Genetically modified alga, sequences and methods thereof

Patent metadata

Field	Value
Publication number	US-11566251-B2
Application number	US-201916700557-A
Country	US
Kind code	B2
Filing date	Dec 2, 2019
Priority date	Dec 3, 2018
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

The present disclosure relates to the fields of biotechnology, molecular biology and genetic engineering. In particular, the present disclosure relates to a genetically modified alga comprising a recombinant cytochrome c6 gene, methods of producing the same and applications thereof. The present disclosure also relates to a codon optimised nucleic acid sequence encoding a cytochrome c6 polypeptide, expression cassette, vectors and host cell thereof. In an embodiment, the present disclosure also relates to a method of increasing biomass and photosynthetic efficiency of algae.

First claim

We claim:

1. A genetically modified alga comprising a recombinant cytochrome c6 gene comprising nucleotide sequence set forth as SEQ ID NO. 1 or a variant having at least 80% identity to SEQ ID NO. 1.

2. The genetically modified alga of claim 1 , wherein the recombinant cytochrome c6 gene is from a different species than the alga which is modified; wherein the genetically modified alga comprises a chloroplast targeting peptide for targeting cytochrome c6 expressed by the recombinant gene to thylakoid lumen of chloroplast of the genetically modified algae.

3. The genetically modified alga of claim 1 , wherein the cytochrome c6 gene is obtained from red algae and is codon-optimized for said modified alga; wherein the red algae is Porphyra yezoensis ; and wherein the genetically modified alga is selected from the group consisting of Chlorella sp., Nannochloropsis sp., Nannochloris sp., Neochloris sp., Pseudoneochloris sp., Chlamydomonas sp., Picochlorum sp., cyanobacteria, diatoms, Spirulina , and any combination thereof.

4. The genetically modified alga of claim 1 , wherein the alga is Chlorella sorokiniana ; wherein said SEQ ID NO. 1 is codon-optimized for Chlorella sorokiniana ; or wherein the genetically modified alga is Chlorella sorokiniana having Accession Number CCAP 211/135.

5. The genetically modified alga of claim 2 , wherein nucleic acid encoding the chloroplast targeting peptide is set forth in SEQ ID. NO. 2 or a variant having at least 80% identity to SEQ ID. NO. 2; and wherein the genetically modified alga has characteristics selected from the group consisting of enhanced production of biomass, enhanced photosynthetic efficiency, resistance to stress, and any combination thereof.

6. A method of obtaining the genetically modified alga of claim 1 , comprising steps of:
a) introducing a nucleic acid, a vector, or an expression cassette comprising recombinant cytochrome c6 gene comprising the nucleotide sequence set forth as SEQ ID NO. 1 or a variant having at least 80% identity to SEQ ID NO. 1 into an algal cell; and
b) selecting modified algal cell expressing the recombinant gene to obtain the genetically modified alga.

7. The method of claim 6 , wherein the recombinant cytochrome c6 gene is from a different species than the genetically modified alga; wherein the nucleic acid, vector, or the expression cassette is introduced in the algal cells by a technique selected from the group consisting of: biolistic, glass bead, carbon whiskers, agrobacterium mediated genetic transformation, and electroporation; and wherein the cytochrome c6 expressed by the recombinant gene is targeted to thylakoid lumen of chloroplast of the genetically modified algae by a chloroplast targeting peptide.

8. The method of claim 6 , wherein the cytochrome c6 gene is obtained from red algae and is codon-optimized for said genetically modified alga, wherein the red algae is Porphyra yezoensis ; and wherein the modified alga is selected from the group consisting of: Chlorella sp., Nannochloropsis sp., Nannochloris sp., Neochloris sp., Pseudoneochloris sp., Chlamydomoas sp., Picochlorum sp., cyanobacteria, diatoms, and Spirulina , and any combination thereof.

9. The method of claim 6 , wherein the modified alga is Chlorella sorokiniana; wherein the recombinant cytochrome c 6 gene comprises a nucleotide sequence set forth as SEQ ID NO. 1 or a variant having at least 80% identity to SEQ ID NO. 1, wherein said SEQ ID NO. 1 is codon-optimized for Chlorella sorokiniana having Accession Number CCAP 211/135.

10. The method of claim 7 , wherein nucleic acid encoding the chloroplast targeting peptide is set forth in SEQ ID. NO. 2, or a variant having at least 80% identity to SEQ ID. NO. 2; wherein the genetically modified alga comprises an expression cassette comprising a recombinant cytochrome c6 gene encoding a cytochrome c6 polypeptide, nucleic acid encoding chloroplast targeting peptide, and an operably linked promoter; and wherein the genetically modified alga has characteristics selected from the group consisting of: enhanced production of biomass, enhanced photosynthetic efficiency, resistance to stress and any combination thereof.

11. A nucleic acid sequence encoding a cytochrome c6 polypeptide comprising sequence as set forth in SEQ ID. NO. 1 or a variant having at least 80% identity to SEQ ID. NO. 1.

12. A vector comprising the nucleic acid sequence of claim 11 and an operably linked promoter.

13. An expression cassette comprising the nucleic acid sequence of claim 11 , nucleic acid encoding chloroplast targeting peptide sequence and an operably linked promoter.

14. The expression cassette of claim 13 , wherein sequence of the nucleic acid encoding chloroplast targeting peptide comprises sequence as set forth in SEQ ID. NO. 2 or a variant thereof having at least 80% identity to SEQ ID. NO. 2; and wherein the promoter comprises sequence as set forth in SEQ ID. NO. 12.

15. A host cell comprising the nucleic acid sequence of claim 11 .

16. The host cell of claim 15 , wherein the host cell is selected from the group consisting of Escherichia sp., Chlorella sp., Nannochloropsis sp., Nannochloris sp., Neochloris sp., Pseudoneochloris sp., Chlamydomonas sp., Picochlorum sp., cyanobacteria, diatoms, dinoflagellates, Spirulina , and any combination thereof.

17. A method of producing algae biomass by culturing the genetically modified alga of claim 1 under conducive condition.

18. A method of enhancing production of biomass or photosynthetic efficiency in alga, said method comprising culturing the genetically modified alga of claim 1 to achieve enhanced production of biomass or photosynthetic efficiency.

19. The method of claim 18 , wherein the method comprises culturing the genetically modified alga at a temperature ranging from about 25° C. to about 35° C. and for a time-period ranging from about 24 hours to about 300 hours; wherein the method optionally comprises culturing the genetically modified alga under light of about 200-2000 μmoles/m 2 /s of photon flux density, and optionally with about 2-5% CO2 and air mix; optionally recovering the algae biomass, and optionally purifying the recovered algae biomass; and wherein the genetically modified alga achieves enhanced biomass production or photosynthetic efficiency relative to corresponding wild-type alga.

20. A method of using the genetically modified alga of claim 1 for production of biomass, high value protein, nutraceuticals, biologicals or biofuel, comprising step of culturing the genetically modified alga under conducive condition.

Assignees

• Reliance Industries Ltd

Inventors

• Banerjee Arun
• Das Gautam
• Patil Sonal Santosh
• Sanyal Sourav
• Patil Smita Dattatraya
• Dasgupta Santanu

Classifications

• C12N15/79 Primary

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• A01G33/00 Primary

  Cultivation of seaweed {or algae} · CPC title

• C07K14/80

  Cytochromes · CPC title

• C12N1/12

  Unicellular algae; Culture media therefor (as new plants A01H13/00) · CPC title

• C07K14/405

  from algae · CPC title