Patent
A genetically enhanced cyanobacterial host cell, Cyanobacterium sp. ABICyano1, is disclosed. The enhanced Cyanobacterium sp. ABICyano1 produces a compound or compounds of interest.
1. A non-naturally occurring ethanologenic Cyanobacterium sp. organism comprising a genetically enhanced plasmid having a polynucleotide sequence identity of at least 85% to SEQ ID NO: 72 and wherein said genetically enhanced plasmid comprises at least one heterologous alcohol dehydrogenase (adh) gene and at least one heterologous pyruvate decarboxylase (pdc) gene wherein said adh gene encodes an alcohol dehydrogenase that has an amino acid sequence identity of at least 60% to the amino acid sequence of the enzyme encoded by nucleotides 2125 to 3135 of SEQ ID NO: 72 and is operably linked to a promoter and wherein said pdc gene encodes a pyruvate decarboxylase that has an amino acid sequence identity of at least 60% to the amino acid sequence of the enzyme encoded by nucleotides 285-1991 of SEQ ID NO: 72 and is operably linked to a promoter, and wherein said organism produces ethanol from about 0.002% (vol/vol) per day up to about 0.07% (vol/vol) per day.
2. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 wherein said organism is capable of producing ethanol at a rate of 0.07% (vol/vol) per day.
3. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 wherein said organism is capable of producing ethanol at a rate of 0.047% (vol/vol) per twelve hours.
4. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 wherein said organism is capable of producing ethanol at a rate of about 0.0201% (vol/vol) per day at about 114 days of growth.
5. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 wherein said organism produces ethanol at about 135 days of growth.
6. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 , wherein said Cyanobacterium sp. organism is Cyanobacterium sp. ABICyano1 deposited in the American Type Culture Collection (ATCC) under ATCC accession number PTA-13311 transformed with said genetically enhanced plasmid, wherein said at least one heterologous adh gene and at least one heterologous pdc gene are expressed in a sufficient amount for the organism to produce ethanol.
7. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 6 , wherein said promoter operably linked to said adh gene is constitutive, and wherein said promoter operably linked to said pdc gene is inducible.
8. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 7 , wherein said promoter operably linked to said adh gene is a promoter having a polynucleotide sequence identity of greater than 90% to a promoter polynucleotide sequence selected from the group consisting of SEQ ID NO: 158 (Prbc*), SEQ ID NO: 9 (PcpcB), SEQ ID NO: 124 (PrpsL*4) and SEQ ID NO: 159 (PrpsL).
9. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 7 , wherein said promoter operably linked to said pdc gene is selected from promoters inducible by the presence or absence of nitrate and/or copper.
10. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 9 , wherein said promoter operably linked to said pdc gene is a promoter having a polynucleotide sequence identity of greater than 90% to a promoter polynucleotide sequence selected from the group consisting of SEQ ID NO: 10 (PnirA), SEQ ID NO: 160 (PnirA*2), SEQ ID NO: 161 (PnirA*4), SEQ ID NO: 35 (Porf0221), SEQ ID NO: 37 (Porf0223), and SEQ ID NO: 38 (Porf0316).
11. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 6 , wherein said promoter operably linked to said adh gene is endogenous to Cyanobacterium sp., and wherein said promoter operably linked to said pdc gene is endogenous to Cyanobacterium sp.
12. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 11 , wherein said promoter operably linked to said adh gene and said promoter operably linked to said pdc gene are selected from promoters comprising polynucleotide sequences that are 90% or more identical to the polynucleotide sequence of any of SEQ ID NOs 9-41.
13. The non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 11 , wherein said genetically enhanced plasmid comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 43, 72, 73, 74, 83, 84 and 106.
14. A method for producing ethanol comprising growing the non-naturally occurring ethanologenic Cyanobacterium sp. organism of claim 1 .
15. The method of claim 14 wherein ethanol is produced from about 0.002% vol/vol ethanol per day up to about 0.07% vol/vol ethanol per day.
16. The method of claim 14 wherein said organism is capable of producing ethanol at a rate of 0.07% (vol/vol) per day.
17. The method of claim 14 wherein said organism is capable of producing ethanol at a rate of 0.047% (vol/vol) per twelve hours.
18. The method of claim 14 wherein ethanol is produced at a rate of about 0.0201% (vol/vol) per day at about 114 days of growth.
19. The method of claim 14 , wherein the genetically enhanced plasmid in said non-naturally occurring ethanologenic Cyanobacterium sp. organism comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 43, 72, 73, 74, 83, 84 and 106.