloned for algae under the control of rbcS2 and or fcpA/35S/ubiquitin/tubulin promoters and 3′rbcS2/fcpA/fcpB terminators, in the plasmids pSI103 and or pPHAT1 (Sizova et. al 2001; Lioudmila, et. al 2000) and for cyanobacteria under the constitutive promoter of the rbcLS operon (Deng and Coleman 1999) in the plasmid pCB4 as well as into various expression vectors, allowing various levels of expressions driven by different promoters, including constitutive, inducible and log phase temporal promoters.
According to yet another embodiment, the pds gene is used as selectable marker with phytoene desaturase inhibiting herbicides.
According to still another embodiment the pds gene is used as a selectable marker with phytoene desaturase herbicides for co-transformations with other genes needed in the algae such as reduced RUBISCO, reduced antennae size, enhanced fluorescence proteins, reduced or enhanced starch or other products, deletion or formation or modified or enhanced lipid biosynthesis, or any other gene that may be desirable in the algae or cyanobacteria. The herbicide resistant gene is either co-transformed with other genes such as genes encoding modified protein and starch synthesis, reduced RUBISCO, reduced antenna size or modified lipid or protein biosynthesis or the genes are constructed together on the same plasmid and are transformed to the desired algae and cyanobacteria. New genes can also be introduced in the background of transgenic algae that were transformed with the mentioned genes
According to yet another embodiment the, ppo gene is used as a selectable marker with protoporphyrinogen oxidase inhibiting herbicides.
According to a further embodiment the ppo gene is used as a selectable marker with protoporphyrinogen oxidase inhibiting herbicides for co-transformations with other genes needed in the algae such as reduced RUBISCO, reduced antennae size, enhanced fluorescence proteins, reduced or enhanced starch or other products, deletion or formation or modified or enhanced lipid biosynthesis, or any other gene that may be desirable in the algae or cyanobacteria. The herbicide resistant gene is either co-transformed with other genes such as genes encoding modified protein and starch synthesis, reduced RUBISCO, reduced antenna size or modified lipid or protein biosynthesis or the genes are constructed together on the same plasmid and are transformed to the desired algae and cyanobacteria. New genes can also be introduced in the background of transgenic algae that were transformed with the mentioned genes.
According to one embodiment of this invention, cultivated algae and cyanobacteria species were rendered resistant to flurochloridone/fluridone by transformation with a resistant type phytoene desaturase (pds) gene under a constitutive promoter. Application of the herbicides flurochloridone/fluridone to the transgenic cultured algae and cyanobacteria results in establishment of the desired algae and cyanobacteria without contamination by other algae and cyanobacteria, because they are killed by fluridone/flurochioridone. Under a preferred embodiment flurochloridone is preferable to fluridone, as lower concentrations could be used.
According to yet another embodiment of this invention, cultivated algae and cyanobacteria species were rendered resistant to butafenacil or flumioxazin by transformation with resistant protoporphyrinogen oxidase (ppo) gene under a constitutive promoter. Application of the herbicide butafenacil or flumioxazin to the transgenic cultured algae and cyanobacteria results in establishment of the desired algae and cyanoacteria without contamination by other algae and cyanobacteria, because they are killed by butafenacil or flumioxazin.
The invention is now described by non-limiting examples. One of ordinary skill in the art would realize that various modifications can be made without departing from the spirit of the invention. The examples below show that the process according to this invention is useful, novel, non obvious and it greatly simplifies the harvest and processing of microalgae and cyanobacteria.
In the various embodiments, algae and cyanobacteria were chosen from the following organisms: Phaeodactylum tricornutum, Amphiprora hyaline, Amphora spp., Chaetoceros muelleri, Navicula saprophila, Nitzschia sp., Nitzschia communis, Scenedesmus dimorphus, Scenedesmus obliquus, Tetraselmis suecica, Chlamydomonas reinhardtii, Chlorella vulgaris, Haematococcus pluvialis, Neochloris oleoabundans, Synechococcus elongatus PCC6301, Botryococcus braunii, Gloeobacter violaceus PCC7421, Synechococcus PCC7002, Synechococcus PCC7942, Synechocystis PCC6803, Thermosynechococcus elongatus BP-1, Nannochloropsis oculata, Nannochloropsis salina, Nannochloropsis spp., Nannochloropsis gaditana, Isochrysis galbana, Aphanocapsa sp., Botryococcus sudeticus, Nannochloris spp., Pavlova spp., Euglena gracilis, Neochloris oleoabundans, Nitzschia palea, Pleurochrysis carterae, Tetraselmis chuii, Nannochloris spp. It is however, clear for one skilled in the art that this list is not exclusive, but that various other genera and species can be used as well.
EXAMPLE 1
Synthesis of Appropriate Flurochloridone and Fluridone-Resistant Phytoene Desaturase Genes
The pds gene was de novo synthesized according to the appropriate codon usage of the desired algae and the desired cyanobacteria or according to general algae and general cyanobacterial codon usage. The synthetic genes harbor the histidine, cysteine or serine amino acid, corresponding to arginine304histidine in Hydrilla. 
The phytoene desaturase gene harboring the histidine mutation (SEQ ID NO:1) was custom synthesized according to the Chlamydomonas codon usage by the GENEART AG, Regensburg, Germany (http://www.geneart.com). The gene was synthesized with a BstBI restriction site on the 5′ and BamHI on the 3′ for direct cloning into pSI103 algae expression vector. The cloning was conducted with algae originating from a large taxonomical cross section of species (Table 1). The algae included: Chlamydomonas reinhardtii, Pavlova lutheri, Isochrysis sp. CS-177, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina CS-190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp. as representatives of all algae species.







TABLE 1







Phylogeny of some of algae used











Genus
Family
Order
Phylum
Sub-Kingdom





Chlamydomonas
Chlamydomonadaceae
Volvocales
Chlorophyta
Viridaeplantae


Nannochloris
Coccomyxaceae
Chlorococcales
Chlorophyta
Viridaeplantae


Tetraselmis
Chlorodendraceae
Chlorodendrales
Chlorophyta
Viridaeplantae


Phaeodactylum
Phaeodactylaceae
Naviculales
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) in the Chromobiota, red lineage algae (Grzebyk et al., 2003).






The Hydrilla phytoene desaturase gene is customized with the histidine mutation according to the codon usage of cyanobacterium Synechococcus PCC7002 (SEQ ID NO:2) and the cloning is conducted with the following cyanobacterial species: Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongatus BP-1.
Resistance to phytoene desaturase inhibiting herbicides is also conferred by transformation with Hydrilla phytoene desaturase with the serine mutation according to the codon usage of Chlamydomonas (SEQ ID NO:3), Hydrilla phytoene desaturase with the serine mutation according to the codon usage of the cyanobacterium Synechococcus PCC7002 (SEQ ID NO:4), Hydrilla phytoene desaturase with the cysteine mutation according to the codon usage of Chlamydomonas (SEQ ID NO:5), and Hydrilla phytoene desaturase with the cysteine mutation according to the codon usage of the cyanobacterium Synechococcus PCC7002 (SEQ ID NO:6).
EXAMPLE 2
Cloning the Resistant Phytoene Desaturase into an Expression Vector
The de novo synthesized pds gene SEQ ID NO:1 was cloned under the control of rbcS2 and or fcpA/35S/ubiquitin/tubulin promoters and 3′rbcS2/fcpA/fcpB terminators, in the plasmids pSI103 and or pPHAT1 (Sizova et al., 2001; Zaslayskaia et al., 2000). For cyanobacteria, the gene (SEQ ID NO:2) is cloned under the constitutive promoter of the rbcLS operon (Deng and Coleman 1999) in the plasmid pCB4 as well as into various expression vectors, allowing various levels of expressions driven by different promoters, including constitutive, inducible and log phase temporal promoters.
The synthetic pds gene with the histidine mutation was cloned using BamHI, BstBI restriction sites into the pSI103 expression vector under the control of rbcS2 promoter (FIG. 1; pSI103-PDS).
The synthetic pds gene was also cloned under the fcpA promoter in pPhaT1 by blunt ligation. The pds was digested from pSI103 by BamHI and BstBI, the pPhaT1 was digested by BamHI followed by filling in and blunt ligation (FIG. 1: pPhaT-PDS).
The synthetic pds gene was also cloned under the cauliflower mosaic virus (CaMV) 35s and 35S omega promoters in pPhaT1 by BamHI and EcoRI and replaced the fcpA promoter (FIG. 1: pPhaT1-35S-PDS).
The synthetic pds gene was also cloned under the ubiquitin promoter in the pSI103 expression vector. The maize ubiquitin promoter accession no. AY342393 was excised from the pUB-AG vector by XbaI and replaced the Rbsc-Hsp70 promoters in pSI103 which was excised by XbaI, BstBI, followed by filling in and blunt ligation (FIG. 1: pSI103-Ubiq-PDS).
The synthetic pds gene was further cloned under the tubulin promoter (XM—001693945) in the pSI103 expression vector. The tubulin promoter was excised from the pKS-aph7′′-lox vector by SacI, EcoRI and replaced the hsp70-rbsC-promoters in pSI103 by digestion with NotI, BstBI followed by filling in and blunt ligation (FIG. 1: pSI103-Tub-PDS).
The synthetic pds gene was also cloned under the actin promoter in the pSI103 expression vector. The rice actin promoter (accession no. EU155408) was excised from the pUB-AG vector by HindIII, SmaI and replaced the hsp70-rbsC promoters in pSI103 which was excised by XbaI, BstBI followed by filling in and blunt ligation (FIG. 1: pSI103-Act-PDS).
EXAMPLE 3
Transformation of the Resistant Phytoene Desaturase into Algae and Cyanobacteria
Algae and cyanobacteria were transformed with the resistant form of phytoene desaturase gene by various methods described below. Importantly, this is the first time microporation transformation method is described and applied to transformation of algal or cyanobacterial cells.
I. Electroporation


    
    
        Fresh algal cultures were grown to mid exponential phase (2-5*106 cells/ml) in artificial sea water (ASW)+F/2 media. Cells were then harvested and washed twice with fresh media. After re-suspending the cells in 1/50 of the original volume, protoplasts were prepared by adding an equal volume of 4% hemicellulase (Sigma) and 2% Driselase (Sigma) in ASW and were incubated at 37° C. for 4 hours. Protoplast formation was tested as a lack of Calcofluor white (Fluka) staining of cell walls. Protoplasts were washed twice with ASW containing 0.6M D-mannitol and 0.6M D-sorbitol and re-suspended in the same media, after which DNA was added (10 μg linear DNA for each 100 μl protoplasts). Protoplasts were transferred to cold electroporation cuvettes and incubated on ice for 7 minutes then pulsed by the ECM 830 electroporator (BTX Instrument Division, Harvard Apparatus, Inc., Holliston, Mass., USA). A variety of pulses were usually applied, ranging from 1000 to 1500 volts, 10-20 ms each pulse. Each cuvette was pulsed 5-10 times. Immediately after pulsing the cuvettes were placed on ice for 5 minutes and then the protoplasts were added to 250 μl of fresh growth media (without selection). After incubating the protoplasts for 24 hours in low light, the cells were plated onto selective solid media and incubated under normal growth conditions until single colonies appeared.

II. Microporation

        Fresh algal cultures were grown to mid exponential phase (2-5*106 cells/ml) in ASW+F/2 media. A 10 ml sample of each culture was harvested, washed twice with DPBS (Dulbecco's phosphate buffered saline, Gibco) and resuspended in 250 μl of buffer R (supplied by Digital Bio, NanoEnTek Inc., Seoul, Korea, microporation apparatus and kit). After adding 8 μg linear DNA to every 100 μl cells, the cells were pulsed. A variety of pulses was usually needed, depending on the type of cells, ranging from 700 to 1700 volts, 10-40 ms pulse length; each sample was pulsed 1-5 times. Immediately after pulsing, the cells were transferred to 200 μl fresh growth media (without selection). After incubating for 24 hours in low light, the cells were plated onto selective solid media and incubated under normal growth conditions until single colonies appeared.

III. Particle Bombardment

        Fresh algal culture were grown to mid exponential phase (2-5*106 cells/ml) in ASW+F/2 media. 24 hours prior to bombardment cells were harvested, washed twice with fresh ASW+F/2 and resuspended in 1/10 of the original cell volume in ASW+F/2. 0.5 ml of the cell suspension is spotted onto the center of a 55 mm Petri dish containing solidified ASW+F/2 media. Plates are left to dry under normal growth conditions. Bombardment was carried out using a PDS 1000/He biolistic transformation system according to the manufacturer's (BioRad Laboratories Inc., Hercules, Calif., USA) instructions using M10 tungsten powder (BioRad Laboratories Inc.) 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 was coated with linear DNA. 1100 or 1350 psi rupture discs were used. All disposables were purchased from BioRad Laboratories Inc., (Hercules, Calif., USA). After bombardment the plates were incubated under normal growth conditions for 24 hours after which the cells were plated onto selective solid media and incubated under normal growth conditions until single colonies appeared.

IV. Glass Beads

        Cells (4×107) in 0.4 ml of growth medium containing 5% PEG6000 were transformed with DNA (1±5 mg) by the glass bead vortexing method (Kindle, 1990). The transformation mixture was then transferred to 10 ml of non-selective growth medium for recovery. The cells were kept for at least 18 h at 25° C. in the light. Cells were collected by centrifugation and plated at a density of 13×107 cells per 80 mm plate. Transformants were selected on fresh SGII ((http://www.chlamy.org/SG.html) agar plates containing 3×10−7M flurochloridone.
    
    


The above described procedures were carried out on the following algae: Chlamydomonas reinhardtii, Pavlova lutheri, Isochrysis sp. CS-177, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina, Tetraselmis suecica, Tetraselmis chuii, and Nannocloropsis sp. as representatives of all algae species Cyanobacterial species Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongatus BP-1 were used as representatives of all cyanobacterial species using a standard protocol as set out in (Golden, et al. 1987). Briefly, cells are harvested by centrifugation and resuspended in fresh growth medium (ASW+F/2 for Synechococcus PCC7002 and Synechococcus WH-7803; and BG-11 for Thermosynechococcus elongatus BP-1) at a concentration of 2-5×108 cells/ml. To one ml of this cell solution the appropriate plasmid construct is added to a final concentration of 2-5 μg/ml. Cells were incubated in the dark for 8 hours followed by a 16 h light incubation prior to plating on fresh media plates containing flurochloridone or fluridone to select for the colonies that grow at the highest rates without affecting algal growth. Plates are incubated under growth conditions adjusted to the preferences of each strain. Flurochloridone or fluridone resistant colonies were visible after 7-10 days. This is modified for each organism according to its needs, based on modifications of standard protocols.
Chlamydomonas cells were transformed with the pSI103-PDS construct, harboring the histidine mutation (corresponding to Hydrilla Arg 304), using the glass beads transformation technique as described above and plated on SGII medium+10−7 M flurochloridone as is shown in FIG. 2.
Nannochloropsis oculata CS179 cells were transformed with the PSI103-PDS construct using the ECM 830 electroporator (BTX Instrument Division, Harvard Apparatus, Inc., Holliston, Mass., USA) as described above. After transformation algae were plated on ASW+F/2 media (http://www.marine.csiro.au/microalgae/methods/) containing 10−7M flurochloridone. After two weeks, algal colonies both from the transformation and the mock were replated on ASW+10−6M flurochloridone plates. This allowed differentiation between false positives and true resistant clones (FIG. 3). This material was used in Example 4.
Isochrysis galbana was transformed by the particle bombardment technique (M10 tungsten powder, 1100 psi rupture discs). After two weeks, algal colonies from both the transformation and the mock were replated on ASW+10−7 M flurochloridone plates. This allowed differentiation between false positives and true resistant clone. 10 stable resistant clones were isolated from the experiments by particle bombardment
EXAMPLE 4
Verification of the Presence of the Synthetic pds Gene Transformed Algae and Cyanobacteria Harboring the Resistant Phytoene Desaturase
The transformants described in Example 3 were replated on fresh agar medium containing either 10−7M (Chlamydomonas and Isochrysis) or 10−6M (Nannochloropsis) of flurochloridone and cells were allowed to generate to visible colonies. After incubation for a week the most resistant colonies were chosen. To ascertain gene transformation PCR analysis was performed on wild type and flurochloridone resistant transformants (FIG. 4).
Chlamydomonas genomic DNA was extracted according to the following protocol: An algal pellet of approximately 5-10 μL in size was resuspended in 50 μL of 10 mM NaEDTA by vortexing, followed by incubation at 100° C. for 5 minutes and mixing by vortex. Cells were centrifuged at 12000 g for 1 minute and resuspended in double distilled water (DDW).
Polymerase chain reaction (PCR) analysis was performed on genomic DNA of wild type and flurochloridone resistant Chlamydomonas colonies using the following primers:









pSI103-1362:
AATGCAAGCAGTTCGCATGC
(SEQ ID NO: 7)


 


PDS reverse:
GGCGATGGTCAGGGTCTG
(SEQ ID NO: 8)






The PCR reaction medium was as follows: Genomic DNA 2 l, primer PDS reverse 1 μl, primer pSI103-1362 1 μl, 12.5 μl PCR mix (RedTaq, Sigma), 8.5 μl DDW and the program set at 94° C. for 5′, and then 35 cycles of 94° C. for 1′, 62° C. for 1′, 72° C. for 2.5′. The PCR samples were separated on 1% agarose gel and viewed under UV light (FIG. 4).
PCR analysis was performed on genomic DNA of wild type and flurochloridone resistant Nannochloropsis colonies (FIG. 5) with the following primers:









PDS F short:
CGTGGTGGCCGTGAACCTGA
(SEQ ID NO: 9)


 


PDS R short:
CGCTGTTGCGGAAGCTGGAG
(SEQ ID NO: 10)






PCR content and program was set as follows: Genomic DNA 2 μl, primer PDS F short 1 μl, primer PDS R short 1 μl, 12.5 μl PCR mix (Sigma), 8.5 μl DDW. 94° C. 5′, and then 35 cycles of 94° C. 30′′, 69° C. 30′′, 72° C. 30′′, PCR samples were separated on 1% agarose gel and viewed under UV light (FIG. 5A) Similarly, the same construct was incorporated into Nannocloropsis CS179 by microporation (FIG. 5B).
A dose response experiment was generated for the wild type Chlamydomonas and for its transformants. The wild types and the transformed algae were plated in SGII medium in 24 well plates with increasing concentrations of flurochloridone of 0.1 μM, 0.3 μM, 0.5 μM. Algae were plated without the herbicide as well as a control (FIG. 6). The most resistant colonies were chosen for further analysis.
EXAMPLE 5
Prevention of Algae and/or Cyanobacteria Contaminants
To address the question of contaminants, the Chlamydomonas pds transformants were mixed with the cyanobacterium Synechococcus PCC7942, which is known to overtake ponds. A Chlamydomonas pds transformant and the cyanobacterium Synechococcus PCC7942 were inoculated alone and in mixed cultures with and without flurochloridone. An example of a mixed culture is shown in FIG. 7. The mixed culture was inoculated in a ratio of 10:1 Chlamydomonas pds transformants: Synechococcus PCC7942 wild type, respectively. The cells were allowed to grow for one week in liquid medium, with daily removal of aliquots. Contaminants versus target Chlamydomonas-cells were counted under the microscope (FIG. 8). While the growth of the resistant pds Chlamydomonas cells was unaffected by the flurochloridone addition (FIG. 8A), the growth of Synechococcus PCC7942 cells was completely inhibited, implying that this potential contaminant is not able to outcompete the resistant Chlamydomonas culture.
EXAMPLE 6
Prevention of Contaminants by the Use of Slow Released Herbicide, Embedded in the Plastic/Polyethylene of the Photobioreactor
A hydrophobic herbicide/biocide such as flurochloridone or fluridone and/or butafenacil flumioxazin is/are applied using a volatile organic solvent such as xylene on the inner side of the polyethylene algae photobioreactor or pond liner, such that if it were fully released into the subsequent growth medium the concentration would reach a final concentration of 10−5 to 10−7M. Transgenically resistant algae or cyanobacteria versus wild-type are inoculated in the treated photobioreactors after the solvent has evaporated. In commercial production, the herbicides can be added during production of the polyethyelene.
Culture aliquots are removed daily and cell densities of wild types and the transformed algae or cyanobacteria are compared. While the growth of the resistant pds transformed algae is unaffected by the addition of flurochloridone or fluridone and/or butafenacil or flumioxazin, the growth of the wild type culture is completely inhibited, implying that the incorporation of herbicide into the bioreactor lining during plastic manufacture provides advantage to the herbicide resistant algae and cyanobacteria.
EXAMPLE 7
Greater Photosynthetic Efficiency of pds Transgenic Algae in Dense Cultures at High Intensites
One of the important parameters indicating the welfare of a photoautotrophic culture is its photosynthetic efficiency. We used the following methodologies to show the photosynthetic activity: Oxygen evolution—using Clark Type electrodes. Variable fluorescence—using PAM (Pulse Amplitude Modulated Fluorometry) We also evaluate dark oxygen consumption, in order to estimate net photosynthetic potential of the algal culture. As part of the photosynthetic evaluation we follow several abiotic parameters that potentially influence the physiological state of the cultures.

    
    
        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 CO2 levels (e.g. ambient; 1%; 5%; 14%-100%). This is coupled with pH tolerance.
        Temperature tolerance. Each culture is tested to attain the optimal temperature. In addition, temperatures are raised to the highest point possible without inhibiting other culture activities.

Photosynthetic Activity: Oxygen Evolution

    
    


Measurements of O2 concentrations were performed using a Clark type O2 electrode (Pasco Scientific, Roseville, Calif.). Twenty mL of cell suspension corresponding to 15 μg chlorophyll/mL were placed in an O2 electrode chamber, at relevant temperature (22° C.). Cells were exposed to various light intensities (i.e. 50, 300 and 1000 μmol photons m−2 s−1). Dark incubations were performed in air-tight vessels to follow dark oxygen consumption.
We compared a wild type culture of Chlamydomonas reinhardtii with its pds transformants. Results are shown in FIG. 9, and indeed reveal that not only was the photosynthesis of the transformants not inhibited but they actually exhibited improved photosynthetic activity, implying an enhanced biomass production.
The overall outcome from the analyses shown in FIGS. 9 and 10 implies that the selected transformants are both resistant to the herbicide and they perform better than wild type in terms of oxygen evolution. Taken together, these transgenic cultures are relevant candidates to be grown at large scale production systems as well as being used as a platform for future transformations.
Fluorescence Measurements
Electron transfer activity of photosystem II is measured by pulse modulated fluorescence (PAM) kinetics using PAM-101 (Walz, 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 by a projector lamp at 50-1500 μmol photons m−2 s−1 as required in different experiments and is used to assess steady state fluorescence (Fs). Maximum fluorescence (Fm) is measured with saturating white light pulses of 4000 μmol photons m−2 s−1 for 1 s. At a worse case scenario, a normal electron transfer activity is expected from transformants in order to be considered as relevant for up-scaling. An improved activity may imply a culture that will perform better than the wild type. The advantage of the method is a quick analysis on a wide array of candidate transformants.
EXAMPLE 8
Synthesis of Appropriate Butafenacil and Protox Resistant Protoporphyrinogen Oxidase Gene
The ppo gene was de novo synthesized according to the appropriate codon usage of the desired algae and the desired cyanobacteria or according to general algae and general cyanobacterial codon usage. The ppo gene with the glycine deletion at position 210 according to the Amaranthus tuberculatus GenBank accession no. DQ386116 (SEQ ID NO:11) was custom synthesized, according to the Chlamydomonas codon usage by the by the GENEART synthesis company (http://www.geneart.com/). The gene was synthesized with the BstBI,EcoRI restriction site on the 5′ and BamHI on the 3′ for direct cloning into pSI103 and pPhaT1 algae expression vectors. Transformation is conducted for the following algae: Chlamydomonas reinhardtii, Pavlova lutheri, Isochrysis CS-177, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina CS-190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp. as representatives of all algae species (see Table 1).
Amaranthus tuberculatus protoporphyrinogen oxidase gene according to the codon usage of the cyanobacterium Synechococcus PCC7002 (SEQ ID NO:12) is transformed in cyanobacteria Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongatus BP-1 as representatives of all cyanobacterial species.
EXAMPLE 9
Cloning the Resistant Protoporphyrinogen Oxidase Gene into an Expression Vector
The de novo synthesized ppo gene is cloned under the control of rbcS2 and or fcpA/35S/ubiquitin/tubulin promoters and 3′rbcS2/fcpA/fcpB terminators, in the plasmids pSI103 and or pPHAT1 (Sizova et. al 2001; Zaslayskaia et. al 2000). For cyanobacteria it was cloned under the constitutive promoter of the rbcLS operon (Deng and Coleman 1999) in the plasmid pCB4 as well as into various expression vectors, allowing various levels of expressions driven by different promoters, including constitutive, inducible and log phase temporal promoters.
EXAMPLE 10
Transformation of the Resistant Protoporphyrinogen Oxidase Gene into Algae and Cyanobacteria
Constructs are transformed using various techniques as described in Example 3. These procedures are carried out on the following algae: Chlamydomonas reinhardtii, Pavlova lutheri, Isochrysis CS-177, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina, Tetraselmis suecica, Tetraselmis chuii, Nannochloris sp. and as representatives of all algae species (Table 1).
Constructs are incorporated into the cyanobacteria Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongatus BP-1 as representatives of all cyanobacterial species, as representatives of all cyanobacterial species using a standard protocol as set out in (Golden, et al. 1987). Briefly, cells are harvested by centrifugation and re-suspended in BG-11 medium at a concentration of 2-5×108 cells per ml. To one ml of this cell solution the appropriate plasmid construct is added to a final concentration of 2-5 μg/ml. Cells were incubated in the dark for 8 hours followed by a 16 h light incubation prior to plating on BG-11 plates containing butafenacil or flumioxazin to select for the colonies that grow at the highest rates without affecting algal growth. Plates are incubated under the standard growth conditions (30° C., light intensity of 100 μmol photons m−2 s−1). Butafenacil or flumioxazin resistant colonies were visible in 7-10 days. This is modified for each organism according to its needs, based on modifications of standard protocols.
EXAMPLE 11
Verification of the Presence of the Synthetic ppo Gene Transformed Algae and Cyanobacteria Harboring the Resistant Protoporphyrinogen Oxidase
The transformants are replated on fresh agar medium containing 10−6M or 10−7M of butafenacil or flumioxazin, concentrations that have been shown to inhibit the wild-type algae growth as is shown in FIG. 11. Cells are allowed to generate to visible colonies. After incubation for a week the most resistant colonies are chosen. To ascertain gene transformation PCR analysis is preformed on wild type and butafenacil or flumioxazin resistant colonies.
A dose response curve is generated for the wild type alga and for the transformants. The wild types and the transformant algae are plated in their medium in 24 well plates with increasing concentrations of 0.1 μM, 0.3 μM, 0.5 μM butafenacil or flumioxazin. Algae are plated without the herbicide as a control. Transformants bearing the gene that confers resistance show ability to grow at higher concentrations than the wild type. The most resistant colonies are chosen for further analysis.
EXAMPLE 12
Prevention of Algae/Cyanobacteria Contaminants
To address the question of contaminants, the transformed algae are mixed with cyanobacteria such as Synechococcus PCC7002 and Synechococcus WH7803, which are known to overtake ponds. The transformant and the cyanobacteria are cultured alone and in mixed cultures with and without butafenacil or flumioxazin. The mixed culture is plated in a ratio of 10:1 transformant: cyanobacteria respectively. The cells are allowed to grow for one week in liquid culture, with daily removal of aliquots. These aliquots are counted under the microscope and the ratio of contaminants vs. algae is calculated. The mixed cultures (of transformants and contaminants) containing the herbicide show decreasing numbers of contaminants, whereas in the mixed cultures without the herbicide the cyanobacteria outcompete the algae.
EXAMPLE 13
Combination of Two Herbicide Types for Prevention of Algae/Cyanobacteria Contaminants
To address the question of contaminants, stacked pds and ppo transformed algae are mixed together with wild type Synechococcus 7002 cyanobacteria, which are known to overtake ponds. The cells are allowed to grow together for 1 day to 1 week in liquid culture, with daily removal of aliquots that are plated either to new liquid culture or on Petri dishes with and without the combination of flurochloridone or fluridone and butafenacil or flumioxazin. Contaminant cyanobacteria vs. target algae are counted. Conditions are optimized for the competing organism, in order to verify that the transformed algae overcome outcompete the contamination under these conditions.
EXAMPLE 14
Prevention of Zooplankton Grazing by High CO2 Concentrations and Herbicide Application
To quantify CO2 effects on plankton survivorship we incubate 81 L vessels of the algae Pavlova lutheri, Isochrysis CS-177, Nannochloropsis oculata CS-179, Nannochloropsis like CS-246, Nannochloropsis salina CS-190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp or cyanobacteria Synechococcus PCC7002, Synechococcus WH-7803, Thermosynechococcus elongatus BP-1 with or without zooplankton (e.g. Arthemia sp.). The control and zooplankton treated containers are bubbled with air, 1%, 5% and 14% CO2 together with specific herbicides that are applied at the appropriate concentrations. Algal cell density and zooplankton counts are done on each treatment over a 10 day period. Throughout the experiment pH is maintained at 7.0-8.0, which allows phytoplankton growth.
There is a near 100% reduction of live zooplankton in the >5% CO2 and the herbicide treatments. Zooplankton are killed by the microtubule-inhibiting herbicides that do not harm the wild type algae and the cyanobacteria such as benefin, butralin, dinitramine, ethalfluralin, oryzalin, pendimethalin, trifluralin, amiprophos-methyl, butamiphos, dithiopyr, thiazopyr, propyzamide, tebutam and chlorthal-dimethyl. Algal density is significantly higher at 5 and 14% compared to ambient and 1% CO2 concentrations. While zooplankton reduced algal growth at low concentrations, in high CO2 there are no differences between batches with and without zooplankton, indicating the inhibition of zooplankton activity. Zooplankton numbers remain low for 7 days after termination of CO2 treatments, implying a significant, long-term impact.
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## Claims

1. A method to maintain a cyanobacterial or algal monoculture free from unwanted species, said method comprising the steps of: a) Transforming a cyanobacterium or an alga with a nucleotide sequence to express resistance against one or more herbicides; b) Cultivating said cyanobacterium or alga expressing resistance against one or more herbicides; and c) adding one or more herbicides into the cultivation medium; wherein said alga or said cyanobacterium is transformed with a nucleotide sequence encoding for a protein conferring resistance to said one or more herbicides, wherein said protein has a sequence encoded according to the nucleotide sequence selected from the group consisting of SEQ ID NOs 1-6, 11 and 12.

2. The method of claim 1, wherein the herbicide is hydrophobic and is applied using a volatile organic solvent on an inner side of a polyethylene algae photobioreactor or pond liner.

3. The method of claim 1, wherein the herbicide is selected from the group consisting of fluorochloridone and fluridone.

4. The method of claim 1, wherein the alga is transformed with the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:5.

5. The method of claim 4, wherein the nucleotide sequence is operably linked to a promoter selected from a group consisting of RbcS, fcpA, 35S, ubiquitin, tubulin and actin promoters.

6. The method of claim 1, wherein the cyanobacterium is transformed with the nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

7. The method of claim 6, wherein the nucleotide sequence is operably linked to a promoter selected from a group consisting of RbcS, fcpA, 35S, ubiquitin, tubulin and actin promoters.

8. The method of claim 1, wherein the herbicide is butafenacil or flumioxazin.

9. The method of claim 1, wherein the alga is transformed with the nucleotide sequence SEQ ID NO:11.

10. The method of claim 1, wherein the sequence is operably linked to a promoter selected from a group consisting of RbcS, fcpA, 35S, ubiquitin, tubulin and actin promoters.

11. The method of claim 1, wherein the cyanobacterium is transformed with the nucleotide sequence SEQ ID NO:12.

12. The method of claim 11, wherein the sequence is operably linked to a promoter selected from a group consisting of RbcS, fcpA, 35S, ubiquitin, tubulin and actin promoters.

13. The method of claim 1, further comprising providing a CO2 concentration of the cultivation media of between 5% and 100%.

14. The method of claim 1, wherein the cyanobacterium is selected from the group consisting of Synechococcus PCC7002, Synechococcus WH-7803, and Thermosynechococcus elongatus BP-1.

15. The method of claim 1, wherein the alga is selected from the group consisting of Chlamydomonas reinhardtii, Pavlova lutheri, Isochrysis CS-177, Nanochloropsis CS-179, Nanochloropsis CS-246, Nanochloropsis salina CS-190, Tetraselmis suecica, Tetraselmis chuii and Nannochloris sp.

16. The method of claim 1, wherein the transformation is performed through microporation.