SYSTEM FOR TRANSFORMATION OF THE CHLOROPLAST GENOME OF SCENEDESMUS SP. AND DUNALIELLA SP.
The present disclosure relates to methods of transforming various species of algae, for example, algae from the genus Scenedesmus and the genus Dunaliella , vectors and nucleic acid constructs useful in conducting such transformations, and recombinant algae, for example, Scenedesmus and Dunaliella produced using the vectors and methods disclosed herein.
1 - 80 . (canceled)
81 . An isolated Scenedesmus sp. or Dunaliella sp. comprising a chloroplast genome that has been transformed with an exogenous polynucleotide sequence, wherein the exogenous polynucleotide sequence comprises a nucleic acid sequence encoding a selection marker protein that is a chloramphenicol acetyltransferase (CAT), an erythromycin esterase (EreB), a cytosine deaminase (codA), a 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) resistant protein, or a betaine aldehyde dehydrogenase (BAD).
82 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the nucleic acid sequence encoding the selection marker protein comprises at least one mutation or modification to create a mutated nucleic acid sequence encoding a mutated selection marker protein with a change in at least one amino acid, wherein the selection marker protein and the mutated selection marker protein have amino acid sequences with at least 95% sequence identity to one another and the selection marker protein and mutated selection marker protein can be used in the same manner.
83 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the nucleic acid sequence encoding the selection marker protein is codon optimized for the chloroplast of Chlamydomonas reinhardtii.
84 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the nucleic acid sequence is a nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 148.
85 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the Scenedesmus sp. is S. dimorphus or S. obliquus.
86 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the Dunaliella sp. is D. tertiolecta.
87 . The isolated Scenedesmus sp. or Dunaliella sp. of claim 81 , wherein the selection marker protein is expressed in the Scenedesmus sp. or Dunaliella sp.
88 . A method of selecting for the expression of a selection marker protein in an isolated Scenedesmus sp. or Dunaliella sp. comprising, (a) obtaining the isolated Scenedesmus sp. or Dunaliella sp. of claim 87 , and (b) determining if expression of the selection marker protein results in either a positive or negative selection of the transformed Scenedesmus sp. or Dunaliella sp.
89 . The method of claim 88 , wherein expression of the selection marker protein results in positive selection of the transformed Scenedesmus sp. or Dunaliella sp., and positive selection is determined if:
(a) the transformed Scenedesmus sp. or Dunaliella sp. grows in the presence of chloramphenicol when the expressed protein is CAT;
(b) the transformed Scenedesmus sp. or Dunaliella sp. grows in the presence of erythromycin when the expressed protein is EreB; or
(c) the transformed Scenedesmus sp. or Dunaliella sp. grows in the presence of DCMU or Atrazine when the expressed protein is DCMU resistant.
90 . The method of claim 88 , wherein expression of the selection marker protein results in negative selection of the transformed Scenedesmus sp. or Dunaliella sp., and negative selection is determined if:
(a) the transformed Scenedesmus sp. or Dunaliella sp. does not grow as well as a wild-type Scenedesmus sp. or Dunaliella sp. in the presence of 5-fluorocytosine (5FC) when the expressed protein is codA; or
(b) the transformed Scenedesmus sp. or Dunaliella sp. does not grow as well as a wild-type Scenedesmus sp. or Dunaliella sp. in the presence of betaine aldehyde when the expressed protein is BAD.
91 . A method of transforming a chloroplast genome of a Scenedesmus sp. or a Dunaliella sp. with at least one exogenous nucleotide sequence, comprising:
i) obtaining the exogenous nucleotide sequence, wherein the exogenous nucleotide sequence comprises a nucleic acid sequence encoding a protein;
ii) binding the exogenous nucleotide sequence onto a particle; and
iii) shooting the exogenous nucleotide sequence into the Scenedesmus sp. or Dunaliella sp. by particle bombardment, wherein the chloroplast genome is transformed with the exogenous nucleotide sequence.
92 . The method of claim 91 , wherein the exogenous nucleotide sequence is at least 0.5 kb, at least 1.0 kb, at least 2 kb, at least 3 kb, at least 5 kb, at least 8 kb, at least 11 kb, or at least 19 kb in size.
93 . The method of claim 91 , wherein the particle is a gold particle or a tungsten particle.
94 . The method of claim 93 , wherein the gold particle is about 550 nm to about 1000 nm in diameter.
95 . The method of claim 91 , wherein the particle bombardment is carried out by a biolistic device.
96 . The method of claim 95 , wherein the biolistic device has a helium pressure of about 300 psi to about 500 psi.
97 . The method of claim 95 , wherein the biolistic device has a helium pressure of at least 300 psi, at least 350 psi, at least 400 psi, at least 425 psi, at least 450 psi, or at least 500 psi.
98 . The method of claim 91 , wherein the exogenous nucleotide sequence bound to the particle is shot at a distance of about 2 to about 4 cm from the Scenedesmus sp. or Dunaliella sp.
99 . The method of claim 91 , wherein the Scenedesmus sp. is S. dimorphus or S. obliquus , or the Dunaliella sp. is D. tertiolecta.
100 . The method of claim 91 , wherein the protein is a chloramphenicol acetyltransferase (CAT), an erythromycin esterase (EreB), a cytosine deaminase (codA), a 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) resistant protein, or a betaine aldehyde dehydrogenase (BAD).
101 . A transformed chloroplast genome of a Scenedesmus sp. or Dunaliella sp. transformed by the method of claim 91 .