US 20100081177A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0081177 A1 Schatz et al. (43) Pub. Date: Apr. 1, 2010 (54) DECREASING RUBISCO CONTENT OF Publication Classification ALGAE AND CYANOBACTERIA (51) Int. Cl CULTIVATED IN HIGH CARBON DOXDE CI2P 7/64 (2006.01) (75) Inventors: Daniella Schatz, Givataim (IL); CI2P I/04 (2006.01) Jonathan Gressel, Rehovot (IL); CI2P I/00 (2006.01) Doron Eisenstadt, Haifa (IL); Shai CI2N I/2 (2006.01) Ufaz, Givat Ada (IL) CI2N 15/74 (2006.01) Correspondence Address: CI2N I/3 (2006.01) DODDS & ASSOCATES 1707 NSTREET NW (52) U.S. Cl. ........ 435/134:435/170; 435/41; 435/252.3: WASHINGTON, DC 20036 (US) 435/471; 435/257.2 (73) Assignee: TransAlgae Ltd, Rehovot (IL) (21) Appl. No.: 12/584,571 (57) ABSTRACT (22) Filed: Sep. 8, 2009 Algae and cyanobacteria are genetically engineered to have Related U.S. Application Data lower RUBISCO (ribulose-1,5-bisphosphate carboxylase/ oxygenase) content in order to grow more efficiently at (60) Provisional application No. 61/191,169, filed on Sep. elevated carbon dioxide levels while recycling industrial CO, 5, 2008, provisional application No. 61/191,453, filed emissions back to products, and so as not to be able to grow on Sep. 9, 2008. outside of cultivation. 38:8 3xxx. :33.33. 3:38 :::::::::: Patent Application Publication Apr. 1, 2010 Sheet 1 of 2 US 2010/0081177 A1 *:::::::: Patent Application Publication Apr. 1, 2010 Sheet 2 of 2 US 2010/0081177 A1 | 8 psi-rics AS s s 8:33:3:$33:38 US 2010/0081177 A1 Apr. 1, 2010 DECREASINGRUBSCO CONTENT OF fructose-1,6-bisphosphate aldolase and spinach triose phos ALGAE AND CYANOBACTERA phate isomerase in cells of a cyanobacterium doubled their CULTIVATED IN HIGH CARBON DOXDE activities and greatly increased photosynthetic efficiency and biomass yields (Kang et al. 2005, Ma et al. 2007). 0006. However, one can only over-express enzymes to a PRIORITY limited extent, as the cell does not have unlimited capacity. Therefore, the existing methods have only a limited capacity 0001. This application claims priority of the U.S. Provi and there is a need to improve these methods to enhance the sional applications No. 61/191,169 filed on Sep. 5" 2008 and use of elevated carbon dioxide concentration in algal cultures. 61/191,453 filed on Sep. 9, 2008. SUMMARY OF THE INVENTION 0007. The instant invention provides a solution to this SEQUENCE LISTING existing problem by reducing the levels of other enzymes to “make room' for the over expressed, rate limiting enzymes 0002 This application contains sequence data provided on such as fructose-1,6-bisphosphate aldolase (ALD), chloro a computer readable diskette and as a paper version. The plast triosephosphate isomerase (TPI) or acetyl CoA car paper version of the sequence data is identical to the data boxylase (ACCase) that enhance sink capacity to utilize fixed provided on the diskette carbon dioxide. 0008 Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the key photosynthetic enzyme that catalyzes FIELD OF THE INVENTION the first step of CO, fixation. The chloroplast localized holoenzyme of plants and algae in Sub-kingdom Viridaeplan 0003. This invention relates to the field of genetically tae, Phylum Chlorophyta (heretofore referred to as green engineering algae and cyanobacteria to grow more efficiently algae) contain eight nuclear genome encoded Small subunits on industrial waste emissions of carbon dioxide and is appli and eight chloroplast genome encoded large subunits. In red cable for use with algae and cyanobacteria cultured in closed lineage algae (Sub-Kingdom Chromobiota, Phylum Hapto bioreactors and covered or open ponds for producing high phyta, Heterokonotophyta, Bacillariophyta and others (Table value products, as well as biofuels. It builds on integrating 1), all Sub-units are encoded in the chloroplast. TABLE 1. Phylogeny of some of algae used Genus Family Order Phylum Sub-Kingdom Chlamydomonas Chlamydomonadaceae Volvocales Chlorophyta Viridaeplantae Nannochioris Coccomyxaceae Chlorococcales Chlorophyta Viridaeplantae Tetraseinis Chlorodendraceae Chlorodendrales Chlorophyta Viridaeplantae Phaeodactylum Phaeodactylaceae Naviculales Bacillariophyta Chromobiota Nannochloropsis Monodopsidaceae Eustigmatales Heterokontophyta Chromobiota Paviova Pavlovaceae Pavlovales Haptophyta Chromobiota Isochrysis Isochrysidaceae Isochrysidales Haptophyta Chromobiota Phylogeny according to: http://www.algaebase.org/browseftaxonomy Note: Many genes that in higher plants and Chlorophyta are encoded in the nucleus are encoded on the chlo roplast genome (plastome) of Chromobiota, red lineage algae (Grzebyk, et al. (2003). principles of genetic engineering, photosynthetic physiology 0009. The present consensus is that even more RUBISCO and biochemistry, chemical engineering of bioreactors, and is needed for efficient photosynthesis, as demonstrated in the waste emission engineering. recent suggestion that elevated RUBISCO would enhance the rate of photosynthesis in algae commercially cultivated for BACKGROUND OF THE INVENTION biofuels (Huntley and Redalje 2007). Counter-intuitively, the inventors of this disclosure chose to “make room' for other 0004. A major breakthrough in the large scale cultivation enzymes by reducing concentration of the RUBISCO of algae and cyanobacteria to produce commercially useful enzyme, typically considered to be rate limiting for CO products was the discovery that many such species could be assimilation in photosynthetic cells; RUBISCO can comprise cultivated with flue gas (up to 80% CO) or even pure CO, up to 70% of the soluble protein in plant cells. whereas most other organisms (plants and animals) are “bio 0010 Photosynthesis produces the sugars needed for the chemically anaesthetized” at CO levels of 5% or higher, biosynthesis of the specific primary and secondary metabo slowing all metabolism. This opened the way for cultivating lites of interest in each case (biofuels, pigments, enzymes, such organisms on CO emissions to the environment (Mu pharmaceuticals, starch, etc.). RUBISCO is the first enzyme rakami and Ikenouchi 1997, Negoro, et al. 1993). in the dark reactions of photosynthesis, “fixing carbon diox 0005 Cyanobacteria have already begun to be genetically ide onto an organic molecule. The later reactions use NADPH engineered to utilize these elevated CO levels by over and ATP generated by the light reactions to reduce the fixed expressing genes encoding rate limiting enzymes of the "dark CO to carbohydrates. RUBISCO was the best enzyme evo reactions” (CO assimilating reactions that utilize NADPH lution could produce pre-antiquity, which was of little matter and ATP from the light reactions) of photosynthesis. Thus, for early in the evolution of earth, e.g. in the Archean era 3.5 example, engineering genes from rice encoding for cytosolic billion years ago when CO concentrations in the atmosphere US 2010/0081177 A1 Apr. 1, 2010 were thought to be at least 100 times more than at present especially adapted to thrive in cultivation with high CO (FIG. 1.4 in Falkowski and Raven, 1997), and the very low levels in the medium, allowing for the over-expression of affinity for CO to RUBISCO was of little consequence. As other, rate limiting enzymes of photosynthesis as well as oxygenous photosynthesis began to remove CO from the enzymes encoding for other desirable traits. Thus, these atmosphere and elevate atmospheric O, the levels of organisms are platforms for further engineering. RUBISCO became limiting, and evolution gradually 0015. In one embodiment the small subunit of RUBISCO increased the levels of RUBISCO in photosynthetic organ is subjected to RNAi suppression and in another it is sub isms to the present high levels. Presently, high concentrations jected to antisense. In yet another embodiment both large and of CO can be inexpensively provided to algae and cyanobac Small Subunits are suppressed by chloroplast transformation teria in culture from industrial combustion of fuels. This will of the rbcLS gene cluster under the control of a mutated lead to much of the RUBISCO being superfluous, and promoter replacing the endogenous promoter and genes. decreasing its content “makes way for over-expressing other 0016. In other embodiments DNA encoding other traits is enzymes needed for photosynthesis as well as releasing either engineered in tandem with the RUBISCO suppression resources for more soluble products. This would be one way simultaneously (co-transformation) or Subsequent to the of sequestering large amounts of carbon dioxide, presently engineering of partial RUBISCO suppression. In such imperative due to the purported relationships between embodiments algae or cyanobacteria with reduced RUBISCO elevated global carbon dioxide levels and global warming. levels are used as a platform for further engineering of other Even though the small subunit of RUBISCO is encoded by a desired traits, with greater efficiency of organism activity. family of genes, the gene products are interchangeable, with These include genes encoding enzymes Such as fructose-1,6- considerable consensus among them. bisphosphate aldolase (ALD), chloroplast triosephosphate 0011. According to this disclosure, reducing RUBISCO in isomerase (TPI) or acetyl CoA carboxylase (ACCase) that green algae can be done by using either antisense or RNAi enhance sink capacity to utilize fixed carbon dioxide. Con technology, both targeting the consensus sequences of the versely, other transgenic traits may be in the algae or cyano Small nuclear