# US 8,232,088 B2 · Genetically engineered herbicide resistance for maintaining axenic cultures

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- Number: US 8,232,088 B2
- Document ID: US8232088B2
- Status: Granted
- Kind: B2
- Application number: 12/584,559
- Filed: 2009-09-08
- Published: 2010-03-18
- Granted: 2012-07-31
- Earliest claimed: 2008-09-05
- Assignees: TransAlgae Ltd
- Inventors: Ofra Chen; Shai Einbinder; Daniella Schatz; Doron Eisenstadt; Jonathan Gressel
- Web page: https://patentgrep.com/patent/US8232088B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12N 9/0004: {Oxidoreductases (1.)}
- C12N 9/001: {acting on the CH-CH group of donors (1.3)}
- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

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.

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 1)

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