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Title:
IMPROVED METHODS FOR MAKING MARKER FREE MICROBIAL HOST CELLS
Document Type and Number:
WIPO Patent Application WO/2016/105405
Kind Code:
A1
Abstract:
Methods for making marker-free genetically enhanced microbial host cells capable of producing a compound of interest are disclosed. Also disclosed are plasmids comprising the genes required to produce the compound of interest in the host cell, and the host cells comprising the plasmids.

Inventors:
DÜHRING ULF (US)
ULICZKA FRANK (US)
DENG MING-DE (US)
Application Number:
PCT/US2014/072290
Publication Date:
June 30, 2016
Filing Date:
December 23, 2014
Export Citation:
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Assignee:
ALGENOL BIOTECH LLC (US)
International Classes:
C12N1/21; C12N15/63
Foreign References:
US20140178973A12014-06-26
US20080050819A12008-02-28
US20060026706A12006-02-02
Other References:
ISHIKAWA, MASAHITO ET AL.: "A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination", BMC MICROBIOLOGY, vol. 13, no. 86, 17 April 2013 (2013-04-17), pages 1 - 10
TAN, XIAOMING ET AL.: "Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 97, no. 14, July 2013 (2013-07-01), pages 6373 - 6382
Attorney, Agent or Firm:
BARKLEY, Sam et al. (16121 Lee Road Suite 11, Fort Myers FL, US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. Ail extrachromosomal plasmid capable of excision by the activity of a site-directed recombinase, said plasmid comprising a polynucleotide encoding at least one site-directed recoinbinase which is operably linked to at least one inducible promoter; at least two

recombmase recognition target sites of at least one of said at least one site-specific recombinase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, and at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombinase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 5 ' end and wherein said cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 3 ' end and wherein said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest is positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette.

2. The plasmid of claim 1 wherein said at least one site directed recombinase is selected from the group consisting ofFlp, Cre, Dre, KD, B2, B3, A, HK022, HPi, γδ, ParA, Tii3, Gin, <pC31, Bxbl and R4.

3. The plasmid of claim 1 wherein said at least one inducible promoter operably linked to said polynucleotide encoding said site-specific recombinase is selected from the group consisting of Porf0221, Porfi)223} Porf0316, PmiA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, PpetJ, PnarB, and PmntC.

4. The plasmid of claim 1 wherein said plasmid has greater than 80% sequence identity to SEQ ID O:! .

5. The plasmid of claim 1 wherein said plasmid is self-repli eating within a host cell.

6. The plasmid of claim I further comprising an origin of replication.

7. The plasmid of claim 6 wlierem said origin of replication is a self-replicating origni of replication.

S. The plasmid of claim 6 wherein said origin of replication has greater than.90% sequence identity to nucleotides 3375 to 3408 of SEQ ID NO: 1.

9. The plasmid of claim 1 wherein said counter-selection marker is selected from the group consisting of galK, niazF, and sacB.

10. The plasmid of claim 1 wherein said positive selection marker is a gene encoding for a protein that confers antibiotic resistance.

11. The plasmid of claim 1 w erein said at least one promoter operabiy linked to said at least one counter-selection marker is selected from the grou consisting of PiiirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, PpetJ, PnarB, PnmtC, Porf022L Porf0223, Porf0316, Porf0128, Port! 486, Porf316 Porf3293, Porf3621, Porf3635, Porfl071, Porfl072, Porfl074, Porfl075, Porfl542, Porfl823, Porf3126, Porf0222, Porf3126, Porf3232, Porf3461. PorB749, Prbc, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, and PcpcB.

12. The plasmid of claim 1 wherein said at least one enzyme catalyzing the production of a compound of interest comprises pyruvate decarboxylase and alcohol dehydrogenase.

13. The plasmid of claim 1 wherein said compound of interest is ethanol.

14. The plasmid of claim 1 wherein expression of said site directed recombinase from said three-component cassette causes the excision of said three-component cassette from said plasmid.

15. The plasmid of claim 14 wherein a portion of said polynucleotide sequence of said 5' recombinase recognition target site and/or said 3' recombinase recognition target site of said site- specific recombinase remains in said plasmid after excision of said three-component cassette.

16. The plasmid of claim 15 wherein said portion of said polynucleotide sequence of said 5 ' recombinase recognition target site, and/or said V. recombinase recognition target site of said, site- specific recombinase remains 'in said plasmid after excision of said three-component cassette wherein said portion can be used as a marker to identify excision of said three-component cassette from said plasmid.

17. A method for making a non-naturally occurring positive selection marker free host ceil capable of the production of a compound of interest comprising,

a) introducing an extraehromosomai plasmid comprising a three-component cassette and a second cassette wherein sard three-component cassette comprises a counter-selection marker, a positive selection marker and a site-directed recombinase operably linked to an inducible promoter, and wherein said three-component cassette has a recognition target sequence for said site directed recombinase at a 5' end and a 37 end, and wherein said second cassette comprises at least one polynucleotide sequence encoding for at least one enzyme catalyzmg the production of a compound of interest in a host cell, and

b) inducing the expression of said site-directed recombinase, and

c) expressing said counter-selection marker in a growth medium, and

d) selecting for host cells that grow on said growth medium of step c), and

e) repeating step d until selected host cells contain a homogenous population of said extraehromosomai plasmid lacking said three-component cassette.

18. The method of claim 17 wherein the growth medium of step c comprises a counter- selection compound of said counter-selection marker.

19. The method of claim 17 wherein step b) is performed before step c).

20. The method of claim 17 wherein said host cell is a cyanobacterium.

21. The nietliod of claim 17 wherein said site directed recombinase is selected from the group consisting of Flp, Ore, Dre, KD, B2, B3, "L HK022, HP1, γό\ ParA, Tn3, Gin, fC31, Bxbl and R4.

22. The method of claim 17 wherein said at least one inducible promoter operably linked to said polynucleotide encoding said site-specific recombmase is an selected from the group consisting of PnirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA. PpetJ, PnarB, PmntG. Porf022L Porfl>223, PorfD316, PorfD128, Porfl486, Porf3164, Porf3293; Porf3621 , Porf3635, Porfl071, Porfl072, Porfl074, Porfl075, Porfl542, Porfl823, Porf3126, Porffl222, Porf3126, Porf3232, Porf3461 , Porf3749, Prbc, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, and PcpcB.

23. The plasmid of claim 17 wherein said counter-selection marker is selected from the group consisting of galK, roazF, and sacB.

24. The plasmid of claim 17 wherein said posi tive selection marker i a gene encoding for a protein that confers antibiotic resistance.

25. The method of claim 17 wherein said at least one enzyme catalyzing the production of a compound of interest comprises pyruvate decarboxylase and alcohol dehydrogenase.

26. The method of claim 17 wherein said compound of interest is ethanol.

27. The method of claim 17 wherein expression of said site directed recombmase from said three-component cassette causes the excision of said three-component cassette from said plasmid.

28. The method of claim 27 wherein one of said 5 ' recombinase recognition target site and said 3' recombinase recognition target site of said site-specific recombinase remains in said plasmid after excision of said three-component cassette.

29. The method of claim 28 w herein said remaining 5T recombinase recognition target site or said remaining 3' recombmase recognition target site can be used as a marker to identify excision of said three-component cassette.

30. A non-naturally occurring cyanobacterium capable of producing a compound of interest comprising an extrachromosomal plasmid capable of excision by the activity of a site-directed recombmase, said plasmid comprising a polynucleotide encoding at least one site-directed recombmase which is operably linked -to at least one inducible promoter, at least two recombinase recognition target sites of at least one .of said at least one site-specific recombinase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, and at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombinase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 5' end and wherein said cassette has one of said at least two recombmase recognition target sites of said site-specific recombinase at a 3' end and wherein said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest is positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette wherein said non-naturally occurring cyanobacterium makes said compound of interest through the steps of;

a) inducing the expression of said site-directed recombinase. and

b) expressing said counter-selection marker in a growth medium, and

c) selecting for host cells that grow on said growth medium of step b), and

d) repeating step e until selected host cells contain a homogenous population of said extrachromosomal plasmid lacking said three-component cassette, and

e) inducing the expression of said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest, and

f) isolating said compound of interest.

31. The non-naturally occurring cyanobacterium of claim 30 wherein said at least one site directed recombinase is selected from the group consisting of Flp, Cre, Dre, KD, B2, B3, λ, HK022, HP1, γδ, ParA, Tn3, Gin, <pC31 , Bxbl and R4.

32. The non-naturally occurring cyanobacterium of claim 30 wherein said at least one inducible promoter operably l ked to said polynucleotide encoding said site-specific recombinase is selected from the group consisting of Porf0221, Porf0223, Porf0316, PnirA, PziaA, PsmtA, PcorT, PnrsB, PniiA, PpetJ, PnarB, and PmntC.

33. The non-naturally occurring cyanobacterium of claim 30 wherein said plasmid has greater than 80% sequence identity to SEQ ID NO: 1.

34. The non-naturally occurring cyanobacterium of claim 30 wherem said piasmid is self- replicating within a host cell.

35. The non-naturally occurring cyanobacterium of claim 30 further comprising an origin of replication.

36. The non-naturally occurring cyanobacterium of claim 35 wherem said origin of replication is a self-replicating origin of replication.

37. The non-naturally occurring cyanobacterium of claim 35 wherem said origin of replication has greater than 90% sequence identity to nucleotides 3375 to 3408 of SEQ ID NOT.

38. The non-naturally occurring cyanobacterium of claim 30 wherem said counter-selection marker is selected from the group consisting of galK, mazF, and sacB.

39. The non-naturally occurring cyanobacterium of claim 30 wherein said positive selection marker is a gene encoding for a protein that confers antibiotic resistance.

40. The non-naturally occurring cyanobacterium of claim 30 wherein said at least one promoter operabiy linked to said at leas one counter-selection marker is selected from the group consisting ofPnirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, PpetJ, PnarB, PmntC, Porf0221, Porf02233 Porf0316, PoriI)128:. PorfI486, Porf3 64, Porf3293:. Porf3621, Porf3635, Porfl071, Porfl072, Porfl074, Porfl075, Porfl542, Porfl823, Porf31265 Porf0222, Porf3126, Porf3232, Porf346L Porf3749, Prbc, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, and PcpcB.

41. The non-naturally occurring cyanobacterium of claim 30 wherem said at least one enzyme catalyzing the production of a compound of interest comprises pyruvate decarboxylase and alcohol dehydrogenase.

42. The non-naturally occurring cyanobacterium of claim '30 wherein said compound of interest is ethanol.

43. The non-naturally occurring cyanobacterium of claim 30 wherein expression of said site directed recombmase from said three-component cassette causes the excision of said three- component cassette from said plasmid.

44. The non-naturally occurring cyanobacterium of claim 43 wherein a portion of said polynucleotide sequence of said 5' recombmase recognition target site and/or said 3' recombmase recognition target site of said site-specific recombmase remains in said plasmid after excision of said three-component cassette.

45. The non-naturally occurring cyanobacterium of claim 44 wherein said portion of said polynucleotide sequence of said 5' recombmase recognition target site and/or said 3' recombmase recognition target site of said site-specific recombinase remains in said plasmid after excision of said three-component cassette wherein said portion can be used as a marker to identify excision of said three-component cassette from said plasmid.

46. A non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanol comprising an extrachromosomal plasmid derived from wild-type p6.8 capable of excision by the activity of a site-directed recombmase, said plasmid comprising a polynucleotide encoding at least one site- directed recombinase which is operably linked to at least one inducible promoter, at least two recombinase recognition target sites of at least one of said at least one site-specific recombinase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, an alcohol dehydrogenase gene, a pyruvate decarboxylase gene, and wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombinase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombinase recognition target sites of said site-speciiic recombinase at a 5 ' end and wherein said cassette has one of said at least two recombinase recognition target sites of said site -specific recombinase at a 3' end and wherein said alcohol dehydrogenase gene and said pyruvate decarboxylase gene are positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette wherein said non-naturaily occurring ethanoiogenie cyanobacterium makes ethanol through the steps of:

a) inducing the expression of said site-directed recombinase, and

b) expressing said counter-selection marker in a growth medium comprising a counter- selectable marker, and

c) selecting for host cells that grow on said growth medium of step b), and

d) repeating step c until selected host cells contain a homogenous population of said extrachroniosonial plasmid lacking said three-component cassette, and

e) inducing the expression of said alcohol dehydrogenase gene and said pyruvate decarboxylase gene in a growth medium, and

i) isolating ethanol from said growth medium of step e).

47. The non-naturally occurring ethanoiogenie Cyanobacterium sp. ABICyanol of claim 46 wherein said site-directed recombinase is flippase.

48. The non-naturaily occurring ethanoiogenie Cyanobacterium sp. ABICyanol of claim 47 wherem said at least two recombinase recognition target sites of at least one of said at least one site-specific recombinase is a flippase recognition target sequence.

49. The non-naturaily occurring ethanoiogenie Cyanobacterium sp. ABICyanol of claim 46 wherem said at least on positive selection marker is a gene encoding for a protein that confers antibiotic resistance.

49. The non-naturaily occurring ethanoiogenie Cyanobacterium sp. ABICyanol of claim 46 wherem said counter- selection marker is galK.

50. The non-naturally occurring ethanoiogenie Cyanobacterium sp. ABICyan l of claim 49 wherem said counter-selectable marker is 2-DOG.

Description:
Improved Methods for Making Marker Free Microbial Host Cells

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0001 j The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: P0037.01.PCT_sequence_listing_ST25, recorded: December 23, 2014, file size; 193 kilobytes).

FIELD OF THE INVENTION

[0002] The present invention relates to genetically enhancing microbes to produce compounds of interest.

BACKGROUND OF THE INVENTION

[0003] Cyanobacteria are prokaryotes capable ofphotoautotrophy. Cyanobacteria can be genetically enhanced to use light and CO? to produce compounds of interest such as biofuels, industrial chemicals, pharmaceuticals, nutrients, carotenoids, and food supplements. Various cyanobacteria! strains have been genetically enhanced to produce compounds of interest. Carbon dioxide that is used by cyanobacteria can be derived from any source, such as a waste byproduct of industrial production. In this way, cyanobacteria can be used to recycle CO? to compounds of interest.

[0004] The cyanobacteria! genus Cyanobact&ium was first established in 1983 (see Rippka et at. (2001), Bergey's Manual of Systematic Bacteriology, Vol. L p. 497-498). Members of the

Cy bact riwn genus are often found in thermal mats (Moro, et al., 2007, Algological Studies, 123: 1-15).

[0005] The transformation of the cyanobacterial genus C anobacterium with genes that encode enzymes that can produce ethanol for biofuel roduction has been described, for example, in U.S. Patent No. 8.846,369 to Piven eial

[0006] Making genetically modified organisms which lack genes conferring antibiotic resistance (ABPv) currently rely on time and labor intensive methods. The making of an AB , or any marker, free host cell requires multiple transformation steps in order to end up with a marker free organism and an engineered genetic modification.

[0007] Flippase ("FLP") is a site-specific DNA recombinase that is derived from the 2 ηι plasmid of Saccharomyces cerevisi e. Flippase recognizes a particular DNA sequence of 34 nucleotides, which is referred to as flippase recognition target sequence ("FRT"). The flippase recognition target sequence consists of a DNA sequence of 34 bp, and consists of a sequence of 8 bp flanked by two inverted repeats of 13 bp, see .Jayara et al., Pwe, Nail. Acad Sci. 82, 5875- 5879 (1985). The 8 bp sequence is referred to as a spacer region and is where DNA

recombination is earned out, see Umlauf S. W. et al, EMBO Journal, 7, 1845-1852 (1988); Lee J. et al., EMBO Journal 18, 784-791, ( 1999).

[0008] Flippase performs the entire process of cleavage, exchanging and binding of a DNA strand between two FRT sequences, see Babineau et al., J. Biol. Chem., 260, 12313-12319 ( 1985). When two FRT sequences having an identical orientation exist within the same circular DNA molecule, a DNA sequence flanked by the two FRT sequences is excised by the FLP recombinase. Previous approaches to generating ABR free cyanobacterial host cells have relied upon generating auxotrophy, have required chromosomal integration of expression cassettes, homologous recombination and'or only the activity of a site-specific recombinase, see, tor example, Duhring, U. et al., WO2012175750; Curtiss, T. et al., WO2008131359; O'Keefe T. et al, US20100304 32 Al ; Xu, Y. WO2012135766; and Tan, X. et al., Appl Microbiol Biotechnol. 97: 6376-6382 (2013). Thus, existing approaches to generating ABR free cyanobacterial host cells require multiple transformation steps and the resulting genotypes are heterozygous for antibiotic resistance markers even after undergoing multiple rounds of selection.

SUMMARY

[0009] Improved methods for creating non-naturally occurring microbial host cells lacking antibiotic resistance markers are presented herein. In an embodiment, a method requiring only one transformation is used to make a non-naturally occurring microbial host cell lacking an antibiotic resistance marker.

[0010] In an embodiment, a flippase gene, an antibiotic resistance marker and a counterselection marker are all part of a three-component cassette having a flippase recognition target sequence at its 5 ' and 3 T end being situated within an extrachromosomal plasmid that encodes for genes responsible for the production of a compound of interest. Upon expression of the flippase gene and the counterselection marker encoded within the cassette and subsequenct addition of a counter-selection compound, the counterselection marker, the antibiotic resistance marker and the flippase gene are all together excised from the extrachromosomal plasmid much more quickly compared to relying solely upon the activity of tlippase. The dual activity of both flippase and the counter-selection marker produces a marker free extechrornosomal plasmid.

[0011] In an aspect, an extracliromosomal plasmid capable of excision by the activity of a site- directed recombinase is disclosed, said plasmid comprising a polynucleotide encoding at least one site-directed recombinase which is operably linked to at least one inducible promoter, at least two recombinase recognition target sites of at least one of said at least one site- specific recombinase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, and at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombinase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 5' end and wherein said cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 3' end and wherein said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest is positioned within the polynucleotide sequence of said plasmid at a position outsid of said polynucleotide sequence comprising said three-component cassette. In an embodiment, the plasmid contains at least one site directed recombinase is selected from the grou consisting of Flp, Cre, Dre, KD, B2, B3, λ, HK022, HP1, γδ, ParA, Tn3, Gin, φ{ ' 1. Bxbl and R4. in another embodiment, the plasmid contains at least one inducible promoter operably linked to said polynucleotide encoding said site-specific recombinase is selected from the group consisting of Porf0221, PorfD223, Porf0316, PnirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, PpetJ, PnarB, and PiiintC. In yet another embodiment the plasmid has greater than 80% sequence identity to p6.8 from ABICyanol . In another embodiment, the plasmid is self-replicating within a host cell. In an embodiment, the plasmid has an origin of replica tion. In an embodiment, the plasmid has an origin of replication that is a self-replicating origin of replication, in yet another embodiment, the plasmid of has an origin of replication that has greater than 90% sequence identity to nucleotides 3375 to 3408 of p6.8. In one embodiment the plasmid of has a counter-selection marker that is selected from the group consisting of gal , mazF, and sacB. In an embodimen the plasmid contains a positive selection marker that is a gene encoding for a protein that confers antibiotic resistance. In an embodiment, the plasmid of contains at least, one promoter operably linked to said at least one counter-selection marker is selected from the group consisting of PnirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, P etJ, PnarB, PmntC, Porf0221, PorfD223, Porf03K\ Porf0128, Porfl486, PortB164,.Porf3293 i Porf3621, Poxf3635, Porfl07l, Porfl072, Porf Q74, Porf 1075, Porf 1542, Porfl82 Porf3 l26, Porl0222, Porf3126, Porf3232, Porf3461. Porf3749, Prbe, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2 f PpsbD. and PcpcB. In another embodiment, the plasmid of claim i wherein said at least one enzyme catalyzing the production of a compound of interest comprises pyruvate decarboxylase and alcohol dehydrogenase. In an embodiment, the plasmid encodes for enzymes that produce a compound of mterest and that compound of interest is ethanol. In another embodiment, the plasmid expresses said site directed recombinase from said three-component cassette and causes the excision of said three-component cassette from said plasmid. hi another embodiment, the plasmid has a portion of said polynucleotide sequence of said 5 J recombinase recognition target site and'or said 3' reconibmase recognition target site of said site -specific recombinase remains in said plasmid after excision of said three-component cassette. In an embodiment, the plasmid of contains a portion of said polynucleotide sequence of said 5' recombinase recognition target site and or said ' recombinase recognition target site of said site-specific recombinase remains in said plasmid after excision of said three-component cassette wherein said portion can be used as a marker to identify excision of said three-component cassette from said plasmid.

[0012] In another aspect, a method for making a non-naturally occurring positive selection marker free host cell capable of the production of a compound of interest comprising; a) introducing an extrachromosomal plasmid comprising a three-component cassette and a second cassette wherein said three-component cassette comprises a counter-selection marker, a positive selection marker and a site-directed recombinase operably linked to an inducible promoter, and wherein said three-component cassette has a recognition target sequence for said site directed recombinase at a 5' end and a 3' end, and wherein said second cassette comprises at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest in a host cell, and; b) inducing the expression of said site -directed recombinase, and; c) expressing said counter-selection marker in a growth medium, and; d) selecting for host cells that grow on said growth medium of step c), and; e) repeating step d until selected host cells contain a homogenou population of said extrachromosomal plasmid lacking said three-component cassette. In anotlier embodiment, the growth medium of step c comprises a counter- selection compound of said counter- election marker , hi another embodiment, step b) is performed before step c). In an embodiment, the host, cell is a cyanobacterium. In an embodiment, the expression of said site directed recombinase from said three -component cassette causes the excision of said three-component cassette from said plasmid.

{0013] la an aspect, a non-naturally occurrin cyanobacterium capable of producin a compound of interest comprising an extrachromosornai plasmid capable of excision by the activity of a site- directed recombmase is disclosed, and said plasmid comprising a polynucleotide encoding at least one site-directed recoiiibinase which is operably linked to at least one inducible promoter, at least two recombmase recognition target sites of at least one of said at least one site-specific recombmase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, and at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombmase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombmase recognition target sites of said site-specific recombinase at a 5' end and wherein said cassette has one of said at least two recoiiibinase recognition target sites of said site-specific recombinase at a 3' end and wherein said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest is positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette wherein said non-naturally occurring cyanobacterium makes said compound of interest, through the steps of: a) inducing the expression of said site-directed recombinase, and; b) expressing said counter-selection marker in a growth medium, and; c) selecting for host cells that grow on said growth medium of step b), and; d) repeating ste c until selected host cells contain a homogenous population of said extrachromosornai plasmid lacking said three-component cassette, and; e) inducing the expression of said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of said compound of interest, and; f) isolating said compound of interest.

[0014] In another aspect, a non-naturally occurring etiianologemc Cyanobacterium sp.

ABICyano comprising an ex tra hromosom 1 plasmid derived from wild-type p6.8 capable of excision by the activity of a site-directed recombinase is disclosed, wherein said plasmid comprising a polynucleotide encoding at least one site-directed recombmase which is operably linked to at least one inducible promoter, at least two recombmase recognition target sites of at least one of said at least one site-specific recombinase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, an alcohol dehydrogenase gene, a pyruvate decarboxylase gene, and wherein a three-component cassette comprises said polynucleotides encoding said at least one site directed recombinase, said a least one counter-selection marker and said at least one positive selection marker and wherein said three-component cassette has one of said at least two recombmase recognition target sites of said site-specific recombmase at a 5 " end and wherein said cassette has one of said at least two recombmase recognition target sites of said site-specific recombmase at a 3' end and wherein said alcohol dehydrogenase gene and said pyruvate decarboxylase gene are positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette wherein said non-naturally occurring ethanologenic cyanobacterium makes ethanol through the steps of: a) inducing the expression of said site- directed recombmase, and; b) expressing said counter-selection marker in a growth medium comprising a counter- selectable marker, and; c) selecting for host cells that grow on said growth medium of step b), and: d) repeating step c until selected host cells contain a homogenous population of said extrachramosornal plasmid lacking said three-component cassette, and; e) inducing the expression of said alcohol dehydrogenase gene and said pyruvate decarboxylase gene in a growth medium, and; i) isolating ethanol from said growth medium of step e). In an embodiment, the non-naturally occurring ethanologenic Cyanobacterium sp. ABICyanol contains a site-directed recombmase that is flippase. In another embodiment, the non-naturally occurring ethanologenic Cyanobacterium sp, ABICyanol cpmtaoms at least two recombinase recognition target sites of at least one of said at least one site-specific recombinase is a fhppase recognition target sequence. In yet another embodiment, the non-naturally occurring

ethanologenic Cyanobacterium sp. ABICyanol contains at least on posiiive selection marker is a gene encoding for a protein that confers antibiotic resistance. In an embodiment, the non- naturally occurring ethanologenic Cyanobacterium sp. ABICyanol contains a counter-selection marker that is galK.

[0015] In an aspect, a non-naturally occurring cyanobacterium capable of producing a compound of interest comprising an extrachromosomal plasmid capable of excision by the activity 7 of a site- directed recombinase is disclosed, said plasmid comprising a polynucleotide encoding at least one site-directed recombinase which is operably linked to at least one inducible promoter, at least two recombinase recognition target sites of at least one of said at least one site-specific recombmase, at least one counter-selection marker operably linked to at least one promoter, at least one positive selection marker, and at least one polynucleotide -sequence encoding for at least one enzyme catalyzing tlie production of a compound of interest wherein a three-component cassette comprises sai d polynucleotides, encoding said at least one site directed recombinase, said at least one counter-selection marker and said at least one positive selection marker and wherein said three -component cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 5" end and wherein said cassette has one of said at least two recombinase recognition target sites of said site-specific recombinase at a 3" end and wherein said at least one polynucleotide sequence encoding for at least one enzyme catalyzing the production of a compound of interest is positioned within the polynucleotide sequence of said plasmid at a position outside of said polynucleotide sequence comprising said three-component cassette.

[0016] In another aspect, an extrachromosomal plasmid capable of excision by the activity of a site-directed recombinase is disclosed wherein said plasmid comprises: a) a three-component cassette comprising i) a polynucleotide encoding said site-directed recombinase which is operably linked to at least one inducible promoter, b) ii) at least two recombmase recognition target sites of said site -specific site-directed recombinase, wherein one of said at least two recombmase recognition target sites of said site-specific recombinase is positioned at the 5' end of said cassette and one of said at least two recombinase recognition target sites of said site- specific recombinase is positioned at the 3' end of said cassette, ; iii) at least one counter- selection marker operably linked to at least one promoter; and iv) at least one positive selection marker; c) at least one self-replicating origin of replication; d) at least one counter-selection marker operably linked to at least one promoter; e) at least one positive selection marker; and f) at least one polynucleotide sequence encoding a polypeptide involved in a biosynthetic pathway that produces a compound of interest, for at least one enzyme cataly zing the production of a compound of interest

[0017] wherein said polynucleotides encoding said site directed recombinase, said counter- selection marker and said antibiotic positive selection marker comprise a three-component cassette which and wherein said three -component cassette has one of said at least two

recombinase recognition target sites of said site-specific recombinase at a 5 "' end and wherein said cassette lias one of said at least two recombmase recognition target sites of said site-specific recombmase at a 3 ' end and wherei said at least one polynucleotide sequence encoding for at least one enzyme catalyzinga polypeptide involved in a biosynthetic pathway that produces the production of a compound of interest is positioned within the polynucleotide sequence of said piasmid at a position outside of said polynucleotide sequence comprising said three-component cassette, and wherein expession of: said site-directed reeoinbinase causes the excision of said three-component cassette from said piasmid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts a piasmid map of a p ABICyanol 6.8 kb endogenous piasmid from

ABICyanol, alternately referred to as p6.8.

[0019] FIG. 2 depicts the polynucleotide sequence of p6.8 and depicts labels of various

polynucleotide sequences.

[0020] FIG. 3 depicts a piasmid map of self-cleaving piasmid #2084 before and after cleavage by an encoded flippase at flippase recognition target sequences.

[0021] FIG. 4 depicts PGR confirmation of cleavage efficiency by flippase after induction by copper addition to ABICyano 1 host cells transformed with #2084.

[0022] FIG. 5 depicts PGR confirmation of complete segregation of a cleaved #2084 achieved upon selection of ABICyanol #2084 cells on 2-DOG plates.

[0023] FIG. 6 depicts confirmation of lack of antibiotic resistance in fully sell-cleaved

ABICyanol #2084 cells on plates with and without kanamycin.

[0024] FIG. 7 depricts VLE corrected ethanol production in ABICyanol host cells containing control #1646 or cut #2084 (ABR-free) plasmids.

[0025] FIG. 8 depicts growth clmactenstics in ABICyanol host ceils containing control #1646 or cut #2084 (ABR-free) plasmids.

[0026] FIG. 9 depicts ethanol production without a VLE correction in ABICyanol host cells containing control #1646 or cut #2084 (ABR-free) plasmids.

[0027] FIG. 10 depicts growth of control (#1938 and #1933) and self-cleaving p!asmid containing (#2151 and #2152) ABIcyanol host cells,

[0028] FIG. 11 depicts ethanol production of control (#1938 and #1933) and self-cleaving piasmid containing (#2151 and #2152) ABIcyanol host cells.

[0029] FIG. 12 depicts total ethanol production per OD in controls (#1938 and #1933) and self- cleaving piasmid containing (#2151 and #21 2) ABIcyanol host cells.

[0030] FIG. 13 depicts a piasmid map of #1646 ^6.8::PnirA-PDC(optl)-TdsrA-Prbc*(optRBS ADH11 l(opt)-TB0011) circular DNA; 12580 BP. [0031] FIG. 14 depicts ' the pol nucleotide sequence of plasmid #1646 and depicts labels of various, polynucleotide, sequences.

[0032] FIG. 15 depicts a plasmid map of #2084 ip6.8::PnirA-PDC(optI3-¾

ADHI 11 (op )-TB0011 -F T-Porf0223 *-flp-TBO011 -PmntC-galK(opt)-TT7-PrbcL-Km* *-FRT) circular D A; 16106 BP.

[0033] FICT. 16 depicts the polynucleotide sequence of plasmid #2084 and depicts labels of various polynucleotide sequences.

[0034] FIG. 17 depicts a plasmid map of a "cut" plasmid #2084 (p6.8::PmrA-PDC(optl)-TdsrA- Pr c*(optRBS)-ADHl 11 (opt)-TBOO 11 -FRY); circular DNA; 1 1266 BP; "cut" meaning from which the three-component cassette has been excised by the activity of the site-specific recombinase encoded therein.

[0035] FIG. 18 depicts the polynucleotide sequence of a cut plasmid #2084 and depicts labels of various polynucleotide sequences.

[0036] FIG. 19 depicts a plasmid map of plasmid #2151 (p6.8::PmrA*2-PDC(optl )-TdsrA- PcpcB-ADH916(opt}-TrbcS-FRT-PoifB223 *-flp-TB0011 -PmntC-galK(opt)-TT7-Pr cL-Km* *- FRT); circular DNA; 16506 BP.

[0037] FIG. 20 depict the polynucleotide sequence of plasmid #2151 and depicts labels of various polynucleotide sequences.

[0038] FIG. 21 depicts a plasmid map of a cut plasmid #2151 (p6.8: :PnirA*2-PDC(optl )-TdsrA- PcpcB-ADH916(opt)-TrbcS-FRT) circular DNA: 1 1667 BP.

[0039] FIG. 22 depicts the polynucleotide sequence of a cut plasmid #2151 and depicts labels of various polynucleotide sequences.

[0040] FIG. 23 depicts a plasmid map of #2152 6.8::ΡθΓί¾316-ΡΟ€(ορΙΙ)-Τά8ΓΑ-ΡοροΒ-

ADHi l l(opt)-TrbcS-FRT-Psm

circular DNA; 16500 BP.

[0041] FIG. 24 depicts the polynucleotide sequence of plasmid #2152 and depicts labels of various polynucleotide sequences.

[0042] FIG. 25 depicts a plasmid map of cut #2152 (p6.8::Porfi)316-PDC(optl)-TdsrA-PcpcB- ADHl l l (opt)-TrbcS-FRT); circular DNA; 11730 BP).

[0043] FIG. 26 depicts the polynucleotide sequence of cut plasmid #2152 and depicts iabeis of various polynucleotide sequences. DETAILED DESCRIPTION

[0044] Presented herein are improved methods for making marker free iiiicrobial host cells that require only a single transformation event. These host cells can be genetically engineered to produce one or more compounds of interest. In one embodiment, the cells produce the compound of interest by encoding one or more polypeptides that are involved in a biosynthetic pathway, hi an embodiment, the methods used to generate marker free host cells are applicable to all microbes, Eukaryotic, Prokaryotic and Archaeal. In another embodiment, the methods disclosed herein are useful in microbes that lack functional and ' or efficient homologous genetic recombination systems. In another embodiment, the methods disclosed herein are useful for creating non-naturally occurring photoautotrophic host cells, such as cyanobacterial host cells.

[0045] The host cells may be deri ved from any type of microbe. In one embodiment, the host cells are derived from an isolated strain of the C anobacierimn genus. A species member of the Cyanobacterium genus is referred to as a Cyanob ct rium sp. and includes but is not limited to several species and strains and have been found in a variety of environments including thermal mats in Italy (Moro, et al, 2007, Algological Studies, 123:1-15). in an embodiment, the marker free host cells are antibiotic resistance AB ) free host cells generated using methods disclosed herein are a species of the Cyanobacterium genus, Cyanobacterium sp. ABICyanol (referred to herein as ABICyanol or ABl) which has been deposited in the American Type Tissue Collection (ATCC) as PTA-13311.

[0046] Genetically enhanced, non-naturally occurring ABR free organisms disclosed herein are useful for the production of compounds of interest including for example, but not limited to ethanol, organic carbon compounds, alcohols, fatty acids, oils, carotenoids, proteins, enzymes, biofuels, nutraceutieals, and pharmaceuticals. Such genetic modifications can be heterologous genes for expression in the host cell in order to establish a foreign metabolic pathway for production of a product of interest. If the compound of interest is ethanol, then, in one embodiment, exogenous pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes on an extrachromosomal plasmid can be introduced into a cyanobacterial host cell for phototrophic production of ethanol.

[0047] Methods for genetically engineering prokaryotes usually rely upon the ability of the microbes to homologously recombhie. However, in some prokaryotic organisms the

recombniation machinery is either non-functional, poorly functional or does not exist. In an embodiment, the present method does not require that a host cell is capable of homologous recombination. Thus, the methods presented herein are useful for engineering those .microbes lacking functional recombination machinery.

[0048] In contrast to Synechococcus PCC7002 and Synechocys s PCC6803, ABlCyanol is very rarely capable of homologou recombination and especially double-crossover events. Thus, implementing strategies that rely upon single or double recombination events in ABlCyanol were either fruitless, highly labor intensive, or took many months to achieve.

[0049] In an embodiment, site-specific recombinases, such as flippase from Sacckaromyces cerevisi e, actively catalyze recombination between two site-specific recognition sites, for example the flippase recognition target sequence. For example, the short 34 bp FRT sit has the sequence 5 '-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3 ' to which the flippase binds to both 13 bp 5-GAAGTTCCTATTC-3' amis flanking the 8 bp spacer. FRT-mediated cleavage occurs just ahead from the asymmetric 8 bp core region (5 * -tctagaaa-3 ! ) on the top strand and behind this sequence on the bottom strand, see Zhu et al, (1995) "Cleavage-dependent Ligation by the FLP Recombmase" J. Biol Chem 270 (39): 23044-54. The subsequent ligation step is also catalyzed be the flippase enzyme itself (Pan et al,, (1993) "Mechanism of cleavage and ligation by FLP recoiiibiiiase: classification of mutations in FLP protein by in vitro complementation analysis" Mol Cell Biol. Jun;13(6):3167-75).

[0050] Counter selectable markers are powerful tools in genetics because they enable a directed selection for removal of a genetic marker rather than its presence (Balkan et al. (2011) "An improved counter-selectable marker system for mycobacterial recombination using g !Kmd 2- Deoxy-Galactose Gene" Jan 1, 2011; 470(1-2): 31-36).

[0051] In an embodiment, counter-selection markers include, but are not limited to, genes such as sacB, galK and mazF. The E. coli g lK gene, encoding the enzyme galactokinase, catalyzes the phosphorylation of galactose to galactose- 1-phosphate. It also efficiently phosphorylates a galactose analogue, 2-deoxy-galactose (2-DOG). The product of this reaction, 2-deoxy-galactose- 1 -phosphate, cannot be further metabolized, leading to buildup of toxic levels and cell death

(Barkan et al, 2011 ). Tims, expression igalK leads to a sensitivity to 2-DOG which is otherwise nontoxic to the cells, hi another embodiment, rnazF is used as a counter-selection marker in the three-component cassette. MazF from is. coli is an endoribonuclease that cleaves mR A at the ACA triplet sequence, and thus acts as a general inhibitor for the synthesis of all cellular proteins (Cheah Y. E., et al. (2013). A novel counter-selection method for markerless genetic modification in Synechocystis sp. PCC 6803. Biotechnol. Prog. 29, 23-30). The small size of the ga!K and mazF genes are also beneficial in genetic manipulations as the chance of uiactivation increases with the size of the gene.

[0052] In an embodiment, an improved method for making marker-free microbial host cells is disclosed winch only uses one transformation step of only one extrachromosomal plasmid for the production of a compound of interest. In one embodiment, the cassette in the extrachromosomal plasrmd comprises a site-directed recombinase, a positive selectable marker, and a counter- selection marker. In one embodiment, the positive selectable marker is an antibiotic resistance gene. In an embodiment, a cassette containing a kanamycm resistance gene, a flippase (ftp) gene and a galK counter-selection marker gene (three component KmR-flp-ga!K) selection cassette is framed by a FRT site on both its 5' and 3 ? ends within an extrachromo soma 1 plasmid containing adh and pdc genes.

{0053] The genes in the cassette may be operably linked to one or more promoters. In one embodiment, the genes in the cassette are under control of the same promoter. In another embodiment, the genes in the cassette are under control of different promoters, hi one

embodiment, one or more promoters are inducible promoters. In an embodiment, the flp gene and galK gene were placed under control of endogenous inducible promoters of ABICyanol that allow for control of expression of either one or both of the genes. This arrangement of the KmR- flp-galK aUkms for a very practical and comparatively rapid procedure that takes only one single transformation via usual conjugational transfer with subsequent flippase action and final counter- selection by GalK and 2-deoxy-ga!actose (2 -DOG) addition to end up with an ABR-free strain. In one embodiment, the ABR-free strain is an ethanologenic ABICyanol strain able to produce ethanol as long and as high as a conventional ABR containing ABICyano 1 producer strain,

{0054] The methods disclosed herein produce marker free host cells quicker than methods currently practiced in the art. In a embodiment, the methods presented herein take about 7 weeks to produce a marker free host, ceil in contrast to the approximately 7 months usin methods currently used in the art. Other advantages of the methods disclosed herein include enabling a high transformation and conjugation efficiency because wild-type strains can be used for the single transformation step. In addition, this technique allows for the use of host cell derivatives coming out of any type of random mutagenesis/ adapted evolution approaches which have altered the genetic sequence of regions that homologous recombination techniques rely upon and which are geneticall uncharacterized yet. In one embodiment, the host cell from which derivatives are obtained is ABICyanol. The methods presented herein are improved over creating auxotrophic strains and complementing by supplying the necessary gene on an extracfaromosomal plasmid at least because conjugation efficiency in an auxotrophic host cell is often substantially reduced.

[0055 j The present method can be used in any microbe, including all cyanobacteria. The present method can use the three-component selec tion cassette in. the place of a commonly used antibiotic resistance cassette. The site-directed recomhinase (e.g. the ftp gene) and the counter-selection marker gene can remain unexpressed, the three component cassette to be used as an antibiotic resistance cassette in the course of normal genetic engineering. When intitial characterization of a strain containing the three component cassette is positive, the entire cassette can be removed later, at any stage of evaluating the strain. Thus, in certain embodiments, the three component cassette acts as a antibiotic resistance marker just as a single component antibiotic resistance marker would but allows for the removal of the entire three-compenent cassette if necessary for the production of a markerless strain is required.

Definitions

10056] Aspects of the disclosure encompass techniques and methods well known in molecular biology, microbiology and cell culture. Laboratory references for these types of methodologies are readily available to those skilled in the art, see, for example. Molecular Cloning: A

Laboratory Manual (Third Edition), Sambrook, I, et al (2001) Cold Spring Harbor Laboratory Press; Current Protocols in Microbiology (2007) Edited by Coico, R, et al, John Wiley and Sons, Inc.; The Molecular Biology of Cyanobacteria (1994) Donald Bryant (Ed.), Springer

Netherlands; Handbook Of Microalgal Culture Biotechnology And Applied Phyeology (2003) Richmond, A.; (ed.), Blackwell Publishing; and "The Cyanobacteria, Molecular Biology, Genomics and Evolution", Edited by Antonia Herrero and Enrique Flores, Caister Academic Press, Norfolk, UK, 2008.

10057] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or pa tent application was specifically and individually indicated to be incorporated by reference.

|0058] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0059] The term "about" is used herein to mean approximately, in the region of, roughly, or around When the term "about" is used in conjunction with a numerical value/range, it modifies that .value/range by extending the boundaries above and below the numerical value(s) set forth, hi general, the ' term "about" is used herein to modify a numerical value(s ' ) above and below the stated value(s) by a variance of 20%.

[0060] It is well known, to a perso of ordinary skill in the art that large plasmids can be

produced using techniques such as the ones described in the US patents US 6,472,184 Bl titled "Method for Producing Nucleic Acid Polymers" and US 5,750,380 titled "DNA Polymerase Mediated Synthesis of Double Stranded Nucleic Acid Molecules", which are hereby incorporated by reference in their entirety.

[0061] Genes are disclosed as a three letter lower case name followed by a capitalized letter if more than one related gene exists, for example mrA. The respective protein encoded by that gene is denominated by the same name with the first letter capitalized, such as MrA, or all letters are capitalized

[0062] The term "FLP recombinase" or "ilippase" or TLP" used herein refers to an enzyme which is encoded by yeast (Saccharomyces cerevisiae) 2 um plasmid and performs site-specific recombination reaction between two FLP recognition sequences (FRT sequences) [Babineau et a]., J. Biol. Chem., 260, 12313-12319 (1985)]. A region flanked by two FRT sequences which are positioned hi the same orientation can be excised by the FLP recombinase.

[0063] The term "FRT sequence" or "flippase recognition target sequence" or "FRT" as used herein refers to the DNA sequence consisting of 34 bp see Jayaram et al., Proc. Natl. Acad. Sci. 82, 5875-5879 (1985).

[0064] The term "spacer region" used herein refers to a 8-bp DNA sequence flanked by two inverted repeats (13 bp) in the above-mentioned FRT sequence. In the present specification, this FRT sequence consisting of 34 bp is especially referred to as a ''wild-type FRT sequence."

[0065] Promoter sequences, which control the transcription of a gene, are given by a capi talized letter "P" followed by the subscripted gene name according to the above described nomenclature, for example for the promoter controlling the transcription of the mrA gene. Promoter sequences may also be referred to without the gene name being subscripted, for example "FmrA".

[0066] Enzynie names can be given in a two or three letter code indicating the origin of the enzyme, followed by the above mentioned three letter code for the enzyme itself, such as

ADHi 1 l(opt) from Lyngbya, SynAdh (Zir ~ dependent Alcohol dehydrogenase from

Synecho ystis PCC 6803), and PDC(optl) (pyruvate decarboxylase from Zymomonas obilis). [0067] The term "Cyanobactena" refers to a member from the group of hotoautotrop ic prokaryotie microorganism . which can utilize solar energy and fix carbon dioxide. Cyanobactena are also referred to as blue-green algae.

[0068] Tire term "teriTuiiator" refers to a nucleic acid sequence, at which the transcription of a rnK A stops. Non-limiting examples are dsrA from Escherichia coli (E. coli), the oop temiinator or the rho terminator.

[0069] The term ^ Cyanobacierium sp." refers to a member of the genus Cyattobacterium, as, for example, characterized by Rippka et aL, 1983. Ann. Microbiol, (hist. Pasteur) 134B: 32.

[0070] The term "BG-1 1" or "BG1 1 " refers to a growth media used for growing cyanobactena 1 species as disclosed in Rippka, R., et al."Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria." (1979) J. Gen. Microbiol. I l l: 1-61.

[0071] The term "mBG-H" or "mBGl i" refers to marine BG11 and in may alternatively be referred to as marine medium. mBGl 1 has from about 30 to about 38 psu (practical salinity units).

[0072] The terms "host cell" and "recombinant host ceil" include a cell suitable for metabolic manipulation including, but not limited to, incorporating heterologous polynucleotide sequences and can be transformed. Host cell and recombinant host cell includes progeny of the cell originally transformed. In particular embodiments, the cell is a prokaryotie cell, such as a cyanobacteria! cell. The term recombinant host cell is intended to include a cell thai has already engineered to have desirable properties and is suitable for further enhancement using the compositions and methods disclosed herein.

[0073] The term "positive selection marker" or "positive marker" as used herein are selectable markers that confer selective advantage to the host organism such s an antibiotic resistance gene which allows a host organism to survive antibiotic selection.

[0074] The term "negative selection marker" or "counterselectable marker" or "counter-selection marker" are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example of a counter-selection marker is galK wherein expression of GalK in the presence of a counter-selection compound, in this case 2-deoxy-galactose, causes the death of a host cell. Other counter-selectio markers are well known in the art and include mazF and sacB, for example.

[0075] The term "positive and negative selectable marker" as used herein means markers that can serve as both a positive and a negative marker by conferring an advantage to the host under one condition, but iahibiting growth under a different condition. An example would be an enzyme that exhibits positive selection by being able to complement an auxotrophy in a first conditio and in a second condition exhibits negative selection by converting a compound to a compound, toxic to the host cell.

[0076] The term "screenabie maker' refers to a marker that when expressed in a host cell confers a first condition and when not expressed in a host cell confers a second condition measurably different from the first condition. An example of a screenabie marker is a gene that produces a coiorometric difference between host cells containing the screenabie marker and host cells that do not contain a functional screenabie marker. A example of a screenabie marker is the lacZ gene that can be used for blue/white screening when grown in the presence ofX-gai and IPTG.

[0077] The term "cassette" or "gene cassette" as used herein refers to a mampulab!e nucleotide sequence or fragment of a nucleotide sequence carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction sites.

[0078] The term "shuttle vector" refers to a vector, such as a piasmid, which can propagate in different host species. For example, a shuttle vector with a cyanohacterial origin of replication can be replicated and propagated in different cyanohacterial genera such as Cyanobacterhm, Synec ococcus, and Synechocystis. Alternatively, or additionally, a shuttle vector may also contain an origin of replication for different phyla of bacteria such as Enterobacteriaceae and Cyanobacteria, so that cloning/genetic enhancements can be performed in E. coli and the recombinant piasmid can be expressed ' maintamed in eyanobacterial hosts. For example, in the latter case, in certain embodiments, the shuttle vector is either a broad host range vector whose origin of replication is recognized by E. coli and cyanobacteria, or a piasmid which contains at least two different origins of replication for the appropriate organism.

[0079] The term " genome" refers to the chromosomal genome as well as to extrachromosomal plasmids which are normally present in wild type cyanobacteria without having performed recombinant DNA technology.

[0080] "Competent to express" refers to a host cell that provides a sufficient cellular environment for expression of endogenous and/or exogenous polynucleotides.

{0081] As used herein, the term "genetically enhanced" refers to any change in the- endogenous genome (chromosomal and plasmidial) of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. Changes to the genome of various organisms disclosed herein are made by the hand of man through the use of various recombmaiit polynucleotide technologies and other techniques such as mutagenesis, for example, included in changes to the genomes are changes in . protein coding sequences or nonprotein coding sequences, including regulatory sequences such as promoters, enhancers or other regulators of transcription.

[0082] The ucleic acids disclosed herein may be modified and Or contain non-natural nucleotide bases.

[0083] As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more ammo acids, but do not affect the functional properties of the protein encoded by the D A sequences. In certain embodiments, changes in one or more nucleotide bases do not change the encoded amino acid. Substantially similar also reiers to modifications of the nucleic acid fragments such as substitution, deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript,

[0084] As used herein, in certain embodiments, homologous nucleic acid sequences are about 60%, 65%, 68%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or even higher identical to nucleic acids disclosed herein.

[0085] The percentage o f identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994 Nucleic Acid

Research 22: 4673-4, 680). A nucleotide sequence or an amino acid sequence can also be used as a so-called "query sequence" to perform a search against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous sequences, winch can also be used in embodiments of this disclosure. Such searches can be performed using the algorithm of Karlin and Altschul (1999 Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993 Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877). Such an algorithm is incorporated in the

NBLAST and XBLAST programs of Altschul et al. (1999 Journal of Molecular Biology 215: 403 to 410). Where gaps exist between two sequences, gapped BLAST can be utilized as described in Altschul et al. (1997 Nucleic Acid Research, 25: 3,389 to 3,402).

[0086] "Recombinant" refers to polynucleotides synthesized or otherwise manipulated in vitro or in vivo ("recombinant polynucleotides") and to methods of using r ecombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems. For example, a cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid. can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a 'Recombinant host cell" or a "recombinant bacterium" or a "recombinant cyanobacteria." The gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant protein." A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

[0087] The term "non-naturally occurring", when used in reference to a microbial organism or microorganism herein is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring str ain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example,

modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and'or other func tional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and

homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon such as regions associated with promoters, for example. Exemplary metabolic

polypeptides include enzymes or proteins within an ethanologenic biosynthetic pathway resulting in the production of ethanol by a non-naturally occurring organism.

[0088] The term "recombinant nucleic acid molecule" includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides).

[0089] The term ''transformation" is used herein to mean the insertion of heterologous genetic material into the host cell. Typically, the genetic material is DNA on a plasmid vector, but other means can also be employed. General transformation methods and selectable markers for bacteria and cyanobaeteria are known in the art (Wiith, Mol Gen Genet. 216: 175-177 (1989);

Koksharova, Appl Microbiol Biotechnol 58: 123-137 (2002). Additionally, transformation methods and selectable markers for use in bacteria are well known (see, e.g., Sambrook et ai, supra).

[0090] The term "homologous recombination" refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity. The term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion). In addition, the recombination can be the result of equivalent onion- equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions. For a description of the enzymes and mechanisms involved in homologous recombination see Court et al., "Genetic engineering using homologous

recombination-" Annual Review of Genetics 36:361-388; 2002.

[0091] The term "non-homologous or random integration" refers to any process by which DNA is integrated into the genome that does not involve homologous recombination. It appears to be a random process in which incorporation can occur at any of a large number of genomic locations.

[0092] The term "vector" as used herein is intended to refer to a nucleic acid molecule

(polynucleotides and oligonucleotides) capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which generally refers to a circular double stranded DNA molecule into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PGR) or from treatment of a circular plasmid with a restriction enzyme.

[0093] Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0094] The term " promoter" is intended to include a polynucleotide segment that can

transcriptionally control a gene of interest, e.g. , a pyruvate decarboxylase gene that it does or does not transcriptionally control in nature, hi one embodiment, the transcriptional control of a promoter results in an increase in expression of the gene of interest. In an embodiment, a promoter is placed 5' to the gene of interest. A heterologous promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter. A promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous

polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used. Promoters may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of an operably linked gene.

[0095] The phrase "operably linked" means that the nucleotide sequence of the nucleic acid molecule or gene of interest is linked to the regulatory sequence(s), e.g., a promoter, in a manner which allows for regulation of expression (e.g.. enhanced, increased, constitutive, basal, attenuated, decreased or repressed expression) of the nucleotide sequence and expression of a gene product encoded by the nucleotide sequence (e.g., when the recombinant nucleic acid molecule is included in a recombinant vector, as defined herein, and is introduced into a microorganism).

[0096] The term "gene" refers to an assembly of nucleotides that encode for a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulator}' sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0097] The term "exogenous" as used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host cell genetic material such as by integration into a host chromosome or as non-chromosomal generic material such as a p!asmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity mat is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly„ the term when used in reference to expression of an encoding nuc leic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

[0098] The term "fragment" refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence substantially identical to the reierence nucleic acid Such a. nucleic acid iragment according to the disclosure may be included in a larger polynucleotide of winch it is a constiaient. Such fragments comprise, or alternatively consist of,, oligonucleotides ranging in length from at least about 6 to about 1500 or more consecutive nucleotides of a polynucleotide according to the disclosure.

[0099] The term "open reading frame" abbreviated as "ORF," refers to a length of nucleic acid sequence, either DNA, cDNA or RNA that contains a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0100] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or stalling point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0101] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0102] The terms "restriction endonuclease" and "restriction enzyme' "" refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.

[0103] The term "expression" as used herein, refers to the transcription and stable accumulation mRNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide. Expression may also be used to refer to the process by which a gene's coded information is converted into the structures and functions of a cell, such as a protein, transfer RNA, or ribosomal RNA.

{0104] An "expression cassette" or "construct" refers to a series of polynucleotide element s that permit, transcription of a gene in a host cell. Typically, the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed. Expression cassettes or constructs may also include, e.g., transcription termination signals, poiyadenylation signals, and enhancer elements.

[0105] The term "codon bias" refers to the fact that differe t organisms use different codon frequencies.

[0106] The term "codon improvement" refers to the modification of at least some of the codons present in a heterologous gene sequence from a triplet code that is not generally used in the host organism to a triplet code that is more common in the particular host organism. This can result in a higher expression level of the gene of interest. The term "codon improvement" can also be used, s nonymously with codon optimization.

[0107] The term "reporter gene' ' refers to a nucleic acid encoding an identifying factor thai ca be identified based upon the reporter gene's effect, in order to determine or confirni that a cell or organism contains the nucleic acid of interest, and/or to measure gene expression induction or transcription.

[0108] The term "selectable marker" or "marker" means an identifying factor, usually an antibiotic, chemical resistance gene, or counter-selection gene, that is abl to be selected for based upon the marker gene's effect, such as resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and or to identify a cell or organism that has inherited the nucleic acid of interest,

[0109] A "heterologous protein" refers to a protein not naturally produced in the ceil.

[0110] An "isolated polypeptide" or "isolated protein" is a polypeptide or protein that is substantiatly free of those compounds that are normally associated tlierewitli in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).

[0111] An "isolated organism" is an organism that is substantially free of other organisms that are normally associated therewith in its natural state.

[0112] The term "homogenous" when used herein in the context of a "homogeneous population ' " is synonymous with "fully segregated" and means isolating a population of cells that contain essentially only one kind of a plasmid. In one embodiment, an isolated population of host cells is considered to be a homogenous population when it contains greater than 99%, greater than 99.5%, greater man 99.9% of a certain type of a kind of plasmid that is a member of a population of lasmids. For example, if greater than 99% of a population consisting of a wild-type p6.8 and a p6.8 derived plasmid containing a three-component cassette is the p6.8 derived plasmid containing a three-component cassette, then a host-cell containing essentially only the the p6.8 derived plasmid containing a three-component cassette is "homogenous" and/or "fully

segregated" with respect to population consisting of a wild-type p6.8 and a p6.8 derived plasmid containing a three-component cassette, hi an embodiment a homogenous population and a fully segregated population contains 100% of a certain type of a kind of plasmid. For example, 100% of a population initially consisting of a wild-type p6.8 and a p6.8 deri ved plasmid containing ' a three-component cassette is only the p6.8 derived plasmid containing a three-component cassette.

[0113] The term " ' tolerate" refers to the ability of an organism to continue to grow after exposure to a condition. In one embodiment, "tolerate" is defined as the ability of an organism to grow after being exposed to an environmental condition after being exposed to the condition for at least 2 hours per day over a time period of at least 7 days. In another embodiment, "tolerate" is synonymous with withstand. In an embodiment ability 7 of an organism to tolerate environmental conditions is refereed to as "hardiness".

[0114] A "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post- translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.

[0115] As used herein, the term "VLE" stands for vapor-liquid equilibrium. VLE is a method of determining ethanol concentration in a medium by measuring the ethanol concentration in a vapor over the medium. VLE relies upon the vapor pressure of ethanol in a medium and other variables such as temperature and exchange of other gasses in the vapor. In one embodiment, ethanol concentration of the vapor phase over the medium is measured by gas ehromotagraphy. In another embodiment, Raman spectroscopy, infrared spectroscopy and other spectrographic analyses may be performed in order to determine the concentration of a compound of interest in the vapor phase over a medium.

[0116] As used herein, the phrase "increased activity" refers to any genetic modification resulting in increased levels of enzyme function in a host cell. As known to one of ordinary skill in the art, m certain embodiments, enzyme activity may be increased by increasing the level of

transcription, either by modifying promoter function or by increasing gene copy number, increasing translational efficiency of an enzyme messenger RNA, e.g., by modifying ribosornal binding, or by increasing the stability- of an enzyme, which increases the half-life of the protein, leading to the presence of more enzyme molecules in the cell. All of these represent non-limiting examples of increasing the activity of an enzyme, see for example, mRNA Processing and Metabolism. " Methods and Protocols, Edited by Daniel R. Schoenberg, Humana Press Inc., Toto a, N.J.; 2004; ISBN 1-59259-750-5: Prokar otic Gene Expression (1999) Baumberg, S_,

Oxford University Press, ISBN 01 9636036; The Biomedical Engineering Handbook (2000) Bronzino, J. D., Springer, ISBN 354O66808X, all of which are incorporated by reference.

{0117] The terms "pyruvate decarboxylase", "Pdc" and "PDC" refer to an enzyme that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. A "pdc gene" refers to the gene encoding an enzyme that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide.

[0118] The terms "alcohol dehydrogenase", "Adh" and "ADH" refer to an enzyme that catalyzes the interconversion between alcohols and aldehydes or ketones. An "adh gene" refers to the gene encoding an enzyme that catalyzes the interconversion between alcohols and aldehydes or ketones.

[0119] The term "pdc/adh" refers to the pdc and adh genes collectively. A "pdc/adh cassette" refers to a nucleic acid sequence encoding a PDC enzyme and an ADH enzyme.

[0120] The term "ethanologenic cassette' " refers to any polynucleotide sequence that encodes for enzymes capable of producing ethanol alone or in combination with other exogenous or endogenou enzymes. In a certain embodiment, an ethanologenic cassette comprises genes encoding for an alcohol dehydrogenase and a pyruvate decarboxylase. In another embodiment, an ethanologenic cassette comprises genes encoding for a bifunctional alcohol/aldehyde

dehydrogenase. In certain embodiments, an ethanologenic cassette comprises genes encoding for enzymes that are part of a biochemical pathway to generate precursors for alcohol

dehydrogenases and pyruvate decarboxylases of an ethanologenic cassette.

[0121] The term "primer" is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesi under suitable conditions. Such primers may be used in a polymerase chai reaction.

[0122] The term "polymerase chain reaction," also termed "PGR," refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PGR involves a repetitive series of temperature cycles with each cycle comprising three stages; denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PGR

oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PGR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids. [0123] The term " 'three-component cassette" as used herein refers to at least three genes that are part of a polynucleotide sequence having a restriction target sequence on both a 5 " and 3' end of the three -component cassette. In an embodiment, the three-component cassette contains a positive selection marker, a counter-selection marker, and a site-specific recombinase. In another embodiment, the positive selection marker is an antibiotic resistance gene. As used herein, and in certain embodiments, a three-component cassette may acutalry contain more than three genes. For example, a three-component cassette may contain a positive selection marker, a site-specific recombinase, a first counter-selection marker, and a second counter selection marker. In an embodiment the first and second counter-selection makers are different from one another. In another embodiment the first and second counter-selection makers are the same as each other. The relative arrangement of a positive selection marker, a counter-selection marker, and a site- specific recombinase can be of any combination within the cassette whether the number be three genes or greater than three genes. In an embodiment, a positive selection marker, a counter- selection marker, and a site-specific recombinase that are part of the three -component cassette are each operably linked to their own promoter. In another embodiment of a three-component cassette, a positive selection marker is operably linked to a first promoter and a counter-selection marker, and a sit -specific recombinase are operably linked to a second promoter. In additional embodiments, all combinations of promoters operably linked to genes of the three-component cassette are possible embodiments.

[0124] Database entry numbers as used herein may be from the NCBI database (National Center for Biotechnology information; http://mvw.ncbi.iilm .nih.gov ) or from the CyanoBase, the genome database for cyanobacteria ((http://bacteria.kazusa.or.jp/ cyanobase/index.html);

Yazukazu et al. "CyanoBase, the genome database for Synechocystis sp. Strain PCC6803: status for the year 2000", Nucleic Acid Research, 2000, Vol. 18, page 72).

[0125] The enzyme commission numbers (EC numbers) cited throughout this patent application are numbers which are a numerical classification scheme for enzymes based on the chemical reactions which are catalyzed by the enzymes.

Growth of ABICyanol

[0126] hi an embodiment, methods disclosed herein are used for making an ABICyanol

markerless host cell useful for the production of a compound or compounds of interest. In comparison to other cyanobacterial species, ABICyano l grows quickly and can tolerate and grow over a large range- of various environmental stresses related to temperature, salinity, light intensity, oxygen levels, pH and the presenc e of contaminants including chemical and microbial contaminants. ABICyanoTs ability to tolerate wide-ranging environmental parameters makes it ideally suited to growth, in cyanobacterial culture systems. ABICyanol can be .genetically enlianced to express endogenous and exogenous genes used for the production of compounds of interest, such as biofuels, and still tolerates and grows over a large range of various

environmental stresses related to temperature, salinity, light intensity, oxygen levels, pH and the presence of various contaminants.

[0127] Methods for cultivation of cyanobacteria in liquid media and on agarose-eontaimng plates are well known to those skilled in the art (see, e.g., websites associated with ATCC). Any of thes methods or media maybe used to culture ABICyanol or derivatives thereof. A number of known recipes for cyanobacterial growth medium can be used, hi an embodiment, BGl 1 medium is used for growing ABICyanol, see Stanier, R.Y., et aL Bacterid. Rev. 1971 , 35: 71-205, which is hereby incorporated by reference.

10128] In an embodiment, the cyanobacterial strain is a fresh water strain, and BGl 1 is used. In another embodiment, the cyanobacteria culture grows best in a marine (salt water) medium, by adding an amount of salt to the BGl 1 medium. In an embodiment, marine BGl 1 (niBGl 1) contains about 35 practical salinity units (psu), see Unesco, The Practical Salinity 7 Scale 1978 and the International Equation of State of Seawater 1980. Tech. Pap. Mar. ScL 1981, 36: 25 which is hereby incorporated by reference.

ABICyanol endogenous plasmids

[0129] ABICyanol contains at least three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate .ABICyanol from other Cyanohacterium species.

[0130] One endogenous plasmid is 6828 base pairs (SEQ ID NO: 1). The 6828 bp endogenous p!asmid is alternatively referred to herein as pABICyanol, p6.8 or 6.8. A plasmid map of the

6828 endogenous plasmid is depicted in FIG. 1. The polynucleotide sequence and descriptors of various portions of the polynucleotide sequence of p6.8 is depicted in FIG. 2.

[0131] Another endogenous plasmid is 35386 base pan's (SEQ ID NO: 2).

[0132] ABICyanol endogenous plasmid p6.8 contains six open reading frames ORF I, ORF 2,

ORF 3, ORF 4, ORF 5, and ORF 6 encoding for polypeptides having sequences as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. respectively. With respect to the nucleotide -sequence: of SEQ ID NO: 1 ofp6.8, ORF 1 consists of nucleotides 594 to 3779, ORE 2 consists .of nucleotides 3815 to 4000, O F 3 consists of nucleotides 4260 to 5024, ORF 4 consists of nucleotides 5350 to 6036, ORF 5 consists of nucleotides 6078 to 6341, ORF 6 consists of nucleotides 6338 to 6586. and the origin of replication consists of nucleotides 3375 to 3408.

[0133] In an embodiment, a modified p6.8 as used herein includes none or any combination of the up to six open reading frames of the e dogenous p6.8.

[0134] As disclosed herein, piasmid 6.8 has been modified in vivo and in vitro for use as a extrachromosomal piasmid vector containing a three-component cassette and genes of interest for the production of compounds of interest.

[0135] In an embodiment, a modified endogenous vector derived from p6.8 from ABICyanolwas developed. The modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyano I itself.

[0136] In certain embodiments, the method provides the p6.8 piasmid and modified vectors comprising sequences of the p6.8 piasmid. hi an embodiment, the modified endogenous vector contains at least one of the following", a recombinant gene that encodes at least one protein involved in a biosynthetic pathway for the production of a compound or a marker protein; and an origin of replication suitable for replication in the host cell. In one embodiment, the gene coding for a replication initiation factor that binds to the origin of replication is present on the modified vector. In another embodiment, the gene codmg for a replication initiation factor that binds to the origin of replication is present in the chromosomes of the host cell. In yet another embodiment, the gene coding for a replication initiation factor that binds to the origin of replication is present in extrachromosomal p!asmids of the host cell, hi one embodiment, the extracliromosomal piasmid is endogenous to the host cell. In another embodiment, the extrachromosomal piasmid is exogenous to the host cell. In one embodiment the host cell is ABICyanol.

[0137] In certain embodiments, a gene codmg for a replication initiation factor that binds to the origin of replication can either be present on the modified vector or can be present in the

chromosomes or other extracliromosomal plasmids of ABICyanol . An origin of replication suitable for replication in ABICyanol and the gene coding for the replication initiation factor binding to that origin of replication ensure that the modified endogenous vector can be replicated in ABICyanol. [0138] In an embodiment the nucleotide sequence of an origin of replication of the modified endogenous plasmid vector can have at least 80%, 90%, and 95% identify -or can be identical to the nuckotides 3375 to 3408 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: I).

[0139] In an embodiment, the sequence of the gene coding for the replication initiation factor has at least 80%, 90%, and 95% identify or is identical to nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb pl smid (SEQ ID NO: I). In an embodiment, the gene coding for the replication initiation factor codes for a protein having at least 80%, 90%, and 95% sequence identify or is identical to the protein coded by nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 1) of ABICyanol. This putative initiation replication factor is thought to bind to the putative origin of replication, thereby ensuring the replication of a plasmid containing the initiation factor in ABICyanol.

[0140] In an embodiment, a modified endogenous plasmid vector can contain a sequence having at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identify to the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO. 1). In another embodiment, the modified endogenous vector contains the entire p6.8 endogenous plasmid from ABICyanol.

[0141] In another embodiment, gene delivery vehicles that are developed using the endogenous 6.8 kb plasmid (or a portion of the plasmid) containing characteristic portions of the endogenous 6.8 kb plasmid may be able to be efficiently transformed into a wide range of cyanobacteria. In an embodiment, characteristic portions of the 6.8 kb endogenous plasmid from ABICyanol include portions that enable it to replicate in a host cell (origin of replication and replication initiation factor, for example) and can be referred to as the backbone of the endogenous 6.S kb plasmid. Such vectors may also be able to efficiently produce heterologous proteins and other compounds of interest in cyanobactenal cultures.

[0142] In another embodiment, modifications starting with the backbone of the 6.8 kb endogenous plasmid from ABICyanol are performed individually or together to increase transformation efficiency, increase the replication rate within the cell, and to increase the production of a desired product from the cyanobacterial cell. Suitable modifications include, for example, insertion of three-component cassettes, selection markers (such as antibiotic resistance genes), recombinant genes or cassettes for the production of a desired compound, and other modifications to increase the expression or stability of the plasmid in the cyaiiohacteriai cell, hi an embodiment, the invention includes cyanobacteria, e.g. ABICyanoi, comprising a modified. p6.8 plasmid having any of these improved characteristics.

[0143] In yet another .embodiment, codon improvement of the at least one recombinant gene is performed for improved expression in the cyanobacterial. host cell. Codon improvement can also be performed by adapting the codon usage of the at least one recombinant gene to the codon usage in Cyanobacteri m sp., in particular ABICyanoi . In an embodiment, the G and/or C wobble bases in the codo s for the amino acids in the at least one recombinant gene can be replaced by A and/or T because the GC content of the genome of ABICyanoi is relatively low at about 36%.

[0144] In an embodiment, only 2% to 6% or 1% to 10% of the codons of variants of recombinant genes are codon improved. Li another embodiment, highly codon improved valiants of recombinant genes, at least 25%, to at least 50%, 65% or even at least 70% of the codons have been changed. In another embodiment, recombinant genes are used which are not codon improved.

Transformation of ABICyanoi

{0145] Methods for producing a genetically enhanced, non-naturally occuring Cyanobacterium sp. and ABICyanoi host cells are disclosed herein. In an embodiment, methods include introducing a recombinant nucleic acid sequence into a cyanobacterial host cell. At least one recombinant gene can be introduced into the host cells through the transformatio of the host cell by an extrachromosomai plasmid. In an embodiment, the extrachromosomai plasmid can independently replicate in the host cell. In another embodiment, at least one recombinant gene can be introduced into the genome of the host cell. In yet another embodiment, at least one recombinant gene is introduced mto the genome of the host cell by homologous recombination.

[0146] In an embodiment, a recombinant nucleic acid sequence can be provided as part of an extrachromosomai plasmid containing cyanobacterial nucleic acid sequences in order to increase the likelihood of success for the transformation.

{0147] In another embodiment, the method for producing a genetically enhanced

Cyanobacterium sp. host cell uses an extrachromosomai plasmid derived from an endogenous plasmid of the host cell to introduce a recombinant nucleic acid sequence into the host cell. This endogenous plasmid can be, for example, an extrachromosomai plasmid derived from the 6.8 kb endogenous plasmid of ABICyanoi . [0148] In another embodiment, a method for producing a genetically enhanced microbial host cell uses an extrachromosomal plasmid derived from an endogenous plasmid of the host cell. I an embodiment, the extrachromosomal plasmid contains a three-component cassette. In another embodiment the extrachromosomal plasmid is also capable of self-replication. In yet another embodiment, the extrachromosomal plasmid contains a toxin-antitoxin cassette.

[0149] In an embodiment, the ABICyanol 6.8 kb endogenous plasmid is used as a backbone for a plasmid vector used for transformation of Cyanohactetium sp. Since this is the endogenous vector from the species, it is likely to be more stable when transformed into the cell than plasmids derived from completely different organisms. In an embodiment, the entire p6.8 endogenous plasmid is inserted into a vector used for transformation. In another embodiment, a sequence of about 50%, 70%, 75%, 80% 85%, 90%, 95%, 98%, 99%, or 99.5% identity to the entire endogenous plasmid sequence is inserted into the extrachromosomal plasmid vector.

[0150] In another embodiment, the p6.8 derived plasmid vector als contains an origin of transfer (oriT) which is suitable for conjugation. In particular, the plasmid vector can contain a combined origin of replication and an origin of transfer (oriVT), which enables replication in

Enterohacteriaceae, in particular E. coli, and which also enables conjugation with, for example, an E. coli donor strain and C anobactemtm sp., in particular ABICyanol as a recipient strain. Such a plasmid vector can be used for triparental mating wherein a conjugatfve plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid, for example a plasmid vector disclosed herein, present in a second bacterial strain into a third recipient bacterial strain, which can be ABICyanol .

[0151] In an embodiment for transforming host cells with p6.8 derived vectors, a shuttle vector expresses a codon-optimized antibiotic resistance gene (ABR), such as codon adapted kanamycin or gentamycin resistance genes. In an embodiment, the shuttle vector is constructed based on a modular basi so that all of the key elements (replication ori, ABR gene and reporter gene) are exchangeable via unique restriction sites thus providing versatile cloning options and facilitating the delivery of genes of interest to target organisms. Other antibiotic resistance genes can be used if desired. For example, gene conferring resistance to ampicillin, chloramphenicol,

spectinomycm or other antibiotics can be inserted into the vector, under the control of a suitable promoter. In some embodiments, the vector contains more than one antibiotic resistance gene.

[0152] In yet another embodiment, the p6.8 derived vector is modified by several factors so that it is capable of efficient replication in multiple types of cyanobacterial species. In an embodiment, recombinant, genes are present on an extrachromosomal plasmid containing a three- component cassette having multiple copies per cell. The plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per cyanofeacteriai host cell. In an embodiment, the recombinant plasmids are fully segregated from the non-recombinant plasmids in tlie presence of a negative selection compound such as an antibiotic. In an

embodiment the three-component cassette containing plasmids are fully segregated from the excised plasmids winch do not contain a three-component cassette by the dual expression of the site-specific recombinase and the counter-selection marker in a medium contaimng a counter- selection compound.

[0153] Exemplar}' methods suitable for transformation of cyaiiobactena include, as non-limiting examples, natural DNA uptake (Chung, et al. (1998) FEMS Microbiol. Lett. 164: 353-361;

Frigaard, et al. (2004) Methods Mol Biol. 274: 325-40; Za g, et al. (2007) J. Microbiol 45: 241- 245), conjugation, transduction, glass bead transformation (Kindle, et al. (1989) J. Cell Biol. 109: 2589-601; Feng, et al. (2009) Mol. Biol. Rep. 36: 1433-9: U.S. Pat. No. 5,661,017), silicon carbide whisker transformation (Dunahay, et al. (1997) Methods Mol. Biol. (1997) 62: 503-9), biolistics (Dawson, et al. (1997) Curr. Microbiol. 35: 356-62; Hallmann, et al. (1997) Proc. Natl. Acad USA 94: 7469-7474; Jakobiak, et al. (2004) Protist 155:381-93; Tan. et al. (2005) J.

Microbiol. 43: 361-365; Steinbretmer, et al. (2006) Appl Environ. Microbiol. 72: 7477-7484

Kroth (2007) Methods Mol. Biol. 390: 257-267: U.S. Pat. No. 5,661,017) electroporation

(Kjaeralff, et al. ( 1994) Photosynth. Res. 41 : 277-283: Iwai, et al. (2004) Plant Cell Physiol. 45: 171-5; Ravindran, et al. (2006) J. Microbiol Metliods 66: 174-6: Sun, et al. (2006) Gene 377: 140-149; Wang, et al. (2007) Appl. Microbiol Biotechnol. 76: 651-657; Chaurasia, et al. (2008) J. Microbiol. Methods 73: 133-141; Ludwig, et al. (2008) Appl Microbiol. Biotechnol. 78: 729- 35), laser-mediated transformation, or incubation with DNA in the presence of or after

pretreaiment with any of poly(amidoamine) dendrimers (Pasupaihy, et al. (2008) Biotechnol. J. 3 : 1078-82), polyethylene glycol (Olinuma, et al. (2008) Plant Ceil Physiol. 49: 1 17-120), canonic lipids (Muradawa, et al (2008) J. Biosci. Bioeng. 105: 77-80), dextran, calcium phosphate, or calcium chloride (Mendez-Alvarez, et al (1994) J. Bacterid. 176: 7395-7397), optionally after treatment of the cells with cell wall-degrading enzymes (Perrone, et al (1998) Mol Biol Ceil 9: 33 1-3365). Biolistic methods (see, for example, Raniesk et al. (2004) Methods Mol. Biol 274: 355-307: Doestcii, et al. (2001) Curr. Genet. 39: 49-60; all of which are incorporated herein by reference in their entireties. Transformation of ABICyanol by conjugation

[0154] la an embodiment transformation of ABICyanol with exogenous polynucleotides is performed after treatment of the exopolysaccharide layer of ABICyanol by the conjugation technique as described in US8846369 to Piven et al.

Selecting for successful transformation of ABICyanol

[0155] In an embodiment, the presence of a foreign gene encoding antibiotic resistance in a ceil is selected by placing putatively transformed cells into a media containing an amount of the corresponding antibiotic and selecting cells that sumve. The selected cells are then gro in the appropriate culture medium to allow for further testing. Using plasmids and methods disclosed herein, after the host cell has been modified and fully segregated, the antibiotic resistance gene, along with other members of the three-component cassette bordered by FRT sites, for example, can be excised from the plasmid to create a markerless host ceil.

Transformation of other cyanobacteria with p6.8 derived lasmids

[0156] In another embodiment, the modified plasmid vector based on the endogenous 6.8 kb plasmid backbone from ABICyanol, in addition to being useful for transformation to other cyanobacteria! and Cyanobacteriwn sp. host cells, is used to transform oilier microbes including Eubacteria, Archaea and Eukaryotes. As an example, a shuttle vector containing the 6.8 kb endogenous plasmid from ABICyano I with a kaiiamycin resistance cassette (Km R ) and the oriVT for replication in E. coli is transformed into Synechococcus PCC 7002 by natural uptake.

[0157] In another embodiment, a modified extrac hromo soma 1 plasmid, based on the endogenous 6.8 kb plasmid from ABICyanol, containing a three-component cassette and genes whose expression products produce a compound or compounds of interest is transformed into other genera of cyanobacteria. Examples of cyanobacteria that can be transformed with

extraehromosomai plasmids containing three-component cassettes disclosed herein include, but are not limited to, Synechocystis, Synechococcus, Acaryochlons, Anabaena,

T ermosyn chococcus, Chamaesiphon, Chroococcus, Cyambium, Dactylococcopsis,

Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcm, Prochloron,

Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus'.. Arthrospira, Borzia, Crma!iwn, Geitlerine , Ha spirulina, Leptotyngbya, Ltnmoihrix, Lynghya, Microcohus, Cyanod tyon, Aphanocapsa, Osci!iatoria, Planktot rix, PmcMorothrix, Pseud n b sna, Spir lim, Starria, Symploca, Trichodesmium, Tyckonema, Anabaenopsis, Aphanizommon, Calothrix, Cyanospira,. Cylindrospermop s, Cylindrospermutn, Nodularia, Nosioc, Chlorogb opsis, FischereUa, Geiilerm, Nostochopsis, Iyeng rieUa,

Siigonema, Rivularia, Scytonema, Tofypothrix, Cyimorhece, Phormidium, Adrianema, and the

Promoters

[0158] In an embodiment, any desired promoter can be used to regulate the expression of the genes for the production of a desired compound in ABICyanol, a counter-selection marker, an antibiotic resistance marker and a site-specific recombinase and may include both endogenous as well as exogenous promoters. Exemplary promoter types include but are not limited to, constitutive promoters, and inducible promoters induced by, for example, nutrient starvation, heat shock, mechanical stress, enwonmental stress, metal concentration, and light exposure.

Additional promoters, both constitutive and inducible, are well-known in the art.

[0159] In an embodiment recombinant genes are placed under the transcriptional co trol (operably linked) of one or more promoters selected from exongenous or endogenous u i.s,

PnlcA, PHMA, PisiA, PpetJ. PpetE, Pa»T, PwtA, PziaA, PsigB, PiriA, PiitpG, PhspA, PcipBI, PhKB, PggpS, PpsbA2,

PpseA, PjiifA, PnarB, *mA, Pisis, PnrsB, PwA, PBB¾A, Ppsts, and Pcriic. In an embodiment, synthetic promoters are used,

[0160] Recombinant genes disclosed herein may be regulated by one promoter, or they can each be regulated by individual promoters. The promoters can be constitutive or inducible. The promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.

[0161] Exemplar " } ' promoters for expression in cyanobacteria include, but are not limited to, Pp«j,

PpsbD- PablA, Pip A, PisiB, PrlxLS. PateA, PsiblA, PisiA, Ppeti, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA- PhipG, PhspA, PclpBl, PiiiiB, PggpS, PpsbA2, PpsaA, PriirA, PnarB, PnriA, PcriiC, and additional metal ion inducible promoters and the like. Examples of constitutive promoters that can be used include, but are not limited to, PA L, PmpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, PspcB. Additional details of these promoters can be found, for example, in PCT/EP2009 060526, which is herein incorporated by reference in its entirety. [0162] In an embodiment, truncated or partially truncated versions of promoters disclosed herein can be used including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start. Furthermore, : introducing nucleotide changes into the promoter sequence, e.g. into the TATA box, the operator sequence and/or the ribosomal bindi g site (RBS) can be used to tailor or improve the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction. For example, the inducible promoter can be ΡΜΙΑ, and can be PnkA from ABICyanol, which is repressed by ammonium and mduced by nitrite. Tins promoter may contain nucleotide changes in either one of the ribosomal binding site, the TATA box, the operator, and the 5'-UTR

(untranslated region).

[0163] In certain embodiments, the present invention includes a polynucleotide composing or consisting of any of the promoter sequences described herein, or variants thereof, including those having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity io the reference promoter sequence.

[0164] In an embodiment, disclosed herein are recombinant genes of a shuttle vector that comprise or are operably linked to an inducible promoter and/or a constitutive promoter. The promoter can be upstream of one gene to regulate that gene, or the promoter can be upstream of several genes so that one promoter regulates the expression of more than one gene. Alternatively, in some embodiments, each recombinant gene can be regulated by a separate promoter. In an embodiment, the promoter can be derived from a cyanobacterial host cell, can be derived from another cyanobacterial species, or can be derived from another organism.

[0165] In an embodiment, a promoter disclosed herein can be an inducible promoter selected from the grou consisting of PwrA, I . and PMB from ABICyanol, for example. In another embodiment, a promoter is a constitutive promoter selected from the group consisting of PI PS L, Pfbc, Pc cB and PpetE which can all be endogenous promoters of ABICyanol, for example.

[0166] In an embodiment, more than one recombinant gene is used in a recombinant vector. In one embodiment, a first and second recombinant gene can be controlled by one promoter, thereby forming a transcriptional operon. hi another embodiment, the first and second recombinant genes are controlled by different first and second promoters.

[0167] In an embodiment, the recombinant gene under control of the promoter is mduced if a sufficiently high culture density of Cyanobacterhim sp. is reached. In the case that the second recombinant gene codes for a protein catalyzing a chemical reaction present in the wild-type Cyanobacterium sp., such as alcohol dehydrogenase, the gene can he under the control of either an inducible or a constitutive promoter because it does not disturb the carbon flux to the same extent as the non-native protein encoded by the first recombinant gene. The second recombinant gene the may be under the control of constitutive promoters such as PrbcL. PpetE, or PipsL, all from ABICyanol, for example.

[0168] In an embodiment, a transcription terminator is present between the first and second recombi ant gene in order to ensure a separate transcriptional control of the first and second recombinant gene and to provide for a high production of a compound of interest, such as ethanol. In certain embodiments, the present invention includes ethanoiogenie cassettes. In an embodiment for an ethanoiogenie cassette used to produce ethanol as a compound of interest, a first recombinant gene encode pyruvate decarboxylase and the second recombinant gene encodes alcohol dehydrogenase. The first recombinant gene (pdc) is under the transcriptional control of a first inducible promoter and the second recombinant gene (adh) is under the transcriptional control of a second constitutive promoter. The first inducible promoter can be selected from, for example, PmrA, PUBA variants Ρη»Α*2, Patera, PITOAH, Pmatc, PS_*A (Pods i¾s), Ρ∞Ω2ΐ, Per»222, Poifo223, P«f03i6, Porf3232, and PorB46i and the second constitutive promoter can be selected from, for example. PrpsL, Prp_i*4, and P cpcB .

[0169] In an embodiment, a non-naturally occunng ABICyanol host cell comprising any of the ethanoiogenie cassettes described herein produces ethanol in quantities of at least 0.016% (v/v) per day in 12h/12h day/night cycles and a photon flux density of 230μΕ m ' 1 during the daylight phase. In certain other embodiments, the transcription of both the first and second recombinant gene encoding the pyruvate decarboxylase enzyme and the recombinant gene encoding the alcohol dehydrogenase enzymes are controlled by the same single promoter. For these embodiments, an niducible promoter may be used.

[0170] In an embodiment, a transcription terminator is present between the first and second recombinant, gene in order to ensure a separate transcriptional control of the first and second recombinant gene and to provide for a high production of a compound of interest, such as ethanol. In an embodiment for an ethanoiogenie cassette used to produce ethanol as a compound of interest, a first recombinant gene encodes pyruvate decarboxylase and the second recombinant gene encodes alcohol dehydrogenase, hi particular embodiments, the first recombinant gene (pdc) is under the ti anscriptional control of a first inducible promoter and the second recombinant gene (adh) is under the transcriptional control of a second constitutive promoter. [0171] Promoter elements disclosed herein may be operably linked with any genes encoding any enzymes useful for the production of compounds of interest by using standard molecular cloning techniques. In further non-limiting examples, the one or more genes coding for a compound of interest are pdc and/or adh. In one embodiment, the pdc gene is operably linked to PMTA or PoirA* ?. In another embodiment, the adh gene is operably linked to Pdx or P. P ;B.

[0172] In an embodiment, multiple combinations of inducible and constitutive promoters of varying strengths are operably linked to all genes necessary for the production of a compound of interest as well as the antibiotic resistance gene, the site-specific recombinase and the counter- selection marker that make up the self-excision cassette within an extrachromosomal plasmid.

Endogenous promoters from ABICyanol

[0173] In an embodiment, promoters used herein can be endogenous to ABICyanol. In another embodiment endogenous promoters from ABICyanol can be modified in order to increase or decrease efficiency and/or promoter strength. In an embodiment, and as described in

DS20140178958, which is hereby incorporated by reference, endogenous promoters used to control the expression of genes on vectors disclosed herein include, but are not limited to promoters for cpcB, nirA, IrtA, mrgA, nblA, ggpS, petJ, ppsA f rnpA mdpstS from ABICyanol.

[0174] In an embodiment, metal-indueib!e promoters may be operably linked to any of the genes necessary for the production of a compound of interest as well as the antibiotic resistance gene, the site-specific recombinase and the counter-selection marker that make up the self-excising three-component cassette within an extrachromosomal plasmid. Metal inducible promoters from ABICyanol include, but are not limited to those previously disclosed in US20140178958, such as promoters for orf0128, orfl486, orf3164, orf3293, orf3621, orf3635, orf3S58, orfl071, orfl072, orfl074, orfl075, orfl542, orfl823, orfl824, or£3126, orf33S9, orf0221 orf0222, orf0223, orf0316, orf3232, orf346L and orf3749.

Codon improvement of recombinant genes

[0175] At least some of the nucleic acid sequences to be expressed in cyanobacterial host cells can be codon improved tor optimal expression in the target cyanobacterial strain. Tire underlying rationale is that the codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Buhner, Nature 325:728-730; 1987). Codon improvement (sometimes referred to as codon optimization or codon adaptation) can be performed to increase the expression level of foreign genes such as antibiotic resistance genes, ethanologenic (or other compounds of interest) cassettes, and any other -expressed genes on a plasrnid, for example.

[0176] In an embodiment, the nucleic acid sequences ' of the recombinant genes are modified so that they will have improved expression in cyanohacteria. For example, the selectable marker gene that confers gentamycin or kanamycin resistance was codon optimized for higher expression in cyaiiobacteria. Additionally, the selectable marker gene that coniers kanamycin resistance was codon optimized for higher expression in cyanobacteria. In an embodiment, as a result of codon improvement the GC % of the antibiotic resistance genes decreased from 40-53% to 33-40%, winch is similar to that of ABICyanol coding genes (about 36% on average). The codon adaptation mdex of the codon improved antibiotic resistance genes is significantly improved from less than 0.4 io greater than 0.8, which is similar to that of ABICyanol endogenous genes.

[01771 Table 1 depicts the codon usage statistics within ABICyanol.

Table 1

Transformed Host Cells

[0178] In an embodiment, genetically enhanced C anobacteri sp. host cells that lack antibiotic resistance markers, in particular ABICyanol host cells, include at least one recombinant gene encoding at least one protein that is involved in a biosyntherie pathway for the production of a compound or marker protein. In certain embodiments, they comprise an ethanologenic cassette. In certain embodiments, the genetically enhanced Cyanohacterium host cells can be used for the production of various compounds of interest by culturing the host cells under harsh conditions of high temperature, high oxygen levels and in the case of the compound being ethanol. under high levels of ethanol in the medium. In an embodiment, a marker protein, or reporter protein, can be a fluorescent protein, such as a red or green fluorescent protein. In an embodiment, a marker protein, or reporter protein, can be a marker gene conferring resistance to a biocide such as an antibiotic which can be used to select for and maintain cultures of Cy nobacterium sp. host cells in the presence of other bacterial contaminating strains.

[0179] In another embodiment, a recombinant gene is present on an extrachromosornal plasmid that can replicate independently from the chromosomes of the Cyanobacterium sp. host cells such as ABICyanol. In an embodiment, the extrachromosornal plasmids described herein are present in high copy numbers in the host cells so that a compound of interest can be produced in a high yield.

[0180] Genetically enhanced Cyanobacterium sp., for example ABICyanol host cells, can include further genetic enhancements such as partial deletions of endogenous genes of

Cyanobactenum sp. or other recombinant genes which can increase the overall yield of the compound being produced by the host cells. For example, if the compound to be produced is ethanol, the genetic enhancements can relate to the knock out of endogenous genes coding for enzymes converting pyruvate or aeetyl-CoA into a reserve or storage compound. In another embodiment, if the compound to be produced is ethanol, the genetic enhancements can relate to the overexpression of enzymes of the glycolysis pathway, Calvin-cycle, amino acid metabolism, the fermentation pathway, the citric acid cycle, and other intermediate steps of metabolism in order to increase the production of ethanol by the Cyanobacterium sp. host cells. Examples of such genetic enhancements are described in PCT patent publication number WO 2009/098089, which is hereby incorporated by reference for this purpose.

[0181] In another embodiment, genetic enhancements of the genes encoding enzymes of the carbon fixation and subsequent carbohydrate metabolism (for example, pathways which compete with an ethanol production pathway) can be genetically enhanced to further increase the production of a compound of interest. Genetic enhancement targets include, but are not limited to, components of the photosystems (antennas and pigment modification), and components of the photosynthetic and respiraiory electron transport systems as well as of the Calvin cycle. Genetic eniiaiicement targets include local and global regulatory factors including, but not limited to, the two component system, sigma factors, small regulating RMAs and antisense As.

{0182] In an embodiment, Cyanobacierium sp. host cells, e.g., ABICyanol host cells, contain knockout mutations of endogenous genes that do not affect the toleration of being cultured in at least one of the following conditions: 1% (v/v) ethanol in the medium for at least 6, 12 or 16 weeks; 48 °C, 50 °C, 53 to 55 °C for at least 2 hours per day over a time period of at least 7 days, purging with 60% to 80% (v/v) oxygen (resulting in oxygen concentrations of up to 1000 μιηοΙ/L in the culture during the day).

Compounds of interest produced by transformed host cells

[0183] hi certain embodiments, a variety of different compounds of interest can be produced using genetically enhanced host cells disclosed herein. Genes involved in the biosynthetic pathway for the production of a compound of interest can be inserted into the vector. Plasmid vectors disclosed herein (e.g., derivatives of 6.8) can be used to carry a gene or genes encoding a polypeptide involved in various biosynthetic pathways that produce a compound of interest in the host cell. The gene or genes encoding the polypeptide involved in biosynthetic pathway producing one or more compounds of interest are loc ated on the extrachromosomal plasmid outside of the cassette comprising the site-specific recombinase, the positive selectable marker, and or the counter selectable marker, hi one embodiment, the gene or genes involved in a biosynthetic pathway that produces a compound of interest are located in a separate cassette from the three -part cassette comprising the site-specific recombinase. the positive selectable marker, and/or the counter selectable marker. In another embodiment, the genes involved in the

biosynthetic pathway for the production of a compound of interest are not excised by the site- specific recombinase encoded by the extrachromosomal plasmid.

{0184] Exemplary compounds of interest include, but are not limited to, organic carbon

compounds, alcohols, fatty acids, oils, carotenoids, proteins, enzymes, biofuels, nutraceuticals, pharmaceuticals, and the like, in one embodiment, the plasmid carries one or more genes that encode for polypeptides that catalyze the production of one or more compounds of interest.

Additional information on compounds that can be produced from host ceils such a cyanobacteria can be found, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526, both of which are incorporated by reference herein in their entirety. [0185] In one non-limiting embodiment, propanol, 1,2-propanediol, 1,3-propanediol, butanol and their isomers are compounds of interest. In . certain embodiments, genes encoding enzymes involved in isopropanol and isohiitaiiol fermentation are incorporated into recombinant vectors and transformed into the host cell. In a further embodiment, the host cell is ABICyanol.

Examples of enzymes involved in isopropanol fermentation include aeetyl-CoA acetyltransferase (EC 2.3.1.9), aeetyl-CoA ' .acetoacetyl-CoA transferase (EC 2.8.3.8), acetoacetate decarboxylase (EC 4.1.1.4) and isopropanol dehydrogenase (EC 1.1.1.80). Examples of enzymes involved in isobutanol fermentation include acetolactate synthase (EC 2.2.1.6), acetolactate reductoisomerase (EC 1.1.1.86), 2 -dihydroxy-3-methylbutanoate dehydratase (EC 4.2.1.9), a-ketoisovalerate decarboxylase (EC 4.1.1.74), and alcohol dehydrogenase (EC 1.1.1.1).

[0186] In another embodiment, ethylene is produced as a compound of interest. In an

embodiment, at least one recombinant gene encodes an enzyme for ethylen formation. Exampies of enzymes involved in the production of ethylene include ethylene forming enzyme 1- aminocyciopropane- 1-carboxyiate oxidase (EC 1..14.17.4), which catalyzes the last step of ethylene formation, the oxidation of I-annnocyclopropane-i-carboxylic acid to ethylene. The substrate for the ethylene forming enzyme is synthesized by the enzyme 1-aminocyclopropane-l- carboxylic acid synthase (EC 4.4.1.14) from the amino acid methionine.

[0187] In another embodiment, the compound of interest is isoprene. In an embodiment the recombinant vector used to transform a cyanobacterial host cell for the production of isoprene includes at least one recombinant gene encoding an enzyme such as isoprene synthase. Isoprene synthase (EC 4.2.3.27) catalyzes the chemical reaction from dimethylallyl diphosphate to isoprene and pyrophosphate.

[0188] In another embodiment, compounds of interest are terpenes and terpenoids. Terpenes are a large and very diverse class of organic compounds, produced primarily by a w de variety of plants, particularly conifers. Terpenes are derived biosyntheticaliy from units of isoprene and are major biosynthetic building blocks in nearly every living organism. For example, steroids are derivatives of the triterpene squalene. When terpenes are modified chemically, such as by oxidation or rearrangement of the carbon skeleton, the resulting compounds are generally referred to as terpenoids. Terpenes and terpenoids are the primary constituents of the essential oils for many types of plants and flowers. Examples of biosynthetic enzymes are farnesyl diphosphate synthase (EC 2.5.1.1), which catalyzes the reaction of dimethylallyl diphosphate and isopentenyl diphosphate yielding farnesyl diphosphate. Another example is geranylgeranyl diphosphate synthase (EC 2.5.1.29), which catalyzes the reaction between transfamesyl diphosphate and isopenten l diphosphate yielding pyrophosphate and geranylgeranyJ diphosphate.

[0189] In an embodiment the compound of interest is hydrogen, and the recombinant genes can,, for example,, encode for hydrogenase. In an embodiment, hydrogenase is an enzyme catalyzing the following reaction: 12H ÷ ÷ I2Xi«di Ke d -» 6 H. + 12Χοχ¾&«3, where X is an electron carrier such as fenedoxiii.

[0190] In an embodiment, examples of compounds of interest include non-ribosomal peptides (NRP) and the polyketides (PK). In another embodiment, alkaloids are compounds of interest.

[0191] In yet another embodinient, vitamins are compounds of interest. Vitamins are organic compounds that are essential nutrients for certain organisms and act mainly as cofactors in enzymatic reactions but can also have further importance, e.g. as antioxidants. In plants, vitamin C can be made via the L-ascorbic acid (L-AA) biosynthetic pathway starting from D-glucose. In an embodiment, recombinant genes encoding for enzymes involved in vitamin C synthesis are disclosed and include hexokinase, ghicose-6-phosphate isomerase, mannose-6-phosphate isomerase, phosphomairnomutase mannose- 1 -phosphate guanylyltransf erase, GDP-mannose-3 ,5- epimerase, GDP-L-galactose phosphoryiase, L- galactose 1 -phosphate phosphatase, L -galactose dehydrogenase, and L-gaIactono-l,4-lactone dehydrogenase.

[0192] In another embodiment amino acids are compounds of interest. Ammo acids as compounds of interest include naturally occurring amino acids as well as amino acid derivatives.

[0193] In an embodiment, lactams are compounds of interest. In another embodiment, ethers are compounds of interest.

[0194] In yet another embodiment, alkanes (also known as saturated hydrocarbons) are compounds of interest. In an embodiment, these genes may be part of the reconibmant vector and include genes encoding for acyl-ACP reductase (EC 1.3.1.9) which converts a fatty acyl-ACP into a fatty aldehyde that may subsequently be converted into an alkane/alkene by an aldehyde decarbonylase (EC 4.1.99.5).

[0195] In an embodiment, biopolymers such as polyhydroxyalkanoates (PHAs) are compounds of interest. The simplest and most commonly occurring form ofPHA is the fermentative production of poly-3-hydroxybutyrate (P3HB) but many other polymers of this class are

produced by a variety? of organisms. PHAs include poly-4-hydroxybutyrate (P4HB),

polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctaiioate (PHO) and their copolymers. In an embodiment, recombinant genes encoding for enzymes involved in P3HB synthesis are part of recombinant vectors. These genes include genes encoding β-ketothiolase (EC 2.3.1.9) that produces acetoacetyl-CoA which is converted to (R)-3-hydro¾¾utyiyl-CoA (3-HBCoA) by NADPH-dependent acetoacetyl-CoA reductase (EC l.J .l .36). The 3HBCoA is subsequently polymerized by poly(3-hydroxyarkanoate) synthase (EC 2.3.1) and is converted to P3HB.

[0196] In an embodiment, esters, including fatty acid esters, are a compoimd of interest. Simple esters with lower chain alcohols (methyl-, ethyl-, n-propyl-, isopropyl- and butyl esters) are used as emollients in cosmetics and other personal care products and as lubricants.

[0197] In an embodiment, Cyanobacterium sp. host cells naturally contain the entire sequences of recombinant genes coding for enzymes used for the production of a compound of interest. In another embodiment, the Cyanobacterium sp. host cell contains the entire sequences of recombinant genes that encode for all of the enzymes used in a cascade of enzymatically catalyzed reactions that results in the production of a compound of interest.

[0198] In an embodiment, a first protein encoded by a first recombinant gene can produce a first intermediate which is further converted by a second protein encoded by a second recombinant gene into a second intermediate, which then in turn is further converted by a third protein encoded by a third recombinant gene into a third intermediate such that a sequence of reactions provide intermediates for the next enzyme leading to the eventual production of a compound of interest. In an embodiment, the recombinant genes encoding for the enzymes that catalyze the sequence of reactions can be introduced into ABICyanol or other Cyanobacterium sp. host cells.

[0199] In an embodiment, the compounds of interest that are produced from recombinant host cells can be removed intermittently and/or continuously as the culture grows, or the compounds can be separated at the end of a batch growth period. The cultures can be grown indoors, or can be grown outdoors in enclosed containers such as bioreactors, or in another suitable type of container. In an embodiment, the 6.8 kb endogenous plasmid vector from ABICyanol is genetically enhanced to include recombinant genes encoding for enzymes that produce a compound of interest.

Production of ethanol in host cells

[0200] hi an embodmieni, a compound of interest is ethaiiol, and the genetic enhancements to the host ceil include transforming the cell with a p6.8 based vector that comprises one or more recombinant genes encoding for an enzyme used in ethaiiol production. In an embodiment the genes are adh and/or pdc. The gene pdc encodes for pyruvate decarboxylase (PDC), which catalyzes the mterconversion ' between pyruvate and acetaldehyde. The gene adh encodes tor alcohol dehydrogenase (ADH) which catalyzes the interconversion between acetaldehyde and. ethanol. Thus, PDC and ADH act in concert to produce ethanoL In another embodiment the gene is adhE which encodes for AdliE enzyme (alcohol dehydrogenase E) which catalyzes the interconversion between acetyl-coenzyme A and ethanoL Any pdc, adh, and or adhE genes, or derivatives thereof, known in the art may be used. In one embodiment, the host cells are transformed with PDC and or ADH111. In another embodiment, the host cells are transformed with PDC and/or ADH916. In another embodiment, the host cells are transformed with codon optimized variants of pdc, adh, and/or adhE genes. In another embodiment, the host cells are C novact titim sp. In another embodiment, the host cells are ABICyanol.

[0201] Ethanol produced by non-naturally occurring host cells can be measured by any means well known in the art. hi an embodiment, ethanol produced by ethanologenic non-naturally occurring ABICyanol organisms is measured using gas chromatographic analysis of a growth media and/or the headspace above a grow media. In another embodiment, the host ceils are a member of the genus Cyanobacterium sp. In another embodiment, the host ceils are ABICyanol. {0202] In an embodiment, PDC activity is measured by a photometric kinetic reaction that can be monitored at 340 nm using a spectrophotometer. Pyruvate is enzymatically converted to acetaldehyde by pyruvate decarboxylase, which is reduced to ethanol by ethanol dehydrogenase under NADH oxidation. In an embodiment, the PDC enzyme activity is related to the protein content and expressed as the specific activity of PDC.

[0203] In particular embodiments, the ADH enzyme is, for example, a Zn _r -d pendent alcohol dehydrogenase such as an alcohol dehydrogenase from Lynghy (ADH111), an alcohol dehydrogenase from Synechococcus (ADH916), Adhl from Zymomonas mobilis (ZmAdh) or the Adh enzyme from Syneehaeystis PCC 6803 (SynAdh encoded by the synadli gene). Alternatively or in addition, the enzyme is an iron-dependent alcohol dehydrogenase (e.g. Adhll from

Z.mobilis). The Zn 2+ -dependent alcohol dehydrogenase can, for example, be an alcohol dehydrogenase enzyme having at least 60%, 70%, 80%, 90% or even more than 90% sequence identity to the amino acid sequence of Zn i+ dependent alcohol dehydrogenase from Synechocystis PCC 6803. Relative to other alcohol dehydrogenases, SynAdh (annotated open reading frame sir 1192 from the Synechocystis PCC 6803 genome) favors higher overall ethanol production because the reduction of acetaldehyde to ethanol is preferred to the reaction from ethanol to acetaldehy e. Thus, in an embodiment, a SynAdh encoding recombinant gene is useful for production of ethanol in a host cell.

{0204] AdliE is an iron-dependent, birunctional enzyme that mterconverts acetyl coenzyme. A to. ethanol. One characteristic of iron-dependent alcohol dehydrogenases (e.g. AdhE and Adhll) is their sensitivity to oxygen. In an embodiment. AdhE used to transform ABICyanol is derived from thermophilic organisms such as Thermosynechococcus elongates BP-1. in another

embodiment, AdhE is from E. coli. In the case of AdhE from E. coli, a mutant was described that exhibited alcohol dehydrogenase activity under aerobic conditions, see Holland- Staley et aL J Bacterid. 2000 Nov; 182 (21):6049-54. The E568K AdhE mutant of the E. coli AdhE was active both aerobically and anae obically. Thus, in an embodiment, site-directed mutants of various AdhE enzymes could impart catalytic function to AdhE enzymes under both aerobic and anaerobic conditions in genetically enhanced ABICyanol host cells.

[0205] In an embodiment, pyruvate decarboxylase can, for example, be from Z momonas mobffis, Zymobacter paJmae or S echaromyces cer visiae. in an embodiment, nucleic acid sequences, protein sequences and properties of ethanologemc enzymes such as alcohol dehydrogenases and pyruvate decarboxylases disclosed herein, can be found within

WO2009098089 as well as US20140178958.

Cassettes

[0206] In most engineered microbial host cells, genes encoding selectable markers are paired with inserted genes of interest in order to miprove the genetic stability of the inserted gene when a give compound is present in the medium. In one embodiment, the selectable marker is an antibiotic resistance gene that allows the transformed cell to grow in the presence of the antibiotic, Any appropriate positive selectable marker can be used, including but not limited to, beta-lactamase which confers ampicillin resistance, the mo gene which confers resistance to kanamycin in bacteria and geneticin in eukaryotic cells, mutant FabI gene (mFabl) from E. coli which confers triclosan resistance to the host, URA3 an orotidine-5' phosphate decarboxylase from yeast, spectinomycin resistance gene, and a chloramphenicol resistance gene. In one embodiment, the cassette comprises specnnomycin, kanamycin, gentamycin, and/or

chloramphenicol.

[0207] The use of antibiotics in large-scale production quantities is prohibitively expensive. Thus, genetically enhanced microbial host cells that do not contain an antibiotic resistance gene, and do nor require the presence of the corresponding antibiotic in order to mainiain the inserted genes is very useful at least for the cost-effective large scale production of a compound of interest.

{0208] A counter-selection marker gene as part of a three component self-excision cassette {KanR,flp, galK) can be created by putting the three component self-excision cassette in between two FRT sites, one 5' and one 3' of the three componen t cassette, as depicted in FIG. 3, for example. Host cells transformed with extrachromosomal plasmids containing a three component self-excision cassette enable a faster counter-selection of non-clea ved plasmids after induc tion of the site -specific recombinase (e.g. FLP) expression and therefore save tune until complete removal of the cassette comprising the ABR marker, the site-directed (e.g. flippase) and counter- selection gene is achieved. Without being bound by theory, the presence of a counter- election marker in a three-component cassette results m a substantial improvement upon any attempt to achieving full segregation while relying only upon the activity ofilippa.se on subsequent isolated clones. In one embodiment, the cassette comprises flippase, galK, and or KmR. I one embodiment, the site-specific recombinase (e.g. flippase), galK, and/or KmR genes are codon optimized for expression in the host cell. In another emobodiment, the host cell is

C ambacterittm sp. In another embodiment, the host cell is ABICyanol.

[0209] In an embodiment of methods disclosed herein, only a single transformation is needed for plasmid introductioa-propagation by a positive selectable marker (e.g. antibiotic selection) in host cells, such as ABICyanol, that will eventually result in the production of a marker free host cell. Using embodiments of methods disclosed herein, there is no need for a second transformation step as the introduced extrachromosomal plasmid contains an inducible site-directed recombinase gene (e.g., _/? >), a counter-selection marker (e.g., galK), and an antibiotic resistance gene (e.g., KmR). This three-component cassette (flp-galK-KmR) has a site directed recombinase recognition target sequence at its 5' end and at. its 3' end. In an embodiment, the flippase gene is then expressed and partially excises the three-component cassette from the extrachromosomal plasmid. However, as shown in FIG. 4 which depicts PCR analysis of both cut and uncut plasmids in a given colony/clone of host cells, without the additional selection pressure created by the presence of the counter-selection compound in the growth media and expressed counter- selection marker, the extrachromosomal plasmid will not readily achieve a homogenous population (would not become fully segregated, would not consist of only host cells with one particular type of plasmid) in a timely manner. Providing selection pressure to the excision activity of the flippase encoded for as part of the three-component ' cassette from the extrachromosomal plasmid enables the facile complete segregation of a extrachromosomal plasmid that does not have an antibiotic resistance gene as well ' as flippase gene and counter- selection gene, but that does contain genes encoding for enzymes responsible for the production of a compound or compounds of interest.

[0210] In an embodiment, the self-excision method for creating an antibiotic marker free host cell takes about six weeks, starting from the day of transformation, compared to about seven months when using a conventional strategies that rely upon complementation of an auxotrophy and require at least three successive transformation steps.

[0211] In an embodiment, the extrachromosomal plasmid containing the three-component cassette and genes responsibl for the production of a compound of interest, is capable of mamtinmg itself within a host-cell. In an embodiment, the extrachromosomal plasmid contains a self-replicating origin of replication that allows the plasmid to maintain its presence in a host cell even without selective pressure, such a containing a positive selection marker.

[0212] In an embodiment, the extrachromosomal plasmid is self-maintaining by using a

toxui''antitoxin system. In an embodiment, the extrachromosomal plasmid contains a gene encoding a first component having a toxic effect on the cyanobacterium, wherein an essential or conditionally essential gene encodes a second component having the ability to suppress the toxic effect on the cyanobacterium. An example of first mid second components are toxin/antitoxin systems. In an embodiment, the toxin/antitoxin system is a Type I system, wherein the antitoxin component is a RNA molecule, or sRNA, which base pan s with the toxi mRNA, thereby inhibiting translation into the component having the toxic effect on the cyanobacterium.

Examples of Type I toxin/antitoxin systems include Hok Sok, FstfRNA II, FlmA FlmB. It is also possible to include a DNA sequence between the first and second part which encodes an mRNA endomiclease recognition site. In this way, the single mRNA chain can be cleaved into separate biocatalyst and antitoxin mRNA chains. This aids intramolecular interactions withm the antitoxin RNA, which typically requires formation of complex secondary structures for its biologic activity. In another variation, the toxin/antitoxin system is a Type Π system, wherein the antitoxin is a protein which inhibits the activity of the toxin. Suitable Type II system examples include CcdB/CcdA, ParE/ParD, MazF/MazE, id' is. In an embodiment, the cyanobacterium comprises at least two or more genes encoding a first component having a toxic effect on the cyanobacterium. In this way, the risk of elimination of the toxin gene by mutation is minimized, which safeguards the self-maintaining selection pressure on the corresponding

bioeatalyst aiitltoxhi fusion product.

Adding a site directed recombinase gene to the cassette

[0213] In one embodiment, the cassette comprises a site-specific recombinase that integrates, excises, and/or inverts nucleotide sequences, hi another embodiment, the site- specific

recombinase includes but is not limited to, tyrosine recombinase, serine recombinase, Cre recombinase, Flp recombinase, gamma-delta resolvase, Tn3 resolvase, <pC31-integrase, Bxbl- intearase. Die. KD. B2, B3, λ. H 022, HP1, γδ. ParA. Gin, and R4 intesrase.

[0214] In an embodiment, a system for making a marker free microbial host ceil does not incorporate a counter-selection marker, hi an embodiment, the site-specific recombinase gene is operably linked to an inducible endogenous promoter and is part of a cassette containing genes of interest that make a compound of interest. In one embodiment, the site- specific recombinase is flippase that is operably linked to an inducible endogenous promoter, hi a further embodiment the inducible endogenouse promoter is Ρ01Ϊ0223 or PsmtA. However, using expression of the a site-specific recombinase (e.g. flippase) gene to achieve complete excision of a two component cassette having an antibiotic resistance gene and the flippase gene, would be very time

inefficient. As a non-limiting example, the cassette comprises a gentamycin resistance marker flanked by FRT site containing the natural flp gene from S. cerevisiae under control of the copper inducible Porfo ii. The transformed host cell hybrids (hybrid populations of cut and uncut plasmids) were selected on chloramphenicol contaming plates and re-streaked several times on BG11 plates containing 15 μΜ CuS04 to induce the flippase expression. Eight to sixteen single colonies were picked and analyzed by PGR after each transfer. Although a partial cleavage was visible after two passages, see FIG. 4, complete cleavage of all plasmid copies required much more time and was finally obtained after eight, transfers.

[0215 j Thus, one issue of using a method that relies upon the antiobiotic resistance marker removal procedure that only depends on the activity of the FLP recombinase is that there is no selection pressure to force the intended segregation process to completion.

Adding a counter-selection marker to the cassette

[0216] Counter selectable markers are powerful tools in genetics because they enable a directed selection for removal of a genetic marker rather than its presence. There are several well described counter-selection markers- known in the art and some of them are also known- to be functional in eyanobacteria. Examples of counter-selectable markers include but are not limited to,. URA3/5-FQA, terAr, sacB,. rpsL, ccdB, pheS, thymidine kinase (TK), 2-DOG, and nutritional requirements. In an embodiment counter-selection markers are galK and mazF. The counter selectable markers may be under the control of one or more operably linked promoters. In one embodiment, the counter selectable marker is operably linked to the PmntC promoter.

[0217] The E. coli galK gene, encoding the enzyme galactokinase, catalyzes the phosphorylation of galactose to galactose- ! -phosphate. It also efficiently phosphorylates a galactose analogue, 2- deoxy-gahctose (2-DOG). The product of this reaction, 2-deoxy-galactose- 1 -phosphate , cannot be further metabolized, leading to buildup of toxic levels and cell death. Thus, expression of ga!K leads to a sensitivity to 2-DOG which is otherwise nontoxic to the cells.

[0218] MazF from E. coli is an endoribonuclease that cleaves mRNA at the ACA triplet sequence, and thus acts as a general inhibitor for the synthesis of all cellular proteins. The small size of the galK and mazF genes are also beneficial in genetic manipulations as the chance of mactivation increases with the size of the gene.

Marker free ABICyanol host cells

[0219] In an embodiment, the host cells are first transformed with a plasmid containing a three- component cassette encoding a site-specific recombinase, an antibiotic resistance enzyme, and a eoimterselection marker. The three-component cassette is flanked by FRT sites on each 5' and 3 " end. Without being bound by theory, once the site- specific recombinase is expressed from the gene in the cassette, it will recognize the site-specific recombinase target sites flanking the cassette and excise it from the extrachromosomal plasmid. In an embodiment, a first ste of a method for making marker free microbial host cells is to segregate the population of host cells containing only a 6.8 derived extrachromosomal plasmid containing production gene cassette and self-cleavage three-component cassette from host cells containing any portion of the wild type p6.8 plasmid driven by addition of the respective antibiotic the introduced ABR marker is conferring resistance to. Any cell not transformed with the modified plasmid will die after exposure to the antibiotic, in an embodiment, a second ste comprises expressing the flippase gene either through the induction of the operably linked promoter, through "leaky" expression of the flippase gene or via constitutive expression of the flippase gene. The flippase gene is not very efficient, and only cuts at about 1-30% of the FRT sites in a given round. Thus, in order to obtain a population of cells comprising solely p6.8 derived plasmids wi th a production gene cassette of interest that lacks the antibiotic resistance gene, multiple rounds of inducing the expression of the flippase gene would have to be carried out, colonies selected according, to their segregation state analyzed by PGR, resulting in a segregation process lasting multiple months which is timely, rather unpredictable and possibly never achievable. These cumulative steps present a major time impediment to the facile engineering of the host cells .

[0220] Without being bound by theory, a two component, selection cassette encoding flippase and an antibiotic resistance enzyme flanked by FRT sites on each 5' and 3' end would probably never lead to a completely cleaved p6.8 plasmid population when located on an extrachromosomal plasmid. This is likely because with increasing cleavage efficiency of the cassette there would be, at the same time, a decreasing flippase gene dosage and thus, consequently, Flp expression and activity would likely decrease. Tins inverse relationship between cleavage efficiency and flippase expression level minimizes the chance for successful full segregation of cut from uncut extrachromosomal plasmids that would contain such a two component selection cassette, but lack a counter-selection component,

[0221] In an effort to impart a selective pressure for having a markerless extrachromosomal plasmid comprising a cassette of interest for the production of a compound of interest in a more predictable and shorter period of time also a counter-selection marker gene is part of the self- cleavage selection cassette and a counter-selective compound can be added to a growth medium to force the selection cassette removal. If flippase has not excised at the FRT sites in the extrachromosomal plasmid, the addition of the counter-selective compound will cause cell death because of the presence of the counter-selective marker in the cells lacking excision. Thus, the addition of a counter-selective compound, such as 2-DOG, causes positive selective pressure on host cells that have a pure population of the p6.8 deri ved plasmid that has been excised by flippase at the FRT sites and negative selection pressure on cells with p6.8 derived plasmids that have not been cut or incompletely cut by the flippase. The selective pressure forces complete segregation of the partially cut p6.8 plasmid population resulting in a pure population of the p6.8 derived plasmid that has been excised by flippase action completely as shown in FIG. 5 which depic ts PCR analysis showing the existing of only a cut #2084 after growing in the presence of 2- DOG. Table 2 depicts constructs with various promoters for pdc, adh and Sippase as well mgalK and KmR genes. [0222] In an embodmient, the plasmids disclosed herein are maintained stably i the host cell. In another embodiment, the plasmids are maintained for at least about 5 days, about 10 days, at least about 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about about 6 months, hi one embodiment, the stably maintained plasmid is an excised plasmid. In one embodiment, the plasmid is p6.8. In another embodiment, the plasmid is derived from p6.8. In an embodiment, p6.8, and plasmids derived therefrom, are maintained stably in the host cell. Thus, the engineered p6.8 derived plasmid is maintained within the host cell even after at least a period of 30 days.

Table 2

[0223] In an embodiment, the three-component cassette is construct #2084 (pABICyanol- 6.8;:PnirA-PDC(optl)-TdsrA-Prbc*{optRBS)-ADHl 1 l{opt)-TB001 l-FRT-Porf0223*-flp- TB0011 -PmiitC-galKCop -TTT-PrbcL-Km^^-FRT), for example.

[0224] In contrast to #2148 - #2151 the flp gene in construct #2152 and #2153 is zinc-inducible in order to avoid possible issues due to co-induction of PDC when PDC and flippase gene are both under control of a copper-inducible promoter (Porf0316 and Porf0223). #2151 uncut has a polynucleotide sequence as depicted in FIG. 21 (SEQ ID NO: 12). #2152 uncut has a polynucleotide sequence as depicted in FIG. 23 (SEQ ID NO: 14).

[0225] The self-cutting cassette can be applied for a broad range of organisms (incl. eukaryotes) with the only requirements for use of the self-excising three-component cassette in microbes being at least one inducible omoter controlling the flippase expression (galK gene can be constitutive or inducible); the endogenous plasmids are self-replicating (e.g. pAQ series of

Synechococ s PCC7002) or shuttle plasmids (e.g. RSFI010 based pVZ321 piasmid for

Synechocystis PCC6803) with stable maintenance in the host cell (w o AB selection) but also YACs, BACs, cosmids and phagemids for eukaryontic host cells: and that the host cells enable sensitivity to a counter-selection marker gene an respective counter-selection compound, for example galK counter-selection (2-DOG + GalK enzyme).

[0226] If the extrachromosomal piasmid that contains the three-component cassette is not stably maintained within the host ceil, known toxin anti-toxin systems may be added to the piasmid in order to mediate/guaranty stable piasmid replication (see, for example, Kopfmann 5., Hess WR„ Toxin-Antitoxin Systems on the Large Defense Piasmid pSYSA of Synechocystis sp. PCC 6803, J Biol Chem. 2013 Mar 8; 288(10): 7399-409).

[0227] If no sensitivity for GalK counter-selection is the case other known counter-selection marker can be applied alternatively: mazF, sacB, etc. In an embodiment, chromosomal manipulations (e.g. deletions/ insertions) can be an additional application for the self-cleavage tlnee-component ABR-eassette {beside the use for self-replicating plasmids).

Extrachromosomal plasmids with ethanologenic gene cassettes and three-component cassettes

[0228] hi an embodiment, ethanologenic gene cassettes are part of an extrachromosomal piasmid that is introduced into the host cells and those host cells are used for the productio of ethanoi. In one embodmient. the host cells are ABICvanol host cells. Ethanologenic cassettes disclosed herein vary in promoters used as well as the source of adh and pdc genes. Any adh and/or pdc genes known in the art may be used. Hie skilled artisan will be able to select the best gene/promoter combination to optimize ethanoi production in a given host cell, in an

embodiment an extrachromosomal piasmid derived from p6.8 contains an ethanologenic cassette and a three-component cassette. For example. FIG. 3 depicts a map of the a #2084 (pABICyanol- 6.8: :PnirA-PDC(optl)-TdsrA-Prbc*(optRBS)-ADH 1 11 (opt)_ter-FRT-PoriD223*-flp-TB0011 - PnmtC-galK(opt)-Tr7 ' PrbcL-Kin^^-FRT) before and after self-excision of the three component cassette by flippase. The genes comprising both the ethanpl cassetted and the three-component cassette can be codon optimized for optimal expression in ABICyano l and can utilize any suitable promoter and other regulatory' sequences.

[0229] Table 3 lists additional extrachromosoniai plasmids derived from p6.8 that contain both an ethano!ogenic cassette and a three-component cassette thai were used for the transformation of ABICyanol as well as the composition of the plasmid after self-excision i.e. "cut". Table 3 also demonstrates the ethanol production in the respectively transformed ABICyanol cells.

Table 3

10230] Figure 7 depicts ethanol production of ABICyano l host cells containing plasmids #1646 uncut #2084 and cut #2084. All three strains produce ethanol with no meaningful difference in ethanol production between cut and uncut #2084. [0231] Figure 8 depicts growth characteristics of ABICyanoi host cells transformed with #1646 and ' #2084 while they are producing ethanoi. There is virtually no effect of the three-component cassette on the growth of ABICyanoi host cells.

[0232] Figure 9 depicts ethanoi production of ABICyanoi host cells, transformed with #1646 and #2084. There is virtually no effect of the three-component cassette on the ethanoi production of ABICyanoi host cells when compared to the control plasmid #1646 whic is the ABR containing counterpart.

[0233] Figure 10 depicts the effect of cell growth on ABICyano i host cells containing

ethanologenic p6.8 derived extrachromosomai plasmids #1933 and #1938 with cut #2151 and cut #2152. As shown in FIG. 10, the growth of cut #2151 and cut #2152 is as good or better than the growth of plasmids #1933 and #1938 that never contained a three-component cassette.

[0234] Figure 11 depicts the ethanoi production of ABICyanoi host cells containing

ethanologenic p6.8 derived extrachromosomai p!asmids #1933 and #1938 with ABR-free cut #2151 and cut #2152. Figure 11 depicts ethanoi production by determining ethanoi concentration in a medium by measuring the ethanoi concentration in a vapor over the medium. As shown in FIG. 11, the production of ethanoi in cu #2151 and cut #21 2 is as good or better than the ethanoi production of ABICyanoi cells #1933 and #1938 containing the kanamycin resistance marker.

[0235] Figure 12 depicts the ethanoi production per OD750 of ABICyano i host cells containing ethanologenic p6.8 derived extrachromosomai plasmids #1933, #1938, and ABR-free cut #2151 and cut #2152. As shown in FIG. 12, the production of ethanoi per OD750 in cut #2151 and cut #2152 is as good or better than the ethanoi production per OD750 of plasmids #1933 and #1938 containing the kanamycin resistance marker.

Kits for producing compounds of interest

[0236] In an embodiment, a kit for producing a compound of interest using genetically enhanced ABICyanoi host cells that are antibiotic marker free includes genetically enhanced ABICyanoi host cells, a vessel for culturing the host cells and a means for illumination of the host cells. In an emhodiment, the host cells of the kit produce ethanoi. and the means for illumination i photosyntheticaily active radiation from the sun. In an embodiment, the means for illumination of the host cells include lamps or light emitting diodes or a combination thereof. The vessel of the kit can be a photobioreactor which is at least partly transparent for the radiation emitted by the means for illumination of the host cells. In particular embodiments, an of the photobioreaetors disclosed in. the PCT application WO 2008.055190 A2, which is hereby incorporated in its entirety by reference, can be used. FuruSermore the kit also can also include means for separating the compoimd, preferably etlianol from die growth medium as, for example, disclosed in the PCT application WO2011/103277 Ai, which is hereby incorporated in its entirety by reference,

[0237] The present disclosure is further described by the following non-limiting examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present disclosure.

EXAMPLES

Example 1: Bacterial strains, growth conditions, selection of transformants, and general procedures

[0238] Escherichia eoli (E. coli) strains HB101 (Proniega), XLIO-Gold (Siratagene), a-select (Bioli e) and NEB Turbo (NEB) were grown in Luria-Bertani (LB) medium at 37°C. Ampiciilm (100 kanamycin (25-50 g ' ml), gentamycin ί15μ§ί η 1) and chloramphenicol (34 ug/ml) were used when appropriate. Cultures were continuously shaken overnight at 200 rpm. AB1 was cultured at 30°C in liquid BG11 fresh water medium on a reciprocal shaker at 150 rpm under continuous illumination of approximately 30 - 40 umol photons nrVs 1 . Cyanobacterial

transformants were selected on solid BG11 medium containing 10 - 20 ug/ml kanamycin, gentamycin or chloramphenicol and were maintained on BG11 plates containing 20 - 100 μ&/κύ kanamycin, gentamycin, chloramphenicol or without antibiotics (ABR-free).

Example 2: Preparation of cyanobacterial culture medium

[0239] BG- 11 stock solution was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO). Stock solutions of the antibiotics, kanamycin (50 mg niL), were purchased from Teknova (Teknova, Hollister, CA). Stock solution of the antibiotic gentamycin (10 mg/niL) was purchased from MP Biomedicals (MP Biomedicals, Solon, OH). Marine BG-l 1 (mBG- 11) was prepared by dissolving 35 g Crystal Sea Marinemix (Marine Enterprises International, Inc., MD) in 1 L water and supplementing with BG-l 1 stock solution.

[0240] Culture conditions for mLvPBR cultivation under standard conditions and inoculation with cell with an OD750 of about 0.5 included the following as depicted in Table 4 below. [0241] Culture conditions for LvPBR cultivation under standard conditions and inoculation with cell with an QD750 of about 0.5 included the following as depicted in Table 5 below.

Table 4

Reactor: 0.4 L gassed mlvPBR

Volume 0.35L

Media: 35 psu aswBG l l, $ mM TES pH 7.3

Cultivation pH 7.3 (TES)

CO; supply 5 % CO j during daylight at 10 mL/min

Aeration Continuous

Light 230 μΕ from 1 side

Temperature Day 30-31° C, Night 2S " C

Table 5

Reactor: L2 L flexible vPBR

^ledia: 35 psu BASW SG-11

i nduction : PorfOSiS 15 μ Cu-EDTA

PnirA *2 17.5 mM M0 3 (BG-11)

Feeding: ½ of NO j consumed addition of SG-11 and phosphate |SOP) pH : 7.3 ÷ 0.01

C0 2 supply ; 15 % pH dependent Into liquid phase

Aeration: 38 ml mirv 1 contiguous

Light: 230 μΕ from one side

Temperatur: 2S ± .' . '= C r ight, 37 ± 1° C day

Example 3: Competent ABICyanol ceils for transformation by conjugation

[0242] Two hundred mL of an exponentially growing culture (OD750 greater than about 0.5 and less than about 1.0) was incubated with NAC for 2 days at 16 °C (end concentration of NAC is about 0.1 ing/mL) without shaking. This pretreatment was followed by several steps to degrade the EPS and to weaken the cell wail. The pretreated culture was pelleted at 4400 rpm and washed with 0.9% NaCl containing 8 mM EDTA.

{0243] For further treatment with lysozyme, the cell pellet was resuspended in 0.5 M sucrose and incubated 60 min at room temperature (RT) with slow shaking (85 rpm). Then, cells were centrifuged and resuspended in 40 mL of a solution containing 50niM Tris (pH 8.0), 10 mM EDTA pH 8.0), -4% sucrose, and 20-40 g mL I soz me. After incubation at rt for 10-15 min, cells were eentrifuged and washed three times using different washing -solutions; i) 30 mM Iris containing 4% sucrose and 1 iriM EDTA: ii) lOQmM Tris containing 2 % sucrose and iii) with BGI 1 medium. All centrifugaiioii steps before Iysozyme treatment were performed at 4400 rpm for 10 min at 10 °C, All eentrifugations after the Iysozyme treatment were performed at 2400 rpm for 5 mm at 4 °C. Resuspended cells were used for conjugation.

Example 4: Transformation of ABICyanol by Conjugation

[0244] Gene transfer to ABICyanol was performed using conjugation. Generated plasmids containing oriVT were used for conjugation. The shuttle vectors were transformed into

ABICyanol following a modified conjugation protocol which includes the pretreatment of ABICyanol to reduce its EPS layer.

[0245] Some plasmids used in this project are listed in Table 4.

Table 4

[0246] For triparental mating E. coli strain J53 bearing a conjugative RP4 piasmid and E. coli strain HB101 harboring the cargo and the pRL528 helper piasmid (for in vivo methylation) were used. E. coli strains were grown in 20 ml LB to exponential growth phase, washed twice with LB medium and resuspended in 200 μΐ LB medium. Then, E. coli strains were mixed for triparental mating, eentrifuged and resuspended in 100 μ! BGI 1 medium.

[0247] One hundred μΐ resuspended cyanobacterial and E. coli cultures were mixed and applied onto a membrane filter (Millipore GVWP, 0.22 μηι pore size) placed on the surface of solid BGI 1 medium supplemented with 5% LB. Petri dishes were incubated under dim light (5 μηιοί photons/m2/sl) for 2 days. Afterwards cells were resuspended in fresh BGI 1 medium and plated onto selective medium containing 10-20 μ§Λη1 kanamyem/ gentamycin/ chloramphenicol, respectively. Selection conditions were: light intensity approximately at 20-40 μηιοί

photons/m2/s I , temperature at approximately 30°C. Once transformants were visible (approx. after 5-7 days), colonies were transferred on new plates containing 10-50 μ§Λη1 kanamycin. Example 5: Making an ABR free eyanobat'terM host cell

[0248] The following method is an example of methods used for making ABR free

cyanobacterial host cells as described herein. In a first step, extrac romosomal plasmids containing an ABR marker and self-cleaving three-component cassettes were introduced into ABICyanol host cells by conjugation techniques discussed in the examples above.

Extrachromosomal plasmids containing an ABR marker and self-cleaving three-component cassettes were successfully introduced into ABICyanol host cells and include: #2148

(pABICyanol -6.8::PmiA*2-PDC(optI) TdsrA-PcpcB-ADHl 1 l(opt)-TrbcS-FRT-Porf0223*-flp- TB0011-PmntC-gal (opt)-TT7-PrbcL-Km^-FRT); #2149 (pABICyanol-6.8::Porf0316- PDC(opil )-TdsrA-PcpcB-ADH 111 (opt)-TrbcS-FRT-PoriI)223 *-flp-TB0011 -PmntC-gal (opt)- TT7-PrbcL-Km**-FRT); #2150 (pABICyanol-6.8::Porf0316-PDC(optl)-TdsrA-PcpcB-ADH 916(opt)-TrbcS-Fin , -Porf0223*-f^

(pABICyano 1-6.8:: PnirA* 2-PDC(opt 1 )-TdsrA-PcpcB- ADH 16(opt)-TrbcS-FRT-Porfl)223*- flp-TB001 l-PnintC-gaIK(opt)-TT7-PrbcL-Kin* *-FRT); #2152 (pABICyano 1 -6.S : :PorfD316- PDC(opt I )-Tdsi A-PcpcB-ADH 111 (opt)-TrbcS-FRT-PsmtA-flp-TB 1002-PrrmtC-galK(opt)-TT7- PrbcL-Km* *-FRT) ; and #2153 (pABICyano 1-6.8: : Porf0316-PDC(opt 1)-Tdsr A-PcpcB-ADH 916f opt)-TrbcS-FRT-PsmtA-flp-TBl 002-PmntC-galK(opt}-TT7-PrbcL-Km :i! *-FRT}.

[0249] In a second step, successfully transformed ABICyanol hybrids were selected and transferred to agar plates containing higher concentrations of kanamycin, such as 50 ug/mL.

[0250] In a third step. ABICyanol cells able to grow on the growth, media from step two were transferred again to agar plates containing higher concentrations of kanamycin, such as 50 $ / ιη11 or greater, in order to achieve complete segregation of ABR containing plasmids firom wild-type p6.8 plasmids.

[0251] In a fourth step, PCR analysis of the extrachromosomal plasmids from, the segregated cells of step 3 was performed to verify that the population of cells obtained from step three was fully segregated, only contained extrachromosomal plasmids containing the three-component cassettes and not containing any copies of the wild-type p6.8 plasmid.

[0252] In a fifth step, flippase expression was induced on agar plates without kanamycin in the fully segregated cells from step 4.

[0253] In a sixth step, PCR analysis was performed on the extrachromosomal plasmids to determine the cleavage efficiency of the expressed flippase. If the percentage of excised plasmids (excision of the three -component cassette) was greater than about 10%, then the method proceeds to a seventh, step.

[0254] la a seventh step, the population of cells demonstrating greater than about Ϊ 0% excision of the three-component cassette were grown on plates containing 0.02% 2-DOG in order to begin the counter-selection process of cells containing uiicleaved ABR-containing (and three- component cassette containing) extrachromosomal plasmids from those cells from which the ABR gene (and three-component cassette) have been cleaved.

[0255] In an eighth step, cells which continue to grow on plates containing 0.02% 2-DOG were analyzed by PCR to verify counter-selection efficiency, to test for the amount of segregation.

[0256] In a ninth step, clones demonstrating greater than 99% cleavage and segregation were transferred to agar plate containing 0.2% 2-DOG to achieve 100% (fully segregated) ABR-free host cells.

[0257] In a tenth step, PCR analysis was performed on the cells able to grow on the 0.2% 2-DOG plates, but unable to grow on plates containing kanamycin.

[0258] An entire process of obtaining an ABR-free ethanologenie cyanobacterial host cell took about six weeks starting from the intial transformation with the extrachromosomal plasmids that contain the three-component cassettes.

Example 6: Determination of ethanol production using gas chromatography

[0259] Two kinds of GC headspaee measurements were performed:

a) GC online vial measurements (applied for clone testing and short-term characterizations of cultures cultivated in GC vials with a duration of up to 72 hours,

b) single GC single measurements (applied for measurements of EtOH concentrations in samples dayly taken from PBR cultures) by measuring the ethanol content after transferring 0,5 mL of tlie PBR cultures into GC vials after certain points of tune of cultivation in the PBR.

GC single measurements do not involve the cultivation of the strains in the GC vials. GC single measurements were performed in order to characterize the long temi ethanol production of strains, which are already known to produce ethanol in sufficient quantities in GC online vial measurements. GC single measurements further differ from GC online vial measurements in the volume of the culture (2 mL in GC online vial and 0.5 mL aliquots taken from a PBR culture in GC single measurements). In single GC measurements only tlie absolute amount of ethanol produced at a certain point of time is detenriined, whereas the GC online vial measurements determines the course of ethanol production during a certain period of time up to 72 hours of growing the cells a GC vial under constant illumination. For GC single measurements the sample was heated to 60 C C in order to transfer all ethanol from the liquid phase to the gas phase for the GC headspace chromatography, which resulted in a disruption of the culture. In contrast to that this 60°C heating step was omitted during GC online vial measurements in order not to destroy the culture and in order to further continue with the culturing of the cells in the GC vial. In the following GC online vial measurements are described.

[0260] GC online vial headspace measurements are performed on a Shimadzu GC-2010 gas chromatograph with Flame Ionization Detector. The detection limit for ethanol quantification is at 0.0005%, hut a calibration has to be done for detecting quantities below 0.001%. The instrument is connected in-line with a Shimadzu PAL LHS2-SHEV1 AOC-5000 autosampier, comprising a gas-tight syringe for transfer of headspace aiiquots from the culture samples to the analytical unit. Specific modifications were indroduced as follows: Each sample tray is exposed with a LED acrylic sheet (length: 230mm, wide: 120mm,. diameter: 8 mm, 24Chip, S4, 530OK), equipped with a dimmer by company Stingl GmbH. Below the sample tray a magnetic stirrer is installed (IKA RQ 5 power ) allow ing for mixing of cultures which are cultivated in GC vials thai stand in the sample tray. The sample trays are penetrating of maximum, so that the GC Vial stands in the Tray. A heating mat between LED acrylic sheet and the magnetic stirrer (MOHR &Co, one heating circuit, 230 V, 200 Watt, length: 250 mm, wide: 150 mm, diameter: ca. 2.5 mm) with a temperature regulator (JUMO dTRON 316) allows for the incubation of cultures in GC vials at specific temperatures. The gas chromatograph is connected to helium earner gas as well as hydrogen and artificial air as fuel gas and oxidize gas, respectively, for the flame ionization detector. Oxidizer air is generated with the generator WGAZA50 from Science Support. The gas chromatograph is equipped with a FS-CS-624 medium bore capillar} ' with a length of 30 m, internal diameter of 0.32 mm and film thickness of 1.8 um from the GC supplier Chromatographic Sendee GmbH.

[0261 ] The ethanol production in the culture has to be induced 1-2 days before the GC online vial experiment is realized by triggering the overexpression of pdc and adh. For induction hybrid cells are harvested from liquid cultures by centrifugation and are resuspended in a sterile tube with mBGi 1 media with additional 50mM TES pH 7.3, 20mM NaHCCb, antibiotics and nitrate until they reached an OD of 2. For the hybrids with nirA promoter the induction is realized by transfer to nitrate containing medium. The clones were incubated on a small shaker at 180 rpm for 4S hours at 28°C. The shaker is armed with a dimmabie light table adjusted to 120 μΕ (300μΕ-0μΕ). After 48 h centrifuge the tube at 20°C for 10 minutes,.4500 rpm and discard the supernatant. The Pellet is resiispended himBGI 1 medium snppl. with 50mM TES pH 7.3, 2-Om NaHCOs, containing nitrate and no antibiotics. For hybrids under control of copper responsive promoters the induction is realized by addition of 3-όμΜ copper, for zmc inducible promoters is the induction is realized by addition of 10μΜ zinc sulfate (heptahydrate) and for hybrids with the petJ promoter the induction is done by transfer to copper-free medium. The clones were incubated on a small shaker at ISO rpm for 24-48 hours at 28°C. The shaker is armed with a dimmabie light table adjusted to 120 μΕ (300μΕ-0μΕ). After 24h - 48h cells were harvested by centrifugation in a 50mL Falcon tube at 20°C for 10 minutes, 4500 rpm and discard the

supernatant. The pellet is resuspended in mBGl 1 medium supplemented with 50 mi*'! TES pH 7.3, 20mM NaHCOs, and appropriate metal ions for induction without antibiotics. The sample will be adjusted to an OD?50 of about 0.7 (+/- 0.1) for 4 replicates. 2 mL are filled in 20 rriL GC vials equipped with a magnetic stir bar (12 mm) in which the lid is not completely tightened. 5 mL pure carbon dioxide is injected for 1-3 days with the 30 mL syringe through the septum, and then the lid tightly closed (gas tight). The tightly closed GC vials are placed into the headspace auto sampler rack which is temperature controlled at a given temperature for example 37°C and are analyzed at the same day. After the GC measurements the final OD750 is determined for the calculation of the ethanol production rate per average ODTSO. The average cell density for each sample is determined by calculating the arithmetic mean of the optical density at the starting point and the optical density at the end point of the process.

[0262] If necessary, reference samples for the calibration of the gas chromatograph can be prepared as 2 mL aliquots with 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mg/mL ethanol in 35 psti sodium cloride. Reference samples are placed into the same 20 mL sample containers with self -sealing silicon septum caps for headspace autosampling. For each reference sample at least six measurements are applied. After the measurements, the resulting peak areas of the reference samples are used for generating two calibration curves, the first in the concentration range from 0.005 to 0.5 mg/mL ethanol and the second one for the concentration range from 0.5 to 10 mg/mL ethanol. The calibration curves hav to fulfill linearity.

[0263] The sample incubation temperature for the GC online measurements in the autosampler is adjusted to a given temperature for example 37 °C. The illumination is set at 90 uE to 50 μΕ, preferably 120 μΕ. The magnetic stirrer is configured for interval mixing of the samples, with cycles of 2 minutes mixing at 400 rpm, followed by 90 minutes without mixing. An automated process follows, wherein after given periods aliquots of 500 L of the headspace; of the samples are automatically drawn with the gas-tight headspace syringe and injected via the injection port into the gas chromatograph. for analysis. Before each headspace autosampling, the mixing is changed for 10 mm to continuous mixing with 750 rpm at 37 ' C incubation temperature. The syringe temperature is set at 70 °C. The fill speed is 250 Τ per second, following an initial lag time of 1 second after the septum of the samples has been pierced by the syringe needle. The injection of the aliquot into the gas chromatograph happens with an injection speed of 500 μΐ, per second. Afterwards, tlie syringe flushes for 3 minutes with air to prevent sample carryover between two injections. The gas chromatograph runtime is 4 minutes and 30 seconds. The injection temperature on the gas chromatograph is 230 °C. The column temperature is 60 °C. Detection is accomplished with the flame ionization detector at 250 °C process temperature. The makeup gas is nitrogen at 30 mL per minute, the fuel gas is hydrogen at 35 mL per minute and the oxidizer gas is artificial air at 400 mL per minute.

[0264] After the final GC online vial measurement, the final optical density at 750 nm of the samples is measured and an average cell density lor each sample is determined by calculating the arithmetic mean of tlie optical density at the starting point and tlie optical density at the end point of the process divided by two. Afterwards, the average ethanol production rate per cell density is calculated.

[0265] The following procedure describes the standard lab conditions under which a 1.2L vPBR is operating as well as the necessary parts, ports, etc, to construct tins 1.2L vPBR. The 1 column vPBR consists of an autoclavable polypropylene flexible film (Profol unsistoffe GmbH;

Germany} with the dimensions of 750 mm total height and a diameter of 50 mm when filled with liquid. The iillmg volume is about 1.2 L leading to a liquid height of about 620 ± 20 mm a d a headspace of about 150 ± 20 mm. Tlie vPBR is equipped with several ports for operation, located on specific positions of tlie vPBR from bottom to top: a sampling port (60 mm from the bottom), a gasin port (130 mm from the bottom), a pH probe port (300 mm from the bottom), D02 probe port (350 mm from the bottom), a mediumin port (650 mm from the bottom) and a gasout port (700 mm from the bottom). The illuminated surface area when illuminated from one side is 0.049m2. Tli standard light conditions is a uniform light field from one side with 230 μηιοΐ m-2 s-1 at the vPBR surface generated by a light panel winch consists of 9 to 12 T5 54W 6500K fluorescent bulbs operating in a 12/12h day/night cycle. Tlie temperature is set to 39 °C ± 2 °C during day and 29 °C ± 2 °C during night. The mixing is realized via the ascending air bubbles through the liquid culture. The gas flow is operating in a constant sparging mode (day and night) with air enriched with 15% C02 introduced on demand via pH control pH setpoint - 7.3, day and night) and a flow rate of 38 mL min-1. The number of holes in the sparging tube for a I 2L vPBR is approx. 50 holes (perforated from both sides) and the spargmg tube length is 220 mm. The standard cell density for starting a cultivation experiment OD750nm - 0.5 mmBGl 1 medium with 35ppt (parts per thousand) salt (about 35 psu) (for strains that do not aggregate under high-light illumination).