IP Library › Granted Patent US 9,862,974
Granted Patent B2
US 9,862,974 · App. 14/654,827 · Granted Jan 9, 2018

Patent

Inventors: Kui Wang (Fort Myers, FL); Tuo Shi (San Diego, CA); Irina Piven (Berlin, DE); Masami Inaba (Berlin, DE); Frank Uliczka (Berlin, DE); Dan Kramer (Berlin, DE); Heike Enke (Berlin, DE); Kerstin Baier (Kleinmachnow, DE); Alexandra Friedrich (Berlin, DE); Ulf Duehring (Berlin, DE)
Assignee: Algenol Biotech LLC
C12P7/06C07K14/195C12N9/0006C12N9/88C12N15/52C12N15/74C12P7/065C12R1/01Y02E50/17
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Quick Facts
Patent No.
US 9,862,974
App. No.
14/654,827
Granted
Jan 9, 2018
Kind
B2
Abstract

A genetically enhanced cyanobacterial host cell, Cyanobacterium sp. ABICyanoI, is disclosed. The enhanced Cyanobacterium sp. ABICyanoI produces a compound or compounds of interest.

Claims (26)

1. A non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism comprising a genetically modified plasmid that is different from the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, comprising a nucleic acid sequence that has at least 60% sequence identity to an endogenous Cyanobacterium sp. ABICyanoI plasmid selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 1 and the nucleic acid sequence of SEQ ID NO: 2, wherein said genetically modified plasmid has a function of expressing a gene of interest in said non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism.

2. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 1 , wherein said genetically modified plasmid comprises a heterologous alcohol dehydrogenase (adh) gene and a heterologous pyruvate decarboxylase (pdc) gene, and wherein said adh gene is operably linked to a constitutive promoter, and wherein said pdc gene is operably linked to a copper inducible promoter selected from the group consisting of Porf0221, Porf0223 and Porf0316.

3. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 1 , wherein said genetically modified plasmid comprises a heterologous alcohol dehydrogenase (adh) gene and a heterologous pyruvate decarboxylase (pdc) gene, and wherein said adh gene is operably linked to a constitutive promoter, and wherein said pdc gene is operably linked to a copper inducible promoter selected from the group consisting of Porf0221, Porf0223 and Porf0316, and wherein production of ethanol by said ethanologenic Cyanobacterium sp. ABICyanoI host cell is from about 0.017 percent ethanol (vol/vol) per OD 750 per day to about 0.031 percent ethanol (vol/vol) per OD 750 per day for up to 30 days.

4. The non-naturally occurring Cyanobacterium sp. ABICyanoI organism of claim 1 wherein said ethanologenic Cyanobacterium sp. ABICyanoI host cell produces up to about 0.049 percent ethanol (vol/vol) per OD 750 per day.

5. A non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism deposited in the American Type Culture Collection (ATCC) under ATCC accession number PTA-13311, and further comprising an ethanologenic pathway and at least one exogenous nucleic acid encoding an ethanologenic pathway enzyme expressed in a sufficient amount to produce ethanol, said ethanologenic pathway comprising a pyruvate decarboxylase, and an alcohol dehydrogenase.

6. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 5 , wherein said organism comprises one, two, or three exogenous nucleic acids each encoding an ethanologenic pathway enzyme.

7. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 5 , wherein said at least one exogenous nucleic acid encoding an ethanologenic pathway enzyme is inserted into a plasmid that is endogenous to Cyanobacterium sp. ABICyanoI, to create an ethanologenic plasmid.

8. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 7 , wherein said ethanologenic plasmid contains a nucleic acid sequence that has at least 70% identity to the nucleic acid sequence of SEQ ID NO: 1.

9. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 7 , wherein said ethanologenic plasmid comprises an origin of replication that has a sequence identity of at least 80% to the sequence of nucleotides 3375 to 3408 of SEQ ID NO: 1.

10. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 7 , wherein said ethanologenic plasmid comprises a heterologous adh gene and a heterologous pdc gene, and wherein said adh gene is operably linked to a promoter, and wherein said pdc gene is operably linked to a promoter.

11. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 10 , wherein said heterologous adh gene is selected from the gene encoding for an alcohol dehydrogenase selected from the group consisting of ADH1520 from Microcystis , ADH553 from Cyanothece , ADH242 from Arthrospira , ADH916 from Synechococcus and ADH1102 from Croococcidiopsis.

12. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 10 , wherein said promoter operably linked to an adh gene and said promoter operably linked to a pdc gene each have a sequence that is at least 90% identical to the sequence of a promoter that is endogenous to Cyanobacterium sp. ABICyanoI.

13. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 10 wherein said promoter operably linked to an adh gene and said promoter operably linked to a pdc gene are selected from the group consisting of PrbcLS, PntcA, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpB1, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PisiB, PnrsB, PlrtA, PmrgA, PpstS, and PcrhC, PpetJ, PpsbD, PnblA, PrpoA, PisiB, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpB1, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PcrhC, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, PcpcB, PhspA, PclpB1, PhliB, PnirA*2, PnirA*3, PnirA*4, PmntC, PrpsL*4, Prbc*, PcpcB, PrpsL*4, PcpcB (SEQ ID NO: 9), PnirA (SEQ ID NO: 10), PlrtA (SEQ ID NO: 11), PmrgA (SEQ ID NO: 12), PnblA (SEQ ID NO: 13), PggpS (SEQ ID NO: 14), PpetJ (SEQ ID NO: 15), PppsA (SEQ ID NO: 16), PrnpA (SEQ ID NO: 17), PpstS (SEQ ID NO: 18), Porf0128 (SEQ ID NO: 19), Porf1486 (SEQ ID NO: 20), Porf3164 (SEQ ID NO: 21), Porf3293 (SEQ ID NO: 22), Porf3621 (SEQ ID NO: 23), Porf3635 (SEQ ID NO: 24), Porf3858 (SEQ ID NO: 25), Porf1071 (SEQ ID NO: 26), Porf1072 (SEQ ID NO: 27), Porf1074 (SEQ ID NO: 28), Porf1075 (SEQ ID NO: 29), Porf1542 (SEQ ID NO: 30), Porf1823 (SEQ ID NO: 31), Porf1824 (SEQ ID NO: 32), Porf3126 (SEQ ID NO: 33), Porf3389 (SEQ ID NO: 34), Porf0221 (SEQ ID NO: 35), Porf0222 (SEQ ID NO: 36), Porf0223 (SEQ ID NO: 37), Porf0316 (SEQ ID NO: 38), Porf3232 (SEQ ID NO: 39), Porf3461 (SEQ ID NO: 40), Porf3749, and (SEQ ID NO: 41).

14. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 10 wherein said promoter operably linked to an adh gene and said promoter operably linked to a pdc gene are selected from the group consisting of Porf0128, Porf1486, Porf3164, Porf3293, Porf3621, Porf3635, Porf1071, Porf1072, Porf1074, Porf1075, Porf1542, Porf1823, Porf3126, Porf0221, Porf0222, Porf0223, Porf0316, Porf3126, Porf3232, Porf3461, and Porf3749.

15. The non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism of claim 10 wherein said promoter operably linked to an adh gene and said promoter operably linked to a pdc gene are selected from a polynucleotide sequence having at least 90% nucleic acid sequence identity to the nucleic acid sequence of a promoter that is selected from the group consisting of PrbcLS, PntcA, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpB1, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PisiB, PnrsB, PlrtA, PmrgA, PpstS, and PcrhC, PpetJ, PpsbD, PnblA, PrpoA, PisiB, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpB1, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PcrhC, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, PcpcB, PhspA, PclpB1, PhliB, PnirA*2, PnirA*3, PnirA*4, PmntC, PrpsL*4, Prbc*, PcpcB, PrpsL*4, PcpcB (SEQ ID NO: 9), PnirA (SEQ ID NO: 10), PlrtA (SEQ ID NO: 11), PmrgA (SEQ ID NO: 12), PnblA (SEQ ID NO: 13), PggpS (SEQ ID NO: 14), PpetJ (SEQ ID NO: 15), PppsA (SEQ ID NO: 16), PrnpA (SEQ ID NO: 17), PpstS (SEQ ID NO: 18), Porf0128 (SEQ ID NO: 19), Porf1486 (SEQ ID NO: 20), Porf3164 (SEQ ID NO: 21), Porf3293 (SEQ ID NO: 22), Porf3621 (SEQ ID NO: 23), Porf3635 (SEQ ID NO: 24), Porf3858 (SEQ ID NO: 25), Porf1071 (SEQ ID NO: 26), Porf1072 (SEQ ID NO: 27), Porf1074 (SEQ ID NO: 28), Porf1075 (SEQ ID NO: 29), Porf1542 (SEQ ID NO: 30), Porf1823 (SEQ ID NO: 31), Porf1824 (SEQ ID NO: 32), Porf3126 (SEQ ID NO: 33), Porf3389 (SEQ ID NO: 34), Porf0221 (SEQ ID NO: 35), Porf0222 (SEQ ID NO: 36), Porf0223 (SEQ ID NO: 37), Porf0316 (SEQ ID NO: 38), Porf3232 (SEQ ID NO: 39), Porf3461 (SEQ ID NO: 40), Porf3749, and (SEQ ID NO: 41).

16. A non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanoI organism comprising a genetically modified plasmid that has at least 60% sequence identity to an endogenous Cyanobacterium sp. ABICyanoI plasmid sequence selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 1 and the nucleic acid sequence of SEQ ID NO: 2, and further comprising an ethanologenic pathway comprising at least one exogenous nucleic acid encoding an ethanologenic pathway enzyme expressed in a sufficient amount for the organism to produce ethanol, said ethanologenic pathway comprising a pyruvate decarboxylase and an alcohol dehydrogenase, wherein said organism produces ethanol from about 0.002% (vol/vol) per day up to about 0.07% (vol/vol) per day.

17. The non-naturally occurring ethanologenic organism of claim 16 wherein said organism is capable of producing ethanol at a rate of 0.047% (vol/vol) per twelve hours.

18. The non-naturally occurring ethanologenic organism of claim 16 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.

19. The non-naturally occurring ethanologenic organism of claim 16 wherein said organism produces ethanol at about 135 days of growth.

20. The non-naturally occurring ethanologenic organism of claim 16 , wherein said plasmid endogenous to Cyanobacterium sp. ABICyanoI consists of the nucleic acid of SEQ ID NO: 1.

21. The non-naturally occurring ethanologenic organism of claim 20 , wherein said organism comprises one, two, or three exogenous nucleic acids each encoding an ethanologenic pathway enzyme.

22. A method for producing ethanol comprising growing a non-naturally occurring ethanologenic organism deposited in the American Type Culture Collection (ATCC) under ATCC accession number PTA-13311, which further comprises an ethanologenic pathway comprising at least one exogenous nucleic acid encoding an ethanologenic pathway enzyme expressed in a sufficient amount for the organism to produce ethanol, said ethanologenic pathway comprising a pyruvate decarboxylase, and an alcohol dehydrogenase.

23. The method of claim 22 wherein ethanol is produced from about 0.002% vol/vol ethanol per day up to about 0.07% vol/vol ethanol per day.

24. The method of claim 22 wherein said organism is capable of producing ethanol at a rate of 0.07% (vol/vol) per day.

25. The method of claim 22 wherein said organism is capable of producing ethanol at a rate of 0.047% (vol/vol) per twelve hours.

26. The method of claim 22 wherein ethanol is produced at a rate of about 0.0201% (vol/vol) per day at about 114 days of growth.

Assignments (1)
CHANGE OF NAME Recorded May 29, 2019
From: ALGENOL BIOFUELS INC.
To: ALGENOL BIOTECH LLC
Reel/Frame 049850/0919 →
Continuity (3)
Provisional Application 61741000 · Dec 21, 2012
Provisional Application 61835294 · Jun 14, 2013
Related Publication 20160053284A1 · Feb 25, 2016