US008232088B2 (12) United States Patent (10) Patent No.: US 8,232,088 B2 Chen et al. (45) Date of Patent: Jul. 31, 2012 (54) GENETICALLY ENGINEERED HERBICIDE (56) References Cited RESISTANCE FOR MANTAINING AXENIC CULTURES U.S. PATENT DOCUMENTS 5,910,626 A 6/1999 Haselkorn 7,285,701 B2 10/2007 Kakefuda (75) Inventors: Ofra Chen, Rehovot (IL); Shai 2007/0074303 A1 3/2007 McCutchen et al. .......... 800,278 Einbinder, Hofit (IL); Daniella Schatz, 2007/0178451 A1 8/2007 Deng et al. Givataim (IL); Doron Eisenstadt, Haifa (IL); Jonathan Gressel, Rehovot (IL) FOREIGN PATENT DOCUMENTS EP O244234 11, 1987 EP O519229 12/1992 (73) Assignee: Transalgae Ltd, Rehovot (IL) WO WO94/25583 11, 1994 WO WO97/02753 1, 1997 (*) Notice: Subject to any disclaimer, the term of this WO WO97,08325 3, 1997 patent is extended or adjusted under 35 WO WO97/28243 8, 1997 U.S.C. 154(b) by 375 days. OTHER PUBLICATIONS Michel, A. etal. 2004. Somatic mutation-mediated evolution of her (21) Appl. No.: 12/584.559 bicide resistance . Molec. Ecology 12:3229-3237. Windhoevel, U. et al. 1994. Expression of Erwinia uredovora Phytoene Desaturase in Synechochoccus . Plant Physiol. 104: (22) Filed: Sep. 8, 2009 119-125. (65) Prior Publication Data * cited by examiner US 201O/OO6881.6 A1 Mar. 18, 2010 Primary Examiner — Albert Navarro (74) Attorney, Agent, or Firm — Graeser Associates Related U.S. Application Data International Inc.; Dvorah Graeser (57) ABSTRACT (60) Provisional application No. 61/191,167, filed on Sep. This disclosure provides herbicide resistant algae and cyano 5, 2008. bacteria. This disclosure also provides a method to cultivate algae and cyanobacteria in axenic cultures without contami (51) Int. Cl. nating species. Moreover, this disclosure provides transgenic CI2N L/12 (2006.01) algal and cyanobacterial cells that are capable of high pro CI2N L/20 (2006.01) duction in high light intensities as typically applied in culti (52) U.S. Cl. .................................. 435/252.1; 435/252.3 Vation. Furthermore, a novel transformation method is pro (58) Field of Classification Search ........................ None vided for algal cells. See application file for complete search history. 16 Claims, 14 Drawing Sheets U.S. Patent Jul. 31, 2012 Sheet 1 of 14 US 8,232,088 B2 RCS Hsp70 PDS RbcS pro pro ter fopA fopA fopB fopB pro PDS ter pro Bie ter 35s POS fopA fopB fopB pro ter pro Ble te Ubiq PDS RbCS pro ter Tub POS RbCS pro ter Act POS RS pro ter FIGURE 1. U.S. Patent Jul. 31, 2012 Sheet 2 of 14 US 8,232,088 B2 U.S. Patent Jul. 31, 2012 Sheet 3 of 14 US 8,232,088 B2 U.S. Patent Jul. 31, 2012 Sheet 4 of 14 US 8,232,088 B2 & U.S. Patent Jul. 31, 2012 Sheet 5 of 14 US 8,232,088 B2 U.S. Patent Jul. 31, 2012 Sheet 6 of 14 US 8,232,088 B2 38: **** 88. s: U.S. Patent Jul. 31, 2012 Sheet 7 of 14 US 8,232,088 B2 U.S. Patent Jul. 31, 2012 Sheet 8 of 14 US 8,232,088 B2 :33: :x: {3::::::::::::::::::::::::::::: U.S. Patent Jul. 31, 2012 Sheet 9 of 14 US 8,232,088 B2 Synechococcus PCC 7942 E G. o d Vo g d v c=0= Synechococcus -- Chlamydomonas pds + Synechococcus - - - Synechococcus + FC •oxes - Chalmydomonas pds +Synechococcus + FC Figure 8B U.S. Patent Jul. 31, 2012 Sheet 10 of 14 US 8,232,088 B2 N E na e O o 2 O 2OO 400 600 800 1OOO 1200 Time sec Figure 9 U.S. Patent Jul. 31, 2012 Sheet 11 of 14 US 8,232,088 B2 2Sec Light intensity umole photons m?sec O 500 1000 1500 0 200 400 600 800 1000 1200 8.00E-09 - 8.00E-09 -- aa Wr 4.00E-09 moWT orpds44 pas 4.00E-09 -iewpd538 -900E-24 : -9.00E-24 -400E-09 8.00E-09 - 8,00E-09 4.00E-09 | --WT i mpds5 4,00E-09 auto WT -9.00E-24 appds22 -9.00E-24 -400E-09 |- -400E-09 --w-wo-no 8.00E-09 i-o-o-o- 4.00E-09 | moWT i mis-pds40 -9.00E-24 -400E-09 | Fig. 10A U.S. Patent Jul. 31, 2012 Sheet 12 of 14 US 8,232,088 B2 ... Light intensity mole-photons m2 sect). Light-intensity, Imole-photons-m?Sect O 200 400 500 800 COO 1200 O 200 400 600 800 000 1200 : 6.00E-09 onwaraisew.....au,ionov wristorievousis starsaw:s 6,OOE-09 normourerrorl.--------...--------...-a-a-ria arm. 200E-09 wT 2.00E-09 | --WT nappds --pds38 s -2.00E-09 m mom -2.00E-09 e 3 -6.00E-09 -6.OOE-09 s – 6.00E-09 O 6.00E-09 T-arror-rearran.--a-ra-race.-- 2.00-09 seasonannammam"r" g| 200E-09 -on-wi 3 weamin WTdid --pds22 o pass i OOE-09 2,00E-9 C : ss -SOOE-09 -6.OOE-9 ; s 6.00E-09 f 2.0E-09 -2.00E-09 OE-09 Figure 10B U.S. Patent Jul. 31, 2012 Sheet 13 of 14 US 8,232,088 B2 F CH 3 f\? O F/ O N o––.h A O O Cl O-CH-CH=CH, Fig. 11A U.S. Patent Jul. 31, 2012 Sheet 14 of 14 US 8,232,088 B2 Om Nannochloropsis oculata us Nannochloropsis like -m Tetraselmis chufi smo e o Tetrasennis Suecica mOm Pavlova luther - isochrysis -- Phaeodactylum tricornutum a Synechococcus Butafenacil concentration (M) Figure 11B US 8,232,088 B2 1. 2 GENETICALLY ENGINEERED HERBCDE gen IX (protogen IX) to protoporphyrin IX (proto IX). Two RESISTANCE FOR MAINTAINING AXENIC different nuclear genes, PPX1 and PPX2, encode plastid and CULTURES mitochondrial PPO isozymes, respectively. When susceptible plants are treated with PPO inhibitors, the substrate of PPO, PRIORITY protogen IX, accumulates and is exported from the organelles into the cytoplasm where herbicide-insensitive peroxidase This application claims priority of U.S. Provisional Patent like enzymes in the plasma membrane convert it to proto IX. No. 61/191,167 filed on Sep. 5, 2008. Proto IX accumulates in the cytoplasm and, in the presence of light, induces the formation of singlet oxygen that is damag SEQUENCE LISTING 10 ing to cell membranes. Herbicides that act by inhibiting protoporphyrinogen oxi This application contains sequence data provided on a dase are widely used to control weeds in a variety of crops. computer readable diskette and as a paper version. The paper The first weed to evolve resistance to PPO-inhibiting herbi version of the sequence data is identical to the data provided cides was Amaranthus tuberculatus, a problematic weed in on the diskette. 15 the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target FIELD OF THE INVENTION sites (Lermontova et. al., 1997; Watanabe et. al., 2001). Evaluation of a PPO inhibitor-resistant A. tuberculatus bio This invention relates to cultivation of algae and cyanobac type revealed that resistance was an incompletely dominant teria. More specifically this invention relates to methods to trait conferred by a single, nuclear gene. Three genes pre maintain axenic cultures. The invention relates to manage dicted to encode PPO were identified in A. tuberculatus. One ment of algal culture by means of genetic modification gene from the resistant biotype, designated PPX2L, con tained a codon deletion that was shown to conferresistance by BACKGROUND OF THE INVENTION complementation of a hemO mutant strain of Escherichia coli 25 grown in the presence and absence of the PPO inhibitor A major problem in the commercial cultivation of algae lactofen. PPX2L is predicted to encode both plastid- and and cyanobacteria in axenic culture in bioreactors or open or mitochondria-targeted PPO isoforms, allowing a mutation in closed ponds is that they can become contaminated by other, a single gene to conferresistance to two herbicide target sites. highly competitive but unwanted species of algae and cyano Unique aspects of the resistance mechanism include an amino bacteria, fungi and bacteria, as well as by rotifers and other 30 acid deletion, rather than a Substitution, and the dual-target Zooplankton that devour the desired species in the cultures. ing nature of the gene, which may explain why resistance to (Sheehanetal. 2004). Fluridone is the only USEPA-approved PPO inhibitors has been rare (Patzoldt et al., 2006; Gressel systemic herbicide that is commonly used for control of and Levy 2006; Tranel et al., 2007). aquatic weeds (but not algae) in large bodies of water. It is a Even if fluridone/flurochloridone and protox-inhibiting noncompetitive inhibitor of the enzyme phytoene desaturase 35 herbicides are known, their use in algal or cyanobacterial (PDS), which is one of the first dedicated enzymes of the plant culture has not been possible because the cultured photosyn carotenoid biosynthesis pathway. Under high light intensi thetic algae or cyanobacteria would also be killed. Moreover, ties, carotenoids stabilize the photosynthetic apparatus by there is an unsolved problem of contamination of algaculture quenching the excess excitation energy; therefore, inhibition ponds and bioreactors with unwanted species such as rotifers of phytoene desaturase decreases colored carotenoid concen 40 and other Zooplankton, which are not controlled by phytoene tration and causes photo-bleaching of green tissues (Böger desaturase or protox-inhibiting algae. This disclosure pro and Sandmann 1998). vides solution to each of these unsolved contamination prob The pds gene was cloned from the herbicide-susceptible as lems. well as from the recently-evolved, herbicide-resistant bio types of the water weed hydrilla Hydrilla verticillata (Lif) 45 SUMMARY OF THE INVENTION Royle.