Methods for Increasing the Stability of Production of Compounds in Microbial Host Cells

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 62/096,383, filed on December 23, 2014, the disclosure of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[002] None

REFERENCE TO SEQUENCE LISTING

[003] This application contains a sequence listing submitted by EFS-Web, thereby satisfying the requirements of 37 C.F.R. § 1.821 -1.825. The sequence listing file, named

"Essential_operon_P0036_02_PCT_ST25", was created on December 22, 2015, and is 191 kb in size.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

[005] Cyanobacteria are prokaryotes capable of photoautotrophy. Cyanobacteria can be genetically enhanced to use light and C0 ₂ to produce compounds of interest such as biofuels, industrial chemicals, pharmaceuticals, nutrients, carotenoids, and food supplements. Various cyanobacterial 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 C0 ₂ to compounds of interest.

[006] The cyanobacterial genus Cyanobacterium was first established in 1983 (see Rippka et al. (2001 ), Bergey's Manual of Systematic Bacteriology, Vol. 1 , p. 497-498). Members of the Cyanobacterium genus are often found in thermal mats (Moro, et al., 2007, Algological Studies, 123: 1 -15). [007] The transformation of the cyanobacterial genus Cyanobacterium with genes that encode enzymes that can produce ethanol for biofuel production has been described, for example, in U.S. Patent No. 8,846,369 to Piven et al.

SUMMARY

[008] The loss of expression of an exogenous pyruvate decarboxylase (pdc) gene, which is one of the production genes in non-naturally occurring ethanologenic cyanobacterial strains, results in loss of ethanol production. The loss of expression can occur quickly, so that by the end of a production run, very little ethanol is being produced. This has been found to be due to mutation based-inactivation ("reversion") of the pdc gene itself, so that the cell doesn't produce the ethanol intermediate. To prevent this, an endogenous gene that is essential to the cyanobacterial cell's survival is modified so that it is tied to the correct expression of the production gene. If the production gene is not expressed, the essential gene is also not expressed, and the cell dies.

[009] In an embodiment, a pdc-nirA operon was made essential to the survival of host cells when grown on nitrate as the nitrogen source. Non-naturally occurring ethanologenic

cyanobacterial strains were created tying nirA expression to pdc expression in an operon in order to increase genetic stability. For these strains, a knockout out of pdc expression causes the loss of NirA activity. Because these strains lack a chromosomal copy of nirA, the lack of expression of the pdc-nirA operon results in cell death.

[010] In another embodiment, using methods described herein, any gene useful for the production of a compound of interest can be made part of an essential operon containing another gene that complements an auxotrophic host cell. The resulting host-cells demonstrate increased genetic stability of the gene useful for the production of a compound of interest.

[011] In an aspect, a non-naturally occurring auxotrophic microbe capable of production of a compound of interest via the expression of exogenous genes is disclosed wherein said microbe contains an extrachromosomal plasmid comprising at least one of said exogenous genes and a gene whose expression complements the auxotrophy of said non-naturally occurring auxotrophic microbe and wherein said at least one of said exogenous genes and said gene whose expression complements auxotrophy form a polycistronic operon wherein said at least one of said exogenous genes and said gene whose expression complements auxotrophy are both operably linked to a promoter such that expression of said gene whose expression complements auxotrophy cannot occur without expression of said at least one of said exogenous genes. In an embodiment, the non-naturally occurring auxotrophic microbe contains a promoter that is an inducible promoter. In another embodiment, the non-naturally occurring auxotrophic microbe contains a promoter that is light inducible. In an embodiment, the non-naturally occurring auxotrophic microbe of contains a promoter that is a constitutive promoter. In another embodiment, a non-naturally occurring auxotrophic microbe is disclosed that is a cyanobacterial host cell. In yet another embodiment, the non-naturally occurring auxotrophic microbe is a microbe that is an auxotroph derived from Cyanobacterium sp. ABICyanol deposited in the American Type Tissue Collection (ATCC) as PTA-1331 1. In another embodiment, the non-naturally occurring auxotrophic microbe contains at least one inducible promoter operably linked to said polycistronic operon has at least 85% identity to a promoter selected from the group consisting of PnirA, PziaA, PsmtA, PcorT, PnrsB, PnrtA, Ppe l, PnarB, PmntC, PorfO221 , PorfD223, Porf0316, PorfD128, Porfl486, PorD 164, Porf3293, Porf3621 , Porf3635, Porfl 071 (PmntC), Porfl072, Porfl074, Porfl 075, Porfl 542, Porfl 823, Porf3126, PorfD222, Porf3126, Porf3232, Porf3749, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, and PcpcB. In another embodiment, the non-naturally occurring auxotrophic microbe contains an extrachromosomal plasmid that has greater than 80% sequence identity to p6.8. In another embodiment, the non-naturally occurring auxotrophic microbe contains an extrachromosomal plasmid that is self-replicating within the microbe. In another embodiment, the non-naturally occurring auxotrophic microbe contains an extrachromosomal plasmid that contains a self-replicating origin of replication that has greater than 90% sequence identity to the origin of replication of p6.8. In another embodiment, the non-naturally occurring auxotrophic microbe has exogenous genes that are pyruvate decarboxylase and alcohol dehydrogenase. The non-naturally occurring auxotrophic microbe contains a gene whose expression complements the auxotrophy of the non-naturally occurring auxotrophic microbe being auxotrophic because it lacks a functional nirA, or narB, or nrtABCD gene. In another embodiment, the non-naturally occurring auxotrophic microbe contains a gene whose expression complements the auxotrophy of the non-naturally occurring auxotrophic microbe and the gene is selected from the group consisting of a urease subunit gene, a urease accessory gene and a urea uptake gene. In another embodiment, the non-naturally occurring auxotrophic microbe has a gene whose expression complements the auxotrophy of the non-naturally occurring auxotrophic microbe and is involved in vitamin B 12 biosynthesis. In an embodiment, the non-naturally occurring auxotrophic microbe is capable of producing a compound of interest which is ethanol. In yet another embodiment, the non-naturally occurring auxotrophic microbe contains a polycistronic operon that has genes linked through intergenic sequences. The intergenic sequences can be derived from exogenous or endogenous sources. In an embodiment, the non- naturally occurring auxotrophic microbe contains a polycistronic operon that has genes linked through intergenic sequences derived from genes whose expression products are part of a photosynthetic apparatus.

[012] In an another aspect, a non-naturally occurring auxotrophic microbe capable of production of a compound of interest via the expression of exogenous genes is disclosed wherein the microbe contains a chromosomal polynucleotide sequence comprising at least one of the exogenous genes and a gene whose expression complements the auxotrophy of the non-naturally occurring auxotrophic microbe and wherein the at least one of said exogenous genes and the gene whose expression complements auxotrophy form a polycistronic operon wherein the at least one of the exogenous genes and the gene whose expression complements auxotrophy are both operably linked to a promoter such that expression of the gene whose expression complements auxotrophy cannot occur without expression of the at least one of said exogenous genes.

[013] In another aspect, a method for producing a compound of interest is disclosed wherein a non-naturally occurring auxotrophic microbe that is capable of producing the compound of interest produces the compound of interest via the expression of exogenous genes wherein the microbe contains an extrachromosomal plasmid comprising at least one of said exogenous genes and a gene whose expression complements the auxotrophy of said non-naturally occurring auxotrophic microbe and wherein the at least one of said exogenous genes and the gene whose expression complements auxotrophy form a polycistronic operon wherein the at least one of the exogenous genes and the gene whose expression complements auxotrophy are both operably linked to an inducible promoter such that expression of the gene whose expression complements auxotrophy cannot occur without expression of the at least one of said exogenous genes, the method comprising inducing the inducible promoter. In an embodiment, the method uses a microbe that is a cyanobacterial host cell. In another embodiment, the method uses a microbe that is an auxotroph derived from Cyanobacterium sp. ABICyanol deposited in the American Type Tissue Collection (ATCC) as PTA-1331 1.

BRIEF DESCRIPTION OF THE DRAWINGS

[014] FIG. 1 depicts a plasmid map of a pABICyanol 6.8 kb endogenous plasmid from

ABICyanol , alternately referred to as p6.8 (SEQ ID NO: 1 ; also depicted in FIG. 2). The plasmid p6.8 contains six open reading frames ORF 1 (SEQ ID NO: 2), ORF 2 (SEQ ID NO: 3), ORF 3 (SEQ ID NO: 4), ORF 4 (SEQ ID NO: 5), ORF 5 (SEQ ID NO: 6), and ORF 6 (SEQ ID NO: 7).

[015] FIG. 2 depicts the polynucleotide sequence of p6.8 (SEQ ID NO: 1) and depicts labels of various polynucleotide sequences.

[016] FIG. 3 is a bar graph depicting ethanol production of different nirA complementation constructs in a GC vial assay. The production is calculated as ethanol percentage per OD7 ₀ per day.

[017] FIGs. 4A, 4B, 4C, and 4D depict average OD750, ethanol, VLE corrected ethanol, and the percentage of revertant cells, respectively, of the nirA complementation strains and control strains in mLvPBRs. FIG. 4A depicts OD7 o of ABICyanol :pAB722 (SEQ ID NO: 8),

ABICyanol :pAB193 (SEQ ID NO: 9), ABICyanol :pAB 194 (SEQ ID NO: 10), and

ABICyanol :pAB 195 (SEQ ID NO: 1 1). FIG. 4B depicts ethanol production (% v/v). FIG. 4C depicts VLE corrected ethanol production. FIG. 4D depicts the percentage of cells that

"reverted" to non-ethanol producing cells over time, for either the control strain (722) or the nirA complementation strain (193).

[018] FIG. 5 is a bar graph depicting qRT-PCR quantitation of nirA gene expression in ABICyano 1 :pAB722 (SEQ ID NO: 8), ABICyano 1 :pAB 193 (SEQ ID NO: 9),

ABICyanol :pAB194 (SEQ ID NO: 10) and ABICyanol :pAB 195 (SEQ ID NO: 1 1). Samples were taken from repressed inoculum and from mLvPBR evaluations on day 2.

[019] FIGs. 6A and 6B depict PDC and ADH specific activity (nmol/min/mg), respectively, for nirA complementation strains in an mLvPBR evaluation for a first 21 day batch.

[020] FIG. 7A is a map of plasmid #TK596 (SEQ ID NO: 12) (TK596\oriVT-narB_up-FRT- PcpcB-Gm**-TB0014-FRT-narB_down), which is an integrative construct designed for the deletion (knockout) of the nitrate reductase gene narB from the cyanobacterial genome.

[021] FIG. 7B is a map of plasmid #1872 (SEQ ID NO:13), (#1872\oriVT-nirA_up-FRT- PcpcB-Gm**-TB0014-FRT-nirA_down), which is an integrative construct designed for the deletion (knockout) of the nitrite reductase gene nirA from the cyanobacterial genome.

[022] FIG. 7C is a map of plasmid #2198 (SEQ ID NO: 14), (#2198\oriVT-ureC_up-FRT- PcpcB-Gm**-TB0014-FRT-ureC_down), which is an integrative construct designed for the deletion (knockout) of the urease gene ureC from the cyanobacterial genome. [023] FIG. 7D is a map of plasmid #2235 (SEQ ID NO: 15) (#2235\oriVT-cob _up-FRT- PcpcB-Gm**-TB0014-FRT-cob _down), which is an integrative construct designed for the deletion (knockout) of the vitamin Bi ₂ gene cobK from the cyanobacterial genome.

DETAILED DESCRIPTION

[024] Presented herein are improved methods for making microbial host cells that have increased genetic stability for the expression of genes responsible for the production of compounds of interest. In an embodiment, the methods used to increase the genetic stability of 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.

[025] In one embodiment, the host cells are derived from an isolated strain of the

Cyanobacterium genus. A species member of the Cyanobacterium genus is referred to as a Cyanobacterium sp. and includes 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, antibiotic resistance gene (ABR) free host cells generated using methods disclosed herein are a species of the Cyanobacterium genus, Cyanobacterium sp. ABICyanol (referred to herein as "ABICyanol " or "AB 1 ") which has been deposited in the American Type Tissue Collection (ATCC) as PTA-1331 1.

[026] Genetically enhanced, non-naturally occurring ABICyanol organisms disclosed herein are useful for the production of compounds of interest, such as ethanol, for example. Such genetic modifications can comprise 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.

Definitions

[027] 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, J., 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 Phycology (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.

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

[029] 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.

[030] 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. In 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%.

[031] It is well known to a person 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 B l 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 entirely.

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

[033] The term "monocistronic" as used herein is an mRNA molecule that contains the genetic information to translate only a single protein chain. [034] The term "polycistronic" as used herein is an mRNA molecule that carries several open reading frames each of which is translated into a polypeptide. Dicistronic or bicistronic mRNA encodes only two proteins and may be referred to as polycistronic.

[035] The term "operon" as used herein is a segment of genomic DNA that contains one or more genes under the control of a single promoter.

[036] The term "essential gene" as used herein is a gene whose presence in the cell is required for the cell to remain viable.

[037] The term "conditionally essential gene" as used herein is a type of essential gene where the presence of the gene in the cell is required under some conditions, but wherein the cell can survive if another condition is present. An example is nutrient utilization genes, where under certain conditions (such as the presence of an additional compound in the medium), another gene can functionally substitute for the missing gene. As used herein, a "conditionally essential gene" can also be referred to as an "essential gene".

[038] The term "essential operon" as used herein refers to the nucleic acid-based linking of a production gene of interest so that it is located on the same mRNA transcript, immediately upstream of an essential gene. Thus, if the production gene is somehow mutated so that it is not expressed, the essential gene is also not expressed, and the cell dies. The genes are located on the same operon, under the control of one upstream promoter.

[039] The term "conditionally essential operon" as used herein is an essential operon system whereby the essential gene is a conditionally essential gene. This allows for the cell to survive when certain conditions are met, but the cell dies when those conditions are not met.

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

"PnirA".

[041] Enzyme 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 SynAdh (Zn ²⁺ dependent Alcohol dehydrogenase from Synechocystis PCC 6803), ZmPdc (pyruvate decarboxylase from Zymomonas mobilis). [042] The term "Cyanobacteria" refers to a member from the group of photoautotrophic prokaryotic microorganisms which can utilize solar energy and fix carbon dioxide. Cyanobacteria are also referred to as blue-green algae.

[043] The term "terminator" refers to a nucleic acid sequence, at which the transcription of an mRNA stops. Non-limiting examples are dsrA from Escherichia coli (E. coli), the oop terminator or the rho terminator.

[044] The term "Cyanobacterium sp." refers to a member of the genus Cyanobacterium, as, for example, characterized by Rippka et al., 1983. Ann. Microbiol. (Inst. Pasteur) 134B: 32.

[045] The term "BG- 1 l " or "BG 1 1 " refers to a growth media used for growing cyanobacterial species as disclosed in Rippka, R. et al., "Generic Assignments, Strain Histories and Properties of

Pure Cultures of Cyanobacteria." (1979) J. Gen. Microbiol. 1 1 1 : 1 -61 .

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

[047] The terms "host cell" and "recombinant host cell" 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 prokaryotic cell, such as a cyanobacterial cell. The term recombinant host cell is intended to include a cell that has already been engineered to have desirable properties and is suitable for further enhancement using the compositions and methods disclosed herein.

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

[049] 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-selection markers are well known in the art and include mazF and sacB, for example.

[050] 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 inhibiting 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 condition and in a second condition exhibits negative selection by converting a compound to a compound toxic to the host cell.

[051] The term "screenable marker" 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 screenable marker is a gene that produces a colorometric difference between host cells containing the screenable marker and host cells that do not contain a functional screenable marker. An example of a screenable marker is the lacZ gene that can be used for blue/white screening when grown in the presence of X-gal and IPTG.

[052] The term "cassette" or "gene cassette" as used herein, refers to a manipulatable fragment of DN A carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction sites.

[053] The term "shuttle vector" refers to a vector, such as a plasmid, which can propagate in different host species. For example, a shuttle vector with a cyanobacterial origin of replication can be replicated and propagated in different cyanobacterial genera such as Cyanobacterium, Synechococcus, 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 performed in E. coli and the recombinant plasmid can be expressed/maintained in cyanobacterial 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 plasmid which contains at least two different origins of replication for the appropriate organism.

[054] 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.

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

[056] 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 recombinant 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.

[057] The nucleic acids disclosed herein may be modified and/or contain non-natural nucleotide bases.

[058] 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 amino acids, but do not affect the functional properties of the protein encoded by the DNA sequences. In certain embodiments, changes in one or more nucleotide bases do not change the encoded amino acid. Substantially similar also refers 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.

[059] As used herein, in certain embodiments, homologous nucleic acid sequences have about 50%, 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 identity to nucleic acid sequences disclosed herein.

[060] Similarly, homologous amino acid sequences can have a sequence identity that is at least, for example, 50%, 60%, 70%, 80% 90%, 95%, 99%, or 99.5% or more identical to the amino acid sequences disclosed herein.

[061] The percentage of 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, which can also be used in embodiments of this disclosure.

[062] Such searches can be performed using the algorithm of arlin and Altschul (1990, Proceedings of the National Academy of Sciences U.S.A. 87:2,264-2,268), modified as in Karlin and Altschul (1993, Proceedings of the National Academy of Sciences U.S.A. 90: 5,873-5,877). Such an algorithm is incorporated in the BLASTN and BLASTX programs of Altschul et al. (1990, Journal of Molecular Biology 215:403-410). Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the BLASTN program. BLAST protein searches are performed with the BLASTX program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997, Nucleic Acids Research, 25:3,389-3,402).

[063] "Recombinant" refers to polynucleotides synthesized or otherwise manipulated in vitro or in vivo ("recombinant polynucleotides") and to methods of using recombinant 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.

[064] 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 strain 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 functional 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.

[065] 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). [066] 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 cyanobacteria are known in the art (Wirth, 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 al, supra).

[067] 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 or non- 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.

[068] 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.

[069] 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 (PCR) or from treatment of a circular plasmid with a restriction enzyme.

[070] 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"). [071] 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. In 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.

[072] 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).

[073] The term "non-natural ly occurring microbe" as used herein included genetically engineered microbes, and also includes microbes demonstrating non-natural characteristics as a result of being exposed to mutagenic conditions.

[074] 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 regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[075] 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 genetic material such as a plasmid. 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 that 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 nucleic 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.

[076] 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 reference nucleic acid. Such a nucleic acid fragment according to the disclosure may be included in a larger polynucleotide of which it is a constituent. 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.

[077] 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.

[078] 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 starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[079] 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.

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

[081] 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 mR A 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.

[082] An "expression cassette" or "construct" refers to a series of polynucleotide elements 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, polyadenylation signals, and enhancer elements.

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

[084] 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 synonymously with codon optimization. Similarly, the term "codon improved gene" refers to a gene that has had its nucleic acid sequence modified so that it has higher expression in an organism.

[085] The term "codon adaptation index" or "CAI" refers to a method of analyzing codon usage bias of an organism. One can alter the CAI of a heterologous gene (by altering the nucleic acid sequence) to result in more efficient translation that leads to a higher level of the protein encoded by the gene. A suitable CAI table for ABICyanol is found in Table 2.

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

[087] The term "selectable marker" or "marker" means an identifying factor, usually an antibiotic, chemical resistance gene, or counter-selection gene, that is able 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. [088] A "heterologous protein" refers to a protein not naturally produced in the cell and that is produced from a heterologous gene.

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

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

[091] 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 of an organism to tolerate environmental conditions is refereed to as "hardiness".

[092] 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.

[093] 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 chromatagraphy. 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.

[094] 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, in 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 ribosomal 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., Totowa, N.J.; 2004; ISBN 1-59259-750-5; Prokaryotic Gene Expression (1999) Baumberg, S., Oxford University Press, ISBN 0199636036; The Biomedical Engineering Handbook (2000) Bronzino, J. D., Springer, ISBN 354066808X, all of which are incorporated by reference.

[095] 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.

[096] 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.

[097] 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.

[098] 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 endogenous 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.

[099] 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 synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.

[0100] The term "polymerase chain reaction," also termed "PCR," refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR 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 PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR 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.

[0101] The term "three-component cassette" as used herein refers to three genes that are part of a polynucleotide sequence having a restriction enzyme target sequence on both a 5' and 3' end of the three-component cassette. In an embodiment, the three-component cassette contains a selection marker, a counter-selection marker, and a site-specific recombinase. In another embodiment, the selection marker is an antibiotic resistance gene.

[0102] The term "photobioreactor" or "PBR" refers to a growing container for the cyanobacterial cultures. A "vPBR" means a vertical photobioreactor. An outdoor, commercial scale vPBR can have a volume of up to 100 liters or more.

[0103] An "LvPBR" or "lab-scale vertical photobioreactor" refers to a lab scale vertical photobioreactor; typically this system holds a culture volume of about 1.2 liters. As used herein, the term "mLvPBR" or "mini lab-scale vertical photobioreactor" refers to a laboratory-sized vertical photobioreactor system; typically this system holds about 0.4 liters of culture.

[0104] Database entry numbers as used herein may be from the NCBI database (National Center for Biotechnology Information; http://www.ncbi.nlm.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).

[0105] 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

[0106] In 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, ABICyanol 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 presence of contaminants including chemical and microbial contaminants. ABICyanol 's ability to tolerate wide-ranging environmental parameters makes it ideally suited to growth in cyanobacterial culture systems. ABICyanol can be genetically enhanced 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.

[0107] Methods for cultivation of cyanobacteria in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g., websites associated with ATCC). Any of these methods or media maybe used to culture ABICyanol or derivatives thereof. A number of known recipes for cyanobacterial growth medium can be used. In an embodiment, BGl 1 medium is used for growing ABICyanol , see Stanier, R.Y., et al., Bacteriol. Rev. 1971 , 35: 171 -205, which is hereby incorporated by reference.

[0108] 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 (mBGl 1) contains about 35 practical salinity units (psu), see Unesco, The Practical Salinity Scale 1978 and the International Equation of State of Seawater 1980. Tech. Pap. Mar. Sci., 1981 , 36: 25 which is hereby incorporated by reference.

ABICyanol endogenous plasmids

[0109] ABICyanol contains at least three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate ABICyanol from other Cyanobacterium species.

[0110] One endogenous plasmid is 6828 base pairs (SEQ ID NO: 1 ). The 6828 bp endogenous plasmid 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, including descriptors of various portions of the polynucleotide sequence of p6.8 is depicted in FIG. 2.

[0111] Although the p6.8 plasmid was used for most of the transformation and evaluation work shown herein, one can also utilize the other two endogenous plasmids, exogenously derived plasmids, or even the chromosomal DNA, for the purpose of this invention.

[0112] The ABICyanol endogenous plasmid p6.8 contains six open reading frames ORF 1 (SEQ ID NO: 2), ORF 2 (SEQ ID NO: 3), ORF 3 (SEQ ID NO: 4), ORF 4 (SEQ ID NO: 5), ORF 5 (SEQ ID NO: 6), and ORF 6 (SEQ ID NO: 7). With respect to the nucleotide sequence of SEQ ID NO: 1 of p6.8, ORF 1 consists of nucleotides 594 to 3779, ORF 2 consists of nucleotides 3815 to 4000, ORF 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.

[0113] 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 endogenous p6.8.

[0114] As disclosed herein, plasmid 6.8 has been modified in vivo and in vitro for use as an extrachromosomal plasmid vector containing a three-component cassette and genes of interest for the production of compounds of interest.

[0115] In an embodiment, a modified endogenous vector derived from p6.8 from ABICyanol was developed. The modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyanol itself.

[0116] In certain embodiments, the present invention includes the p6.8 plasmid and modified vectors comprising sequences of the p6.8 plasmid. In 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 ABICyanol .

[0117] In certain embodiments, a gene coding for a replication initiation factor that binds to the origin of replication can either be present on the modified endogenous vector or can be present in the chromosomes or other extrachromosomal 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 .

[0118] 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% identity or can be identical to the nucleotides 3375 to 3408 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 1).

[0119] In an embodiment, the sequence of the gene coding for the replication initiation factor has at least 80%, 90%, and 95% identity or is identical to nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 1). In an embodiment, the gene coding for the replication initiation factor codes for a protein having at least 80%, 90%, and 95% sequence identity 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 ABlCyanol . 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 ABlCyanol .

[0120] 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% identity 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 ABlCyanol .

[0121] 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 ABlCyanol 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 6.8 kb plasmid. Such vectors may also be able to efficiently produce heterologous proteins and other compounds of interest in cyanobacterial cultures.

[0122] In another embodiment, modifications starting with the backbone of the 6.8 kb endogenous plasmid from ABlCyanol are performed individually or together to increase transformation efficiency, increase the replication rate or plasmid copy number 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 cyanobacterial cell. In an embodiment, the invention includes cyanobacteria, e.g.

ABlCyanol , comprising a modified p6.8 plasmid having any of these improved characteristics.

[0123] In yet another embodiment, codon improvement of 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 at least one recombinant gene to the codon usage in Cyanobacterium sp., in particular ABlCyano l . In an embodiment, the G and/or C wobble bases in the codons for the amino acids in at least one recombinant gene can be replaced by A and/or T because the GC content of the genome of ABlCyanol is relatively low at about 36%. [0124] In an embodiment, only 2% to 6% or 1% to 10% of the codons of variants of recombinant genes are codon improved. In another embodiment, highly codon improved variants 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 ABICyanol

[0125] Methods for producing a genetically enhanced, non-naturally occurring Cyanobacterium sp. and ABICyanol 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 transformation of the host cell by an extrachromosomal plasmid. In an embodiment, the extrachromosomal 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 into the genome of the host cell by homologous recombination.

[0126] In an embodiment, a recombinant nucleic acid sequence can be provided as part of an extrachromosomal plasmid containing cyanobacterial nucleic acid sequences in order to increase the likelihood of success for the transformation.

[0127] In another embodiment, the method for producing a genetically enhanced

Cyanobacterium sp. host cell uses an extrachromosomal 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 extrachromosomal plasmid derived from the 6.8 kb endogenous plasmid of ABICyanol .

[0128] 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. In 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.

[0129] In an embodiment, the ABICyanol 6.8 kb endogenous plasmid is used as a backbone for a plasmid vector used for transformation of Cyanobacterium 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.

[0130] In another embodiment, the p6.8 derived plasmid vector also 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

Enterobacteriaceae, in particular E. coli, and which also enables conjugation with, for example, an E. coli donor strain and Cyanobacterium sp., in particular ABICyanol as a recipient strain. Such an plasmid vector can be used for triparental mating wherein a conjugative 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 .

[0131] 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 improved kanamycin or gentamycin resistance genes. In an embodiment, the shuttle vector is constructed based on a modular basis 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, genes conferring resistance to ampicillin, chloramphenicol, spectinomycin 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.

[0132] 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 cyanobacterial host cell. In an embodiment, the recombinant plasmids are fully segregated from the non-recombinant plasmids in the 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 which do not contain a three-component cassette by the dual expression of the site-specific recombinase and the counter-selection marker in a medium containing a counter- selection compound. Exemplary methods suitable for transformation of cyanobacteria 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; Zang, 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; Steinbrenner, 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

(Kjaerulff, et al. (1994) Photosynth. Res. 41 : 277-283; Iwai, et al. (2004) Plant Cell Physiol. 45: 171-5; Ravindran, et al. (2006) J. Microbiol. Methods 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 pretreatment with any of poly(amidoamine) dendrimers (Pasupathy, et al. (2008) Biotechnol. J. 3: 1078-82), polyethylene glycol (Ohnuma, et al. (2008) Plant Cell Physiol. 49: 1 17-120), cationic lipids (Muradawa, et al. (2008) J. Biosci. Bioeng. 105: 77-80), dextran, calcium phosphate, or calcium chloride (Mendez- Alvarez, et al. (1994) J. Bacteriol. 176: 7395-7397), optionally after treatment of the cells with cell wall-degrading enzymes (Perrone, et al. (1998) Mol. Biol. Cell 9: 3351-3365). Biolistic methods (see, for example, Ramesh, et al. (2004) Methods Mol. Biol. 274: 355-307; Doestch, et al. (2001) Curr. Genet. 39: 49-60; all of which are incorporated herein by reference in their entireties.

Knockout of essential genes in Cyanobacteria

[0133] There are many essential genes, as well as conditionally essential genes, in the

cyanobacterial genome, in which their complete removal would be likely to cause cell death. In an embodiment, these essential genes are removed from their original location on the

chromosomal DNA and are placed on an extrachromosomal plasmid downstream of a gene of interest (such as the ethanol production gene pdc). As shown in Example 3, the knockout of several different essential (or conditionally essential) genes of ABICyanol was conducted by use of double homologous recombination. Integrative plasmids were prepared to target (and delete) a specific gene. The integrative plasmids contained long flanking regions homologous to the upstream or downstream region of the target DNA. An antibiotic resistance marker was used between the flanking regions. Upon transformation, cells could be selected with gentamycin to select for cells having successfully gone through the double homologous recombination (and thus "knockouts" for the target gene. After multiple selection rounds to be sure that all copies of the chromosome contained the specific knockout, the cyanobacterial cells were ready to test. Using this method, as shown in Example 3, gene knockouts of essential genes narB, nirA, ureC and cobK were performed in ABICyanol . Additionally, other suitable means of deleting target essential genes that are known in the art can be used.

Transformation of ABICyanol by conjugation

[0134] In an embodiment, transformation of ABICyanol with exogenous polynucleotides is performed by the conjugation technique as described in Elhai and Wolk, 1988 by using a helper plasmid pRL528.

Transformation of other cyanobacteria with p6.8 derived plasmids

[0135] In another embodiment, the modified plasmid vector based on the endogenous 6.8 kb plasmid backbone from ABICyano l , in addition to being useful for transformation to other cyanobacterial and Cyanobacterium sp. host cells, is used to transform other microbes including Eubacteria, Archaea and Eukaryotes. As an example, a shuttle vector containing the 6.8 kb endogenous plasmid from ABICyanol with a kanamycin resistance cassette ( m ^R ) and the oriVT for replication in E. coli is transformed into Synechococcus PCC 7002 by natural uptake.

[0136] In another embodiment, a modified extrachromosomal 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

extrachromosomal plasmids containing three-component cassettes disclosed herein include, but are not limited to, Synechocystis, Synechococcus, Cyanobacterium, Acaryochloris, Anabaena, Thermosynechococcus, Chamaesiphon, Chroococcus, Cyanobium, Dactylococcopsis,

Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron,

Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria,

Xenococcus, Arthrospira, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Cyanodictyon, Aphanocapsa, Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaenopsis, Aphanizomenon, Calothrix, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Chlorogloeopsis, Fischerella, Geitleria, Nostochopsis, Iyengariella,

Stigonema, Rivularia, Scytonema, Tolypothrix, Cyanothece, Phormidium, Adrianema, and the like.

Promoters

[0137] 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, environmental stress, metal ion concentration, and light exposure. Additional promoters, both constitutive and inducible, are well-known in the art.

[0138] In an embodiment, recombinant genes are placed under the transcriptional control (operably linked) of one or more promoters selected from exogenous or endogenous PrbcLS, PmcA,

PnblA, PisiA, Ppetl, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpBl, PhliB, PggpS, PpsbA2, PpsaA,

PnirA, PnarB, PnrtA, PisiB, PnrsB, PirtA, PmrgA, Ppsts, and Pcrhc In an embodiment, synthetic promoters are used.

[0139] 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.

[0140] Exemplary promoters for expression in cyanobacteria include, but are not limited to, Ppeu,

PpsbD, PnblA, PrpoA, PisiB, PrbcLS, PntcA, PnblA, PisiA, Ppetl, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG,

PhspA, PcipBi , PhliB, PggpS, P _P sbA2, PpsaA, PnirA, PnarB, PnrtA, Pcrhc, and additional metal ion inducible promoters and the like. Examples of constitutive promoters that can be used include, but are not limited to, PrbcL, PrnpA, PrpsL, PrpoA, PpsaA, PpsbA2, PpsbD, PcpcB. Additional details of these promoters can be found, for example, in PCT/EP2009/060526, which is herein incorporated by reference in its entirety. [0141] 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 binding 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 PnirA, and can be PnirA from ABICyanol , which is repressed by ammonium and induced by nitrite. This promoter may contain nucleotide changes in either one of the ribosomal binding site, the TATA box, the operator, and the 5'-UTR

(untranslated region).

[0142] In certain embodiments, the present invention includes a polynucleotide comprising or consisting of any of the promoter sequences described herein, or variants thereof, including those having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the reference promoter sequence.

[0143] 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.

[0144] In an embodiment, a promoter disclosed herein can be an inducible promoter selected from the group consisting of PnirA, PnrtA, and PnarB from ABICyanol , for example. In another embodiment, a promoter is a constitutive promoter selected from the group consisting of PrpsL, PrbcL, and PcpcB which can all be endogenous promoters of ABICyanol , for example.

[0145] 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. In another embodiment, the first and second recombinant genes are controlled by different first and second promoters.

[0146] In an embodiment, the recombinant gene under control of the promoter is induced if a sufficiently high culture density of Cyanobacterium 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 be 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 then may be under the control of constitutive promoters such as PrbcL, PcpcB or PrpsL, all from ABICyanol , for example.

[0147] 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 certain embodiments, the present invention includes ethanologenic cassettes. In an embodiment for an ethanologenic 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. 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, PnirA, PnirA Variants PnirA*2, PnirA*3, PnirA*4, PmntC, PsmtA (Porf3126), Porf221 ,

Porf222, Porf223, Porf3 i6, Porfi232, and Porf34 ₆ i and the second constitutive promoter can be selected from, for example, PrpsL, PrpsL*4, Prbc*(optRBS), and PcpcB.

[0148] In an embodiment, a non-naturally occurring ABICyanol host cell comprising any of the ethanologenic 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 inducible promoter may be used.

[0149] 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.

[0150] 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 ABlCyanol

[0151] In an embodiment, promoters used herein can be endogenous to ABlCyanol . In another embodiment endogenous promoters from ABlCyanol can be modified in order to increase or decrease efficiency and/or promoter strength. In an embodiment, and as described in US

2014/0178958, 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, narB, nrtA, copA, mntC, smtA, rbcL, rpsL, IrtA, mrgA, nblA, ggpS, petJ, ppsA, rnpA, and pstS from ABICyano 1.

[0152] In an embodiment, metal-inducible 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 ABlCyanol include, but are not limited to those previously disclosed in US 2014/0178958, such as promoters for orf0128, orfl486, orf3164, orf3293, orf3621 , orf3635, orf3858, orfl071 , orfl 072, orfl074, orfl 075, orfl 542, orfl 823, orfl 824, orf3126, orf3389, orf0221 , orf0222, orf0223, orf0316, orf3232, orf3461, and orf3749.

[0153] Several suitable promoters which are endogenous to Cyanobacterium sp. ABlCyanol are listed in Table 1 , below:

Table 1: Cyanobacterium sp. ABlCyanol endogenous promoter sequences

PnrtA TATTATTTI CGTTTATATGCAGATTTAGAATAAACAAAATTCATTTA 18 CTGCAAATTTTCAAAAAAATGTGACTAAACATACAAAATAAAGAAA AAATAAAGTTTTAAATTTATGTACATCAAACTTAAGAAATGTTTAAA TTACTTAGAAATTTATAGTTC

Porl3461 TTTA TATA TAAACTCGAATAAAATTATCAATATAAAGTCAA ACT ATA 19 (petJ) TCTATCCTATTTTAACTGCTATTGGTAAGTCCCTTAATTAGTGTTGGG

GTGAATAGATTTTAAAAGGGCAAACCCCCCTTTATCCTCCCTCGAGA

GGGGGGAGGGCAAAAGGCAAGGGGCAAGGGAAAAATTAAGAATTA

AGAATTAAAAACTCCGAACACCTGTAGGGGCGAATAGCCATTCGCT

TCCCCTCATCCCCCCATCTCCCCAACACCCTAAGCCCCTACTCGTTAC

TCATTTATTTACATCATTTATTTACATCATTAAGAAAAGTAACAAATT

TTGACAAGTAGTCTTTTGACAGGAAAAAGCAAATTCTCGAAGATGA

AAACAATAGAAAAAAATTCAATCTTACAGTAACGATGAAAAAACTT

TTAGGCTTAATT

PnarB TGTCTCAAAAAGACAGGTTTTTTTTATGAAAGTAATAAGAAATAAGT 20

AGAAGTGAGGAGTTGGAAAGATAGGATTAAGAATTAGGAGTTAACT

ATTTTCATrrCTTTATTCTTCCATTGCCCATTGAGAAATCATATCTAAA

ATCAGCAACGCCAAATTTTAGATGCAAAATAACCATAAATAAAATG

CAGAAAAAAGAATACTTTAGATCTTCCGTATCAGAAGATACATTTCT

TAACAAAATCTGGTGACAAGATTAAACACACGAAATCCGAGGTTTT

ATATATTGATTAGTCCTAG

Porfl071 ATTCTGTGAATTGATTAGATTTGAGGTTTTTTAAGAGGTTGATTACCT 21 (PmntC) TGCCTCCAAAAAAATCATAACACACTAATGCTCTATATGAAAGGGCT

TTAGACCCATAGGTTTTTGAGAAAAAAACTTGCTAACTCTCGGACAA

TGTCAGCATAACTAAAGTCAATTCTTTTCGTACTTTATAATTGTCTAT

AATTTAATATACAACTGTTCTGAAACTAGTTTTTCTCTACATTCCTTA

GTTTTATCTGAGTAAGGTTGCTTGTAACTTAACTTCGGTTGGGCCTAA

AAATATCCGATTAGGAGCAGGTGTCAGACTTTAATTAATTATTAATT

ATTAATTGCTTATTGCCAACCCTCGGCGACACCACTTTTTCATCAGCC

CCAGATAAAGATTGATGTTTTAGTTTTGTTTCTT1TTATCCCCTAATT

CAACTAATACAAGTAAAACTAAGGTTGTTTATCAAAAATGATGGTTG

ATGTTTGGGTAAA Π 1 AAGATATTATGAAAAGAAAATGAATAAAA

AATGAAAAATCTTT

Porf0221 GAATATCTCATCCTTAGCTTCTACTTATACCTTCAGCATAGTTAAAAA 22

TCATCCCTTTATTGATGGTAATAAAAGAACAGGTTTTATTAGTGGAG

TAACCTTTTTAATGCTCAATGGTTCTCACTTTACTGCTTCTGAAGTGG

AAGTAGTACATATCATCCAAACCTTAGCTAGTGGCAGAATTACCGAG

GAAGAATTACAACAATGGTTCGTAAGGAAAAGTAAGCAGATGAATA

ATTAAAGCATCATTTCATCCTCATTTCATATTCTCCTGTCACCATGGT ATGGAAGATTAGGTAAAAATGAGGAAAAAGTTTATT

Porfl)223 ATACATGGTTGGTTCACTGACTTTTACCCCAGTTTTCTCTTTGAACAA 23

TTGGCATAACTCTGAAAAAATCAGATCGGGCTTTTGTTGAATTATTT

GTTC A ATC A A AGC A A A ACCGTG ATTGTCTA I ^' ll ^" I CTTTTTTTTCCC AC

CACTCATAGATAAAAATTTATCCCGAACTCAGGTTATATTAAGTTCG

GATGATCACTTAAGATAATTGATCAGATTGGTTAAGATAGAGAAAA

ATTCTTTTTCATAGTGATTTCATAATTGATAGTTACAATAACGATTAT

TATTTAGTAAAAAGATTTTCAAATC

Porfl)316 TGGTCAAGTTACTATATGTTTAGAAACAACAAAAAAAGAAGTCATTA 24

TAAAAATAATTGATACAGGAATTGGCATTAATAAAGAAGAACAAAA

ATTAATTTTTAATCGTTTTTATCGAATCAATAAAGCAAGAAATAGAG

AGAAAGGCAGTTGCGGATTAGGTTTAGCTATTGCAAATGCGATCGCG

CTTAATCATGGTGGTAGAATAATTTTAGAAAGTCAAGAAAATCAAG

GCAGTATTTTTACCGTTTATTTACCGAAAATCATTTCATCCTAATTTC

ATATTCTTTTGACAGAATCAAAGGTAAAGATAAAAAGAGAGAAACA

GTC

Porf0128 CCTCAACTACAAGTTCTTTTATATATTACTTTAACCTGAGTTTTGGAT 25

AAGCTGAAAGCATTATTTTCTCGTAGTCAGAAAACCTTATAGCTTCT

TAGAAATAACGATAAAATTACCTTAATCCGAACTGACGTTAAATATA

TTCACCCCTATCACCCCAAAACCCTAAGCCCCTACTTCCCCCTTTCCC

TTCATCACCTCATCCCCCCATCCCCTAACACTTAACCTTATTCTTTAT

TCTTAAACCGAACTGAGGTGAAGTTGCAGAATACCCATGGGGGGTT

ACAGCATTGTAGAAAAATAAATATTCTTTCATTATTAAGGTTGTTTG

GTAAAAATATGTGAAAACCCTAATAATT

Porfl486 GGGGACAGACATATTTTTATCATAATGGTAAATTCATAATAATTTTA 26

GACTTTTTTTTGCAAAAATTAATCTCACTCTCTTCTTTCCCTATCTCCC

ATTGTTTCTTATATCCCAATGCCCCAATACCCAAAGCTCAGAAAATA

GGTATTAGCGAAGAGGTGTTGATCCCCTCCCCTAGCAAAATATACTC

CTATATAGTAAAGTGAGAAAGTGAAGAAATAAGATCAAGTTCGCAA

TTT

Porf3293 TTGACGATTGTATTGACTTACGCCAAATGGCTTACCCTCATAGTGAA 27

TAGTTGATAATTAAGAATTAAAAATCCCGTTCACGACAGAAGGGAG TGTAAGAGCCTTCGGTGCGAACTCTCATCTTCCCTGAAACCTGACAC CTGAAACCTGACACCTGAAACCTGACACCTCATCTCCCTAATCCCCT AATTTTAATGAAAAAATACCCTGAGTGGGCATTGAAAAAAAAGAAA AGTTGTTCGACTATGAAATAAGAATTCTGCACTTCGTGAGAAAAAAG GAAATGAAAT

Porfi621 CTATTTAACTAGGAAAAGGTAAAGTTAAAAGGACAAGGGTAAATAA 28

TTAAAAATTAAGAATTAAGAACTTCTAACTCTCATTACTCATTACTTA

TTTCCTCCTCTCACCCCTTCTCCTGATCACCTCTTCTCCTCAATACTCG

GAACTCATTTCCCCATGGTGTGACACTCAAATCAAAAGTCTGTTATT

GACTTTCAGATGAAATATTACTATGATAACAATATCCCCCCTATGGG

TATATAAAAATATGAGCGATATTAGTTAAAAATCAAATTTGGATTTT

TTTTCTGAAAATATTTTAAGATTAAGTAAAGATAAGTAAAGAAATTA

TAAGCAATTTTGTTAAATCATACC

Porf3635 CTCACACTGAAAATATTGCCACAAGAAATAAAGATCAAGCAATAAT 29

CCTGACTAAAAAGGAATAAAGTAATTATCCTTTTCCTGATATGTTAT

CTGACTTGTTGTTTCTTAGTCATGTTCCTTCCATTTTTATTTTTGTTTTT

ATCATTTTTATTACAAAAATTTCTTAATAGGGCTAAAGCATTTAGTTA

GTTTTTTAGCTCTCAACAAGTTGACTAATCAATATAATGCCCTAAGTT

AATTTGCCCTTGGTTTGACGGAGGATATTGGAAAAAAGAAACTTCTC

GTTGTATTTCACAGGGAAAAGGGGGAAATTTTATTAATAACTAAACA

ATAGAAAATAATTATTTATTTATATTATTTTGTGAACAAATGTTCAAG

TAGATATAAGCCTAAAGGTCTGAAATTATTATTAGACAATCAATTGA TTCAGAGGTAATAGTTTTTTACTTAAAAATATTTTTTCAAAATTATCC CCTATTTGGGTATTGAAAAATAAATAAATTCAAGTAATAATATACAG AATAAAGGAAAATCTAATCTTAAAAATTTTGTGTGTGAGGAATTGAA A

PorB164 CAAATCACGAGAATTTATGTAGGGACTATTTTGGGTTGACGGTGGAG 30

AGTATGTCGCCCTTGAATTATGACCCGAAGATGAAGATGTCGGGGA

GGTGGAAGGACGGTCTTTAAGAGGTTTAACATCAAAGTTGGTCATAA

TCTCTGTCCCTGTTTGATAACTACTATTTAATTTTGAGTTGTTTTAGGT

ACATCAAAATACCCAAATCCTTACTCTCCCCTCAATATACAACAAAA

AAAACTTTTTGATTCACTTTAGTCATAAAAATTAGAATTTATCTACCG

AAATATTACATAAATGTAATGTATATATTTTCTGATTTATTCCGTGTG

AGCCATGATTCATAATTTATAATTCATAATTTCTAAATATGCCCCTAC

AATGGATATAGAATGTCATTTTAATTATAGGTATCATAATCGTGGTA

GTTACTCCGGAAAAAACTATTGAATCAAATTCAGTCTCACCTGCTAC

AGATAGAGTAGCCGTTATTCTT

Porfl072 CTACAGGGGCAAGATTTGGCGGAAATCTATATGTGGATTCTCTTTCA 31

AGTGAAGAAGGTGCAGTGCCGACTTATCTGGACTTATTAGAATACGA

TATTCGCACTATTACTAATGGTTTGTTAGCAGGAGTGAACAATTAAA

AATTTTTTCCTAATTGACGAATAAAAAATCAATGTCAACTAATAGTT

AACAATACTCTCTGAAAACCAAAAATTGTCAACCAAAACATAACAT AATTTTTACCCAAAAACCTCATTTATAAACTTTAAGGATAAAATCAA TG

ΡΟΓΠ074 GGGATTAGAGAGTTCAAAGTTAGGAATGAGGTGTCAGGTTTTAGGTT 32

TCAGGTTTAGGGGAGCAATGAGAAAGAGGTTTCAGGTTTCAGGTGTC

AGGTTGCAGGTGTCACAGGTGATGAGGGGATGGGGGATGAGGGGGA

AACAAGTAAGTAATAAGTGTTCGGAGTTTTTAATTCTTAATTCTTAAT

TTTTCCTTTGCCTCTTGCCTTTTGCCTTGTCTTAATTACTAATTTCTAA

TTAAAATGATTGTGTTTTCTAGTTTAGTCTCATGGTTACTTGAACCCT

TACAGCATAGTTTT

Porf01075 TTACAAACGGCGGGAATTATTATGGTAGTAGCGATGTTAGTAACCCC 33

GGGTGCGATCGCATATTTACTTACAGATCGTTTTGATCAAATGTTAA

TCTTATCAATAGTTAGTAGTGTTCTATCTTGTGTTTTAGGCACTTATT

TAAGTTATCATTTTGATGTTTCTACGGGGGGAAGTATTGTCGTTTTAA

TGACCATAATTTJTATTTTAGCGATGATTTTTGCTCCTAAATATGGCA

TCATCAATCAAAATACCAAAATATATTCTGCTTAACTTGTTTACTGAT

ACTTCAAATAATCATATAACCTATCTTCCGAGTTAAAAATAATGGAT

ATTATCCAACTGAGGTCGAGAATAGAGTTTCTTTTTTGATAGAATTTT

TTTACACCAGTTATTCATTACTATCATGGGATAAT

ΡΟΓΠ542 TAATATAGTGATTATTATAAATGCAATGTGAATCAAACCTATATTTT 34

ACCGTACATTGACCATGGAACTTAATTTGAGGTGATTAGTAGAGGGT

GCGATCGCCCTATTTGTCAAATAATAAAGATAACATTTGACATTGCT

GATTGAAGACATAAAACACAGAAAAAATCAGGTAAAAATATAAAGC

TAAAGTCTAAATATGGTTTACTTTTGCCTTCGACTTACAACAAAAAA

TCATAGCTAGAATCACCAACGCCTAATATTTTATTTAGCTGAAATTTT

GGGATGAACTTTTTGTAAAAATCGGGGGTCTAAAAATATAGCAACC

ACGATATTAAATAACTGAGTGATTATTTTAATCTATTGGGGGCTTATT

AACTAAATACTTGCATTTTTATGGAGGGTTTTAATT

ΡΟΓΠ823 AAAGATTATTTTCTACAGAAGCAACCCTTTCATCTTCCGAATTTTCAG 35

G A ATTTCCTGC 1 "I ^" 1 ^" 1 GTTTCTG A ATATT AGC AT AGGCGGCTTTTGCCC

ACTCTAAAGAAGGTTGAGACTGAATTTCTGAGGTTTCAGAAGGAGC

ATTAGATTGTTTATCTTCAACAACAGGAGGTTTTTGTTCAATATTTTC

CTTATrrCTCTTTTTTACGGCGAAACCAATTAAACATAATGATTGTGCA

TAAATATTCGTTAATATATTGTAACCCTAGAAAGGAATCGGTTTCAG

GTTTATCCCCAGAGAATGTGAACCTTTACAGAAAGTAAAAAGTCTAA

AATCGTAGCAACAATAAATCACAGAAATTGAG

Porf0222 GCGATTATCAACCACGAAAACATACAATTATTATCAAACCTGCTGAG 36

AAATTATCCACAGAAATAGATGTTTCTGCGAAGGGAAAATGGGCTTT TCATTGCCATTTAATGTATCACATGGATGTGGGAATGTTTCGGACTA

TTAATGTTATTTCCTAAAAAATAATAGTATTAAAGCCTAAAATTTTTA

TAAAAAAATTCATGTCTTTTATTAGGGTGAGCATTCTTCCTTTATGTC

TCCTTATTTTACCTCTTTAGAGGTAACTACAAACTTAATCAAAAAATT

TAGATAATTAATTATATCA

Porf3232 CATCTTTACTTTTGACTAACATTTCATAGGTATCATGACGAAAATTTT 37

TTAGTCTGTTATATTTGTTCATGTAGAGAGATTTTAATTTGTGATTAT

TTTATTTTCTCTCTATTTTTCTTTTTTGTCTTGTCCTTCCTCATTTTTCT

CTACATTTAGTCTAAACTACAGCTCTTTAATCTTCAGTTTCTCTTTCCT

CCTCTTCCTCATCAAGGTAATCATCCCAATTAATATCTTCTTCTTGTT

CTAATTTGGGTTGAGATTGTTGTTTATCAATCATATTTCATACTCCTA

AAACTTTCTTACTTATTTATCAGTTACTTTTTACCCATTTATGCAATA

GTGTAGAAATTTTTTTCGATCGAGTTAATTAATTTTTATTTCAACCAT

ATCTAAATAATTCTTGATGGACATTCTAGTTAACTAGAAGGTTTAAG

CTAAAAATAATTATTGATATTGCCTTCGGTATAACTAACTATATCCA

GAGAAAAAG

Porf3749 CTCAAGAGATAGTTAAAAAACAAATAGCTTTAGTCTATCAATTAATC 38

GAATTATTTTTACAAACAAATTTTCATAAACCCATAGAACTAGAGGA

GGAAGTTATTTATGTTTAAAAATCTAAAAGAGTTTTATATTCCCCTA

AAACCCCCTTAGTAAGAGTGACTTTTTTCATCATTTGCCTGTAAATTC

TCCTCTTTTAATAAGAGAGCTAGGGTGTTTTAAAAGAGGATTTTATT

GCTTTCCAATTCTAACTACTTCAAAAACTTATTTTATACTCAATAATT

TATTAATCAAGAGGAAATTACC

PrbcL TCGAGCGCTCGTTCCGCAAAGCGGTACGGAGTTAGTTAGGGGCTAAT 39 (Prbc) GGGCATTCTCCCGTACAGGAAAGAGTTAGAAGTTATTAATTATCAAC

AAT CTCCTTTGCCTAGTGCATCGTTACCTTTTTAATTAAAACATAAG

GAAAACTAATAATCGTAATAATTTAACCTCAAAGTGTAAAGAAATGT

GAAATTCTGACTTTTATAACGTTAAAGAGGGAAAAATTAGCAGTTTA

AAATACCTAGAGAATAGTCTGGGGTAAGCATAGAGAATTAGATTAG

TTAAGTTAATCAAATTCAGAAAAAATAATAATCGTAAATAGTTAATC

TGGGTGTATAGAAAATGATCCCCTTCATGATAAGATTTAAACTCGAA

AAGCAAAAGCCAAAAAACTAACTTCCATTAAAAGAAGTTGTTACAT

ATA ACGCTATAAAGAAAATTTA TATA TTTGGAGGATACCAAC

PrnpA AATAGTTGATAATTACTCGTTACTCATTACTCACTTAAACCTGCCACC 40

TGATACCTGCCACCTCTCCCCCCATCACCTCATCCCCTCAACATTCCG

AACCCCTTGACACTTTGAACTAAAATTGTATTAAAGTGCAAATCTGG

ACGGGGTTAACCAGTGTGACTTATAATAGTAAACGCTGTTTTTTATA

ATAAATAAGCTAAATATTTAAAAACTATGAGTAAATATACACTAAAT

GGTACTAGACGTAAGCAGAAAAGAACCTCCGGTTTCCGCGCCCGTA TGAGAACCAAAAATGGTAGAAAAGTAATTCAAGCTCGTCGTAATAA

GGGTAGAAAAAGATTAGCAGTATAAAATTACTGTTAAATAAGGAAG

CTAAGTTTAGCATTTITAAGTTTGATATTACTAATCATTAAATTTACTG

TGAAATATAGGTGGGACTACCATCAAAGCATCGACTGAAACGGCGT

TTAAATTTCCAATCTGTTTATCAACAGGGTATTCGCCGCTCTAGTCGT

TATTTTATTGTCCGAGGGTTACGG

PrpsL CTCCGCTTAAAAAATTTCATTTTTCGATCAAAAAAGACAAATTATTA 41

CTAATTAGCTCATGGCAATAAATAATCAGTAGTAATCTGTTTTCACA

TTTTATTGTTAATTTTTATTATTGCTAATATCAACCTTTTCTACTTCTG

CTTAATATTTTATTTATGCTCAATGGGAAAATCTGAAATAAGATTGA

GAACAGTGTTACCAATAGAAGTATTTAAGGTTTAAAGCATACCTTAA

AGATAACATTTTTTTTTGAAAAGAGTCAAATTATTTTTGAAAGGCTG

ATATTTTTGATATTTACTAATATTTTATTTATTTCTTTTTCCCTTAAAA

TAAGAGCTAAATCTGTTTTTATTATCATTTATCAAGCTCTATTAATAC

CTCAACTTTTTCAAGAAAAAATAATAATAATTTTTCCCTCTATTCTCA

TGACCTTTTAGGAAAATTAATTTTAGAAAAACTATTGACAAACCCAT

AAAAAATGAGATAAGATTATAGATTGTCACTGGTATTTTATACTAGA

GGCAAATTATATTTATATATACAAAAATGCTGTATAAAAAACATCT

PrpoA AGTAAAGATTATCACCAACATCTGAAACCTGACTTCATCAACTGAGG 42

AAATAACCACTGTGGCTGTGTTTAAAATCGACTGCGTAGCAAGTAAA ACTCAAAAAAATCAAGGTCAATACGGAAAGTTTGTGCTTGAACCCTT AGAAAAAGGACAAGGCATAACT

PpsaA 43

CTACATCAACTAATCAAAAGTTAAGAAAAAAGATAGAAACGCCCAT

GAATATTAAAGATTAATCTGTGTCCTTTAACTTTTTATCCCCTTAAAA

GAGCATAACTAAAACATTGATAGATTTTATAAAGAAAAGTAACAAA

ATCTTGACTTAAATGAGAAAGGATTAAAAACCAAAGCCTTATCTGAG

GGAATGTTAAACAAATTTTAAATATTGTTAAGCAAGAACCACAATGG

TGACAAATAGCCCTTATCATCTTCAGTAATGTAGTAGTTTAAGTATTT

GTCGAGAGAGGAATCCCTC

PpsbA2 GATCGAATTTTTGACTATTTAATAATTTCTTTACTATTCATAATATCT 44

CAAAAGACTTCTATCTTTTTAAGTAAACTACCTCCTCTAAGAATAAA

CACTTATTGACTATATTCCTTTTTAGTTATAAAATGGCATTTAAAGTT

ACTCAAAATATTTGCAATCATTCTACAAAACATAGTGTATTTCCTTGT

ATTAAGCGTATTGTGTCCTGTTAGATAATGTAGGAAAGATTGTGAGT

TGATAGGTGATAAATACATAACTCATTAGACAACAAGATAAAGTTGT AGGAGTTCTAAATT

PpsbD AAGAGTTTGGCATTTTTATTGGTAAGACTATTCTGAGAAAAATGTGA 45

CAATTTGTTAAAATATTTGCTAGAAATAGAAAAAGTAATTTGGCAAA GATACTTAAATCGTATCGAAAAACGGAGTTACATTAACTCTAACTCA TGCTATATTAAGAAAAGTTAATTGCAGATCAGTATTATTGCTGAGTA GCAGTGCCGTCTCCAATAATATAAAGAGAGACAATATAAAAGTAAA ACTTGACAAGTTAAAAAAAGAAAGATT

PcpcB AACTTTAGATATTCGTAGTTGGCAATGTCGTAAATGCGGAACAATAC 46

ATGGAAAACATATAGATTTGTAATGAGAAAAAGTGTAAACAAATAT

TAAGAAAAAGATCAGAAAAATTTAACAACACGTAATAAAAAAATGC

GTCACTACGGGTTATAAATTTACATGAAAGGTTAAAACACTTTTCTG

AGACGATTTTGATAAAAAAGTTGTCAAAAAATTAAGTTTCTTTACAA

ATGCTTAACAAAAACTTGGTTTTAAGCACAAAATAAGAGAGACTAA

TTTGCAGAAGTTTTACAAGGAAATCTTGAAGAAAAAGATCTAAGTA

AAACGACTCTGTTTAACCAAAATTTAACAAATTTAACAAAACAAACT

AAATCTATTAGGAGATTAACTACA

Codon improvement of recombinant genes

[0154] At least some of the nucleic acid sequences to be expressed in cyanobacterial host cells can be codon improved for optimal expression in the target cyanobacterial strain. The underlying rationale is that the codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Bulmer, 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 plasmid, for example.

[0155] In an embodiment, the nucleic acid sequences of the recombinant genes are modified so that they will have improved expression in cyanobacteria. For example, the selectable marker gene that confers gentamycin or kanamycin resistance was codon optimized for higher expression in cyanobacteria. Additionally, the selectable marker gene that confers 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%, which is similar to that of ABICyano l coding genes (about 36% on average). The codon adaptation index (CAI) of the codon improved antibiotic resistance genes is significantly improved from less than 0.4 to greater than 0.8, which is similar to that of ABlCyanol endogenous genes.

[0156] In an embodiment, the CAI of a recombinant gene is codon improved for enhancing translation by having a codon adaptation index (CAI) of >0.60, preferably >0.70, most preferred >0.80 based on the codon usage table of the host cell. Table 2, below, depicts the codon usage statistics within ABlCyanol .

Table 2

Glu GAA 0.787 60690 53.756

Glu GAG 0.213 16451 14.571

Gly GGA 0.324 22709 20.1 14

Gly GGC 0.125 8720 7.724

Gly GGG 0.151 10542 9.338

Gly GGT 0.401 28065 24.859

His CAC 0.251 4859 4.304

His CAT 0.749 14516 12.858

He ATA 0.195 18334 16.239

He ATC 0.19 17872 15.83

He ATT 0.616 57964 51.342

Leu CTA 0.088 10776 9.545

Leu CTC 0.058 7129 6.314

Leu CTG 0.033 4040 3.578

Leu CTT 0.1 16 14162 12.544

Leu TTA 0.571 69559 61.612

Leu TTG 0.133 16235 14.38

Lys AAA 0.836 59396 52.61

Lys AAG 0.164 1 1694 10.358

Met ATG 1 20093 17.797

Phe TTC 0.172 8420 7.458

Phe TTT 0.828 40450 35.829

Pro CCA 0.169 7746 6.861

Pro CCC 0.275 12613 1 1.172

Pro CCG 0.066 3012 2.668

Pro CCT 0.491 22560 19.982

Ser AGC 0.088 6435 5.7 Ser AGT 0.306 22393 19.835

Ser TCA 0.14 10217 9.05

Ser TCC 0.102 7465 6.612

Ser TCG 0.044 3196 2.831

Ser TCT 0.321 23473 20.791

Thr ACA 0.26 15649 13.861

Thr ACC 0.236 14251 12.623

Thr ACG 0.083 5024 4.45

Thr ACT 0.42 25340 22.445

Trp TGG 1 14964 13.254

Tyr TAC 0.187 7364 6.523

Tyr TAT 0.813 31912 28.266

Val GTA 0.28 18541 16.423

Val GTC 0.1 17 7778 6.889

Val GTG 0.184 12184 10.792

Val GTT 0.419 27713 24.547

End TAA 0.63 2495 2.23

End TAG 0.22 848 0.76

End TGA 0.15 591 0.53

Transformed ABICyanol

[0157] In an embodiment, genetically enhanced Cyanobacterium 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 biosynthetic pathway for the production of a compound or marker protein. In certain embodiments, they comprise an ethanologenic cassette. In certain embodiments, the genetically enhanced Cyanobacterium 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 Cyanobacterium sp. host cells in the presence of other bacterial contaminating strains.

[0158] In another embodiment, a recombinant gene is present on an extrachromosomal plasmid that can replicate independently from the chromosomes of the Cyanobacterium sp. host cells such as ABICyanol . In an embodiment, the extrachromosomal 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.

[0159] Genetically enhanced Cyanobacterium sp., for example ABICyanol host cells, can include further genetic enhancements such as partial deletions of endogenous genes of

Cyanobacterium 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 acetyl-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.

[0160] 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 respiratory electron transport systems as well as of the Calvin cycle. Genetic enhancement targets include local and global regulatory factors including, but not limited to, the two component system, sigma factors, small regulating RNAs and antisense R As.

[0161] In an embodiment, Cyanobacterium 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 μπιοΙ/L in the culture during the day).

Compounds of interest produced by ABICyanol

[0162] In certain embodiments, a variety of different compounds of interest can be produced using genetically enhanced ABICyanol host cells. Plasmid vectors disclosed herein (e.g., derivatives of p6.8) can be used to carry a gene or genes involved in various biosynthetic pathways that produce a compound of interest in the ABICyanol cell. 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. Additional information on compounds that can be produced from cyanobacteria can be found, for example, in PCT/EP2009/000892 and in PCT7EP2009/060526, both of which are incorporated by reference herein in their entirety. Genes involved in the biosynthetic pathway for the production of a compound of interest can be inserted into the vector.

[0163] In one 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 isobutanol fermentation are incorporated into recombinant vectors and transformed into ABICyanol . Examples of enzymes involved in isopropanol fermentation include acetyl-CoA acetyltransferase (EC 2.3.1 .9), acetyl-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), 2,3-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 ).

[0164] In another embodiment, ethylene is produced as a compound of interest. In an

embodiment, at least one recombinant gene encodes an enzyme for ethylene formation. Examples of enzymes involved in the production of ethylene include ethylene forming enzyme 1 - aminocyclopropane-l -carboxylate oxidase (EC 1.14.17.4), which catalyzes the last step of ethylene formation, the oxidation of 1 -aminocyclopropane-l -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. [0165] 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.

[0166] 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 wide variety of plants, particularly conifers. Terpenes are derived biosynthetically 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 transfarnesyl diphosphate and isopentenyl diphosphate yielding pyrophosphate and geranylgeranyl diphosphate.

[0167] 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 ⁺ + 12X _re duced→ 6 H ₂ + 12X ₀ xidized, where X is an electron carrier such as ferredoxin.

[0168] In an embodiment, examples of compounds of interest include non-ribosomal peptides (NRP) and the polyketides (P ). In another embodiment, alkaloids are compounds of interest.

[0169] In yet another embodiment, 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, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1 -phosphate guanylyltransferase, GDP-mannose-3,5- epimerase, GDP-L-galactose phosphorylase, L-galactose 1 -phosphate phosphatase, L-galactose dehydrogenase, and L-galactono-l,4-lactone dehydrogenase. [0170] In another embodiment amino acids are compounds of interest. Amino acids as compounds of interest include naturally occurring amino acids as well as amino acid derivatives.

[0171] In an embodiment, lactams are compounds of interest. In another embodiment, ethers are compounds of interest.

[0172] In yet another embodiment, alkanes (also known as saturated hydrocarbons) are compounds of interest. In an embodiment, these genes may be part of the recombinant 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).

[0173] In an embodiment, biopolymers such as polyhydroxyalkanoates (PHAs) are compounds of interest. The simplest and most commonly occurring form of PHA 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), polyhydroxyoctanoate (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-hydroxybutyryl-CoA (3HBCoA) by NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.1.36). The 3HBCoA is subsequently polymerized by poly(3-hydroxyalkanoate) synthase (EC 2.3.1) and is converted to P3HB.

[0174] In an embodiment, esters, including fatty acid esters, are a compound 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.

[0175] 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.

[0176] 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.

[0177] In an embodiment, the compounds of interest that are produced from recombinant ABICyanol 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.

Production of ethanol in ABICyanol

[0178] 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.

[0179] In an embodiment, a compound of interest is ethanol, and the genetic enhancements to ABICyanol include transforming with a p6.8 based vector that comprises one or more recombinant genes encoding for an enzyme used in ethanol production. In an embodiment, the genes are adh and pdc. The gene pdc encodes for pyruvate decarboxylase (PDC), which catalyzes the interconversion between pyruvate and acetaldehyde. The gene adh encodes for 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 AdhE enzyme (alcohol dehydrogenase E) which catalyzes the interconversion between acetyl-coenzyme A and ethanol.

[0180] Ethanol produced by non-naturally occurring ABICyanol organisms can be measured by any means well known in the art. In 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 growth media.

[0181] In an embodiment, the pdc-encoding gene is from Zymomonas mobilis. In a further embodiment, the pdc-encoding gene is codon optimized for optimal expression in the host organism, such as ABICyanol . The pdc gene can be the codon optimized Zymomonas mobilis sequence of SEQ ID NO: 47. In another embodiment, the pdc gene can have a sequence identity of at least 85%, 90%, 95%, 97%, 99%, 99.5%, or greater in identity to SEQ ID NO: 47. [0182] The PDC protein can be, for example, the Zymomonas mobilis PDC sequence (SEQ ID NO: 48). The gene encoding PDC can also be derived from other organisms. The PDC enzyme can have a sequence identity of at least 85%, 90%, 95%, 97%, 99%, 99.5%, or greater in identity to SEQ ID NO: 48.

[0183] 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.

[0184] The ADH enzyme can be derived from numerous source organisms, and can be codon- optimized for optimal expression in the host cell, if desired. An exemplary adh gene from Synechocystis PCC 6803 is shown in SEQ NO: 49. The corresponding protein sequence is shown in SEQ ID NO: 50. A codon optimized version of the adh gene is present in SEQ ID NO: 51. The corresponding protein sequence is shown in SEQ ID NO: 52. Another example of an alcohol dehydrogenase gene that can be used is derived from Lyngbya sp. The codon-optimized DNA sequence is shown in SEQ ID NO: 53, wherease the corresponding protein sequence is shown in SEQ ID NO: 54.

[0185] In particular embodiments, the ADH enzyme is, for example, a Zn ²⁺ -dependent alcohol dehydrogenase such as Adhl from Zymomonas mobilis (ZmAdh) or the Adh enzyme from Synechocystis PCC 6803 (SynAdh encoded by the synadh 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 ²⁺ dependent alcohol dehydrogenase from Synechocystis PCC 6803. Relative to other alcohol dehydrogenases, SynAdh (annotated open reading frame slrl 192 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 acetaldehyde. Thus, in an embodiment, a SynAdh encoding recombinant gene is useful for production of ethanol in a host cell.

[0186] AdhE is an iron-dependent, bifunctional enzyme that interconverts 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 elongatus 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. (2000), J. Bacteriol. 182:6049-6054. The E568 AdhE mutant of the E. coli AdhE was active both aerobically and anaerobically. 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.

In an embodiment, pyruvate decarboxylase can, for example, be from Zymomonas mobilis, Zymobacter palmae, or Saccharomyces cerevisiae. Nucleic acid sequences, protein sequences and properties of ethanologenic enzymes such as alcohol dehydrogenases and pyruvate decarboxylases disclosed herein, can be found, for example, in International Patent Application Publication No. WO2009098089, International Patent Application Publication No.

WO2014198964, U.S. Patent No. 9,157,101 , and U.S. Patent No. 9,127,297.

Non-naturally occurring, genetically-stable ethanologenic cyanobacterial host cells

[0187] Genetic instability of ethanologenic cyanobacterial strains plays a key role in the loss of productivity of strains in indoor and outdoor bioreactors. Increased genetic stability helps keep production potential stable during scale up and throughout the duration of production in bioreactors. It also offers the ability to dilute cultures, often multiple times, after they enter the ethanol production phase, which saves on capital costs, scale up efforts, and material costs, and is a feasible outdoor strategy.

[0188] Ethanologenic cyanobacterial strains show a heavy bias for revertant cells in which the pdc is inactivated. In an embodiment, the pdc, or other gene responsible for the production of a compound of interest, may be inactivated through mutations in an operably linked promoter. In an embodiment, the pdc, or other gene responsible for the production of a compound of interest, may be inactivated through mutations in the gene. In an embodiment, the pdc, or other gene responsible for the production of a compound of interest, may be inactivated through mutations in the gene resulting in insertions and/or deletions. In an embodiment, the pdc, or other gene responsible for the production of a compound of interest, may be inactivated through mutations that result in truncation of the gene. In another embodiment, inactivation of the pdc, or other gene responsible for the production of a compound of interest, is by transposon insertions, primarily by the transposon ABIcyanol_orf0173 in the promoter or pdc coding sequence. [0189] One method for mediating this type of reversion event is to couple the expression of pdc with an essential gene for selection against non-producer mutants. Transposon insertion in the promoter that drives pdc expression leads to the loss of expression of both pdc and the essential gene while transposon insertion in the pdc coding sequence will separate the promoter of the operon from the essential gene coding sequence. In both cases, the essential gene will not be expressed due to transposon inactivation. As a result, non-producer mutants will not survive. Cyanobacterial cells have many genes that are essential to the cell's survival. In an embodiment, one or more of these essential genes can be chosen to be modified to produce more genetically stable cyanobacterial production cells.

Knockout of an Essential Gene

In an embodiment, a gene that is essential to the host cell survival can be deleted from its original location in a cell ("knockout") using DNA technology, such as double homologous

recombination, or by other means. Because the endogenous essential gene is often present in the cell in multiple copies, a full "knockout" of all of its copies in a cell is typically employed. The essential gene can then be linked to a production gene of interest. Thus, typically, the gene is still present in the cell, but has been moved to a new location with different neighboring genes. In an embodiment, the production genes are located upstream of the essential gene in one operon, so that they must be transcribed correctly in order for the essential gene to be transcribed. This allows the cells containing active production genes to reverse the competitive disadvantage that they may have.

Knockout of Conditionally Essential Gene nirA

[0190] In an embodiment, the essential gene encodes nitrite reductase (nirA). ABICyanol host cells are made auxotrophic for nitrate assimilation by knocking out a chromosomal copy of nirA which encodes for nitrite reductase. Thus, in an embodiment, nirA is encoded for on

extrachromosomal plasmids as an essential gene operon fused to pdc through various naturally and non-naturally occurring intergenic sequences. Loss of nirA expression blocks the nitrate assimilation pathway. This makes the cell unable to grow on nitrate as the sole nitrogen source. Moreover, nitrite derived from nitrate assimilation is cytotoxic. This cytotoxicity makes the selection pressure against cells that do not express active nitrite reductase from an exogenous nirA even stronger. In an embodiment, a knockout of the nirA gene (ABICyanol _orfl)468) was made in ABICyanol and labelled as AB0057. AB0057 is unable to grow on nitrate. To complement AB0057, multiple constructs were made in which pdc was expressed in an operon with nirA. The constructs had different intergenic sequences present between pdc and nirA. In one embodiment, different intergenic sequences between the pdc and nirA genes on the operon were tested to improve expression of the second gene in the operon, nirA.

Addition of Intergenic Sequences to Improve Expression of Downstream Genes in the Operon

[0191] In polycistronic ethanologenic cassettes, genes located downstream of pdc often have different expression strengths depending on the intergenic sequence. Translation of the proteins encoded by the second and subsequent genes in an operon are affected by the neighboring genes in the operon and by the intergenic sequences in between the genes, see Pfleger et al., (2006) "Combinational engineering of intergenic regions in operons tunes expression of multiple genes" Nat. Biotech. 24: 1027-1032. Pfleger et al. described a method for tuning the expression of multiple genes within operons in E. coli by generating libraries of tunable intergenic regions (TIGRs), recombining various post-transcriptional control elements and screening for the desired relative expression levels. TIGRs can vary the relative expression of two reporter genes over a 100-fold range and balance expression of three genes in an operon that encodes a heterologous mevalonate biosynthetic pathway, resulting in a seven-fold increase in mevalonate production. In an embodiment, a TIGR (p70rg-15) that leads to high expression of a second gene was used in an essential gene operon that contained a ribosome-binding site (RBS) that was replaced with a cyanobacterial RBS, such as that of PnirA*2. This intergenic sequence is referred to herein as a synthetic intergenic sequence (SIS).

[0192] Intergenic sequences from ABICyanol operons that encode for naturally abundant proteins were used to improve the expression of genes in engineered operons. In an embodiment, genes that encode proteins used in the photosynthetic apparatus provide useful intergenic regions to help improve expression of all genes in an engineered operon. In one embodiment, intergenic sequences are derived from the alpha and beta subunits of phycocyanin, encoded by the cpcB- cpcA operon. Because cpcB and cpcA form heterodimers, the second gene (cpcA for the alpha subunit) of the operon should be translated as efficiently as cpcB, the first gene (beta subunit). The intergenic region cpcB-cpcA is 67 bp long. As with cpcBA, the intergenic sequence from psaAB is also useful for improving the expression of genes in an engineered polycistronic operon.

Construction and Analysis of the Essential Gene Operon pdc-nirA

[0193] The nirA knockout strain AB0057 was complemented with plasmids bearing the pdc gene tied to nirA such that expression of pdc was required for expression of nirA. The three constructs evaluated for increases in genetic stability have different intergenic sequences between the PDC and nirA in order to improve the expression of genes in operon form (Table 3). Three different intergenic sequences were used: 1) cpcBA intergenic sequence from ABICyanol (pAB193), 2) psaAB intergenic sequence from ABICyanol (pAB194) and 3) a synthetic intergenic sequence from E. coli using the optimized RJBS from PnirA*2 (pAB 195). These three constructs were transformed into AB0057 and evaluated for ethanol production and growth characteristics. Figure 3 depicts ethanol production of different nirA complementation constructs. pAB193 produced a similar level of ethanol as the control strain pAB722, while pAB194 and pAB195 produced slightly less ethanol than the control.

[0194] Once the constructs were evaluated in GC vials, they were then prepared for evaluation in mLvPBRs. For mLvPBR evaluation of genetic stability and ethanol production, each candidate strain was streaked out to isolate single colonies of three strains with the lowest reversion percentages. These three clones were used as biological replicates in mLvPBRs.

[0195] mLvPBRs were sampled three times a week for OD and ethanol percentages, and the VLE-corrected ethanol was calculated from the percentage of ethanol produced (FIGs 4A, 4B and 4C). For all four strains (three isolates and a control pAB722 which is derived from p6.8 and does not contain a pdc-nirA fusion operon), the growth in the first 21 -day batches as measured by OD750 was similar (FIG. 4A). pAB193 did show better growth than the other two test strains and the control pAB722. The ethanol produced during the first batch was also similar among the four strains (FIGs 4B and 4C). However, after the first 20: 1 dilution step, pAB 194 and pAB 195 appeared to experience a lag in growth. Due to this lag in growth, only pAB722 and pAB193 were carried through the second dilution and into the third batch. However, even in the second 21 -day batch, the control pAB722 was showing faster growth and lower ethanol production, indicating an increasing revertant percentage within these mLvPBRs. All three replicates of pAB193, in contrast, showed similar growth and ethanol production in all three 21 -day batch cultures. Table 3

[0196] The strains were also assayed for gene expression using qRT-PCR. Previous experiments have indicated that RNA levels peaked about 2 days after nitrate induction and steadily decline thereafter in systems driven by the PnirA*2 promoter. To assay the gene expression of the nirA, samples were taken from the repressed inoculum and from the mLvPBRs on day 2. Gene expression analysis shows an increase in gene copy number from the inoculum to day 2 for all three constructs (FIG. 5). There is also an increase in nirA expression in the control pAB722, which is to be expected with the switch from urea to nitrate as the nitrogen source. This data shows that nirA expression is induced by nitrate in all nirA complementation/genetic stability strains.

[0197] PDC and ADH activity were measured weekly in the first 21 -day batch for all strains, see FIGs 6A and 6B, respectively. PDC activity increased from the inoculum samples to day 7, then leveled off over the remaining 14 days, see FIG. 6A. PDC levels were highest in pAB 195 cultures. pAB722 (SEQ ID NO: 8) and pAB194 ((SEQ ID NO: 10) cultures had similar levels of PDC, whereas pAB 193 (SEQ ID NO: 9) showed the lowest levels of PDC over time. The higher PDC activity exhibited by pAB 195 could be manifesting as slower growth that is seen in these cultures, while the lower PDC activity of pAB 193 could be allowing this strain to grow faster than the others. Interestingly, pAB 193 grew to a higher cell density while maintaining ethanol production at a level as high as the control, implying higher carbon fixation in pAB 193. [0198] For ADH activity levels (FIG. 6B), all of the cultures showed similar activities, with the highest activity shown earlier on in the batch culture. The adh gene in these constructs is driven by PrbcL, which is responsive to light conditions. In a non-limiting embodiment, the highest activity being earlier in the batch culture can be due to an increase in culture density over time, so that less light is able to penetrate, resulting in lower ADH activity.

[0199] Thus, in an embodiment, genetic stability of ethanologenic cyanobacterial host cells has been improved, resulting in an increase in the duration of ethanol production.

Multiple production genes can be present in the essential operon

[0200] In another embodiment, more than one gene of interest can be placed upstream of the essential gene in the essential gene operon. For example, both pdc and adh can be placed directly upstream of the nirA gene, so that all of the genes involved in ethanol production are expressed under the control of one promoter upstream of pdc. Thus, both pdc and adh would be expressed in order for the essential gene nirA to be expressed. Example 8 shows the construction of this system to produce ethanol. The method can be used to produce other desired compounds, such as, for example, 1 ,2-propanediol or 1 ,3-propanediol, by placing one to several of the production genes upstream of the essential gene, where all of the genes are controlled by the same promoter. The number of genes that can be placed upstream of the essential (or conditionally essential) gene can be, for example, from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Other essential genes

[0201] In an embodiment, other genes can be made essential and then are engineered to become tied to the expression of a gene responsible for the production of a compound of interest.

[0202] In an embodiment, a non-naturally occurring ABICyanol host cell bearing a nitrate reductase (narB) knockout can be made. Example 3 describes the construction of an integrative plasmid, as well as the transformation and selection method, that can be used to create a narB knockout strain. The narB gene can then be inserted into an operon of a plasmid that contains the pdc-adh ethanologenic construct. The narB gene can be placed immediately downstream of the pdc gene, so that it is not expressed if the pdc gene is not expressed. Similar to the method used for the nirA knockout and complementation system described above, the narB system can be utilized in cyanobacteria to create ethanologenic strains that are more stable. [0203] In one embodiment, an ethanologenic vector contains a "pdc-urease" operon driven by a copper inducible promoter in an ABICyanol host bearing a urease gene knock-out. An active urease requires at least seven genes: ureA, ureB and ureC encode the subunits (alpha, beta and gamma) while ureD, ureE, ureF and ureG encode for accessory peptides required for the assembly of the Ni ²⁺ metal locenter. In an embodiment, ureA, ureB or ureC gene could be knocked out on the chromosome and linked to pdc to form an operon in an ethanol cassette. Example 3 describes the construction of an integrative plasmid, as well as the transformation and selection method, that can be used to create the ureC knockout strain.

[0204] In an embodiment, a non-naturally occurring ABICyanol host cell bearing a urease gene knock-out could be grown in a nitrate-containing medium with a low level of copper to repress ethanol production. Ethanol production can be induced by dilution into a medium containing a high level of copper and urea as the sole nitrogen source. In an embodiment, cells that will have lost ethanol production capacity because of transposon inactivation of the pdc-urease operon will not be able to survive in medium containing primarily only urea as a nitrogen source.

[0205] In another embodiment, cobK, a gene used for vitamin B12 biosynthesis, would be knocked out in a host cell and biosynthesis is restored by an essential operon containing a pdc gene and cobK. ABICyanol contains an open reading frame (ABICyanol_orf3616) whose knock out can create a vitamin B 12 auxotrophy in ABICyanol . Example 3 describes the construction of an integrative plasmid, as well as the transformation and selection method, that can be used to create a cobK knockout strain.

[0206] For complementation and in order to further increase genetic stability, a bicistronic essential operon can complement the auxotrophy. All complemented strains will be tested for the ability to grow without vitamin B 12 supplementation and to make ethanol. Thus, in an embodiment, the essential operon approach using an ABICyanol -cobK knockout strain complemented with a plasmid such as pABICyanol_6.8::Porf0316-pdc-cobK, could significantly increase genetic stability of an ethanologenic ABICyanol host cell.

The essential operon can be located on the chromosomal DNA

[0207] In another embodiment, the host cell can be modified so that the chosen essential gene remains present on the chromosomal DNA, but the production gene of interest is inserted directly upstream of the essential gene, so that the the production gene and the essential gene are on one operon, under the control of a promoter upstream of the production gene. The production gene insertion can be performed, for example, using homologous recombination. A selectable marker can be inserted along with the production gene to allow for full segregation of the multiple chromosomal copies.

Kits for producing compounds of interest

[0208] In an embodiment, a kit for producing a compound of interest using genetically enhanced ABICyanol host cells that are antibiotic marker free includes genetically enhanced ABICyanol host cells, a vessel for culturing the host cells and a means for illumination of the host cells. In an embodiment, the host cells of the kit produce ethanol and the means for illumination is photosynthetically 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, any of the photobioreactors disclosed in the PCT application WO 2008/055190 A2, which is hereby incorporated in its entirety by reference, can be used. Furthermore the kit also can also include means for separating the compound, preferably ethanol from the growth medium as, for example, disclosed in the PCT application WO 201 1/103277 Al , which is hereby incorporated in its entirety by reference.

[0209] 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

[0210] Escherichia coli (E. coli) strains HB 101 (Promega), XL I O-Gold (Stratagene), a-select (Bioline) and NEB Turbo (NEB) were grown in Luria-Bertani (LB) medium at 37°C. Ampicillin (100 μg/ml), kanamycin (25-50 μg/ml), gentamycin (15μg/ml) and chloramphenicol (34 μg/ml) were used when appropriate. Cultures were continuously shaken overnight at 200 rpm.

ABICyanol was cultured at 30°C in liquid BG1 1 fresh water medium on a reciprocal shaker at 150 rpm under continuous illumination of approximately 30 - 40 μηιοΐ photons/m ^"2 s ^"1 .

Cyanobacterial transformants were selected on solid BG1 1 medium containing 10 - 20 μg/ml kanamycin, gentamycin or chloramphenicol and were maintained on BG1 1 plates containing 20 - 100 μg/ml kanamycin, gentamycin or chloramphenicol. Example 2

Preparation of cyanobacterial culture medium

[0211] BG-1 1 stock solution was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO). Stock solutions of the antibiotics spectinomycin (100 mg/mL) and kanamycin (50 mg/mL) were purchased from Teknova (Teknova, Hollister, CA). Stock solution of the antibiotic gentamycin (10 mg/mL) was purchased from MP Biomedicals (MP Biomedicals, Solon, OH). Marine BG-1 1 (mBG-1 1) was prepared by dissolving 35 g Crystal Sea Marinemix (Marine Enterprises International, Inc., MD) in 1 L water and supplementing with BG-1 1 stock solution. mBG-1 1(10) was prepared by dissolving 35g Instant Ocean in 1L water and supplementing with BG-11 stock solution. Vitamin Bi ₂ (Sigma Aldrich) was supplemented to mBG-11 to achieve a final concentration of 1 g/L, as needed.

Example 3

Chromosomal knockout of target cyanobacterial essential genes

[0212] Knockout of target essential genes of ABlCyanol was conducted with the help of integrative plasmids. The oriVT based plasmids contain flanking regions homologous to the up- and downstream region of the target gene of ABIcyanol . A gentamycin resistance gene is positioned in between the flanking regions in order to integrate the gentamycin resistance marker via double homologous recombination into the genome.

[0213] In order to knockout narB, nirA, ureC and cobK, ABlCyanol was conjugated with E. coli bearing the integrative constructs TK596 (narB) (SEQ ID NO: 12), #1872 (nirA) (SEQ ID NO: 13), #2198 (ureC) (SEQ ID NO: 14) and #2235 (cobK) (SEQ ID NO: 15). These integrative plasmids were inserted via double-crossover integration in the genome of ABlCyanol , to result in a complete deletion (knockout) of the gene. Selection was conducted on BG1 1 plates containing gentamycin 10 μg/ml. Obtained clones were tested by PCR for double crossover integration of the gentamycin resistance marker.

[0214] Knockout of narB: Positive clones of ABlCyanol ::TK596 were picked and streaked out for several rounds on nitrate free BG1 1 agar plates containing urea/ammonia (3 mM each) and increasing concentrations of gentamycin up to 200 μg/ml in order to force segregation. Deletion of the narB gene was verified by PCR, followed by agarose gel analysis, to determine the segregation status using specific PCR primers to detect the nirA gene. [0215] Knockout of nirA: Positive clones of ABlCyanol ::#1872 were picked and streaked out for several rounds on nitrate free BGl 1 plates containing urea/ammonia (3 mM each) and increasing concentrations of gentamycin up to 200 μg/ml in order to force segregation. Deletion of the nirA gene was verified by PCR, followed by agarose gel analysis, to determine the segregation status using specific PCR primers to detect the nirA gene.

[0216] Knockout of ureC: In order to delete the ureC gene in ABlCyanol, the integrative plasmid #2198 was inserted via double-crossover integration in the genome of ABlCyanol . Positive clones of ABlCyanol ::#2198 were transferred several rounds onto BGl 1 plates with increasing concentrations of gentamycin up to 200 μg/ml. Segregation status was verified by PCR with specific primers to detect the ureC gene. Complete deletion of ureC was successfully achieved

Knockout of cobK: Partial knockout of the cobK gene in ABlCyanol was achieved after double- crossover integration of plasmid #2235 into the genome of ABlCyanol. Positive clones of ABlCyanol ::#2235 were transferred several rounds on BGl 1 plates containing 40 μg/L vitamin B12 and gentamycin 75 μg ml. However, complete segregation of the cobK deletion was not achieved in ABlCyanol , indicating that cobK is essential under the tested selection conditions despite vitamin B12 addition.

Example 4

Transformation of ABlCyanol by Conjugation

[0217] Gene transfer to ABlCyanol was performed using conjugation. Generated plasmids containing oriVT were used for conjugation. The shuttle vectors were transformed into

ABlCyanol following a modified conjugation protocol which includes the pretreatment of ABlCyanol to reduce its EPS layer. Some plasmids used in this project are listed in Table 4, below. '

Table 4

[0218] For u ⁿ iparental mating, E. coli strain J53 bearing a conjugative RP4 plasmid and E. coli strain HB101 harboring the cargo and the pRL528 helper plasmid (for in vivo methylation) were used (Table 2). 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, centrifuged and resuspended in 100 μΐ BGl 1 medium.

[0219] For each conjugation, 25 ml of exponentially growing cyanobacterial culture (OD7 ₅₀ nm >0.5 <1) was incubated with N-acetylcysteine for 24 hours at 16°C in the dark (end

concentration: 0.1 mg/ml) without shaking. This pretreatment was followed by several steps to degrade the EPS and to weaken the cell wall: the pretreated culture was pelleted at 4,600 rpm and washed with 0.9% NaCl containing 8 mM EDTA. After a second wash step with 0.5 M NaCl, the cell pellet was resuspended in 0.5 M sucrose and incubated 30-60 min at 28°C with slow shaking (80-90 rpm). Cells were washed with 20 ml 50 mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 4% sucrose and 10 μg/ml lysozyme. After incubation at RT for 10-15 min, 20 ml TES was added to reduce/stop lysis. Then, cells were centrifuged and washed twice using i) 50 mM Tris containing 2% sucrose and 1 mM EDTA, and ii) BGl 1. All centrifugation steps were performed at 3,200 rpm for 10 min at 4-10°C. Resuspended cells were used for conjugation.

[0220] One hundred μΐ resuspended cyanobacterial and E. coli cultures were mixed and applied to a membrane filter (Millipore GVWP, 0.22 μπι pore size) placed on the surface of solid BGl 1 medium supplemented with 5% LB. Petri dishes were incubated under dim light (5 μπιοΐ photons/m ^"2 s ^"1 ) for 2 days. Cells were then resuspended in fresh BGl 1 medium and plated onto selective medium containing 10-20 μg/ml kanamycin/ gentamycin/ chloramphenicol,

respectively. Selection conditions were: light intensity approximately at 20-40 μπιοΐ photons/m ^"2 s ^"1 , temperature at approximately 30°C. Once transformants were visible (approx. after 5-7 days), colonies were transferred on new plates containing 10-50 μg/ml kanamycin /gentamycin

/chloramphenicol. Example 5

Plasmids containing pdc-nirA operons

[0221] Respective intergenic sequences and nirA were cloned into an ethanologenic plasmid backbone using PCR-based cloning techniques and the Invitrogen GENEART® Seamless Cloning and Assembly Kit from Life Technologies (Carlsbad, CA, USA). The ethanologenic plasmid backbone used was pAB722 (SEQ ID NO: 8), a pABICYAN01 -6.8-based vector constructed, with the following description: pABICYAN01_6.8::PnirA*2- pdc(ABICYAN01optl)-TdsrA-Prbc*(optRBS)-synADH-Toop containing a kanamycin resistance marker. The three intergenic sequences used were the intergenic sequence from the operon cpcB-cpcA (ABICYAN01_orf2472-2471), the intergenic sequence from the operon psaA-psaB (ABICYAN01_orf3243-3241) and a synthetic intergenic sequence (SIS) known as p70rg-15 containing a cyanobacterial RBS (Pfleger et al., 2006) (see Table 2 for construct description).

[0222] Construct plasmid maps were designed using DNAStar software and pAB722 as the vector. pAB722 contains a Sac\ cutting site located at 22 bp downstream from the stop codon of the pdc sequence and immediately upstream of the dsrA terminator.

[0223] The PCR template for producing the nirA sequence was pABl 83 plasmid, a

pABICYAN01-6.8-based vector based on AB0419 in which the nirA is expressed in an operon with a codon optimized pdc gene (pdc-ABICyanol(optl). The PCR template for each intergenic sequence was chemically synthesized by IDT DNA Technologies as either a single-stranded oligo or a gBlock. Respective oligo or gBlock sequences can be referred to in Table 5. PCR primers were designed for each respective template following manufacturer's guidelines for the Invitrogen GENEART® Seamless Cloning and Assembly Kit from Life Technologies (see Table 6 for PCR primers and associated sequences). PCR products were generated and gel purified for the nirA sequence and each respective intergenic sequence. Subsequent PCR reactions were carried out to combine respective inserts into one larger resulting insert (cpcBA-H/ ^' , psaAB- nirA and S\S-nirA) and the resulting PCR products for the combined inserts were gel purified. The pAB722 plasmid was digested with Sacl-HF restriction digest enzyme from New England Biolabs according to manufacturer's instructions to produce the linearized plasmid.

[0224] Following the manufacturer's protocol for the Invitrogen GENEART® Seamless Cloning and Assembly Kit from Life Technologies (Carlsbad, CA, USA), the reaction was carried out for each respective combined insert as listed above with the AB0419 vector digested with Sacl. The resultant constructs were transformed into E. coli XLI O-Gold Ultracompetent Cells from Agilent Technologies. Transformed E. coli cultures were plated and quadrant streaked onto LB agar plates with 50 μg/ml kanamycin (LKBm50). Plates were incubated at 37°C overnight.

Following overnight incubation, 20 colonies per construct were picked and patched onto fresh LB m50 agar plates and allowed to incubate overnight at 37°C. Colony PCR was carried out on 8 clones of each construct and subsequent plasmid mini-preps were carried out on 3 positive clones of each construct. Plasmids were sequenced. One clone of each construct was confirmed, chosen and transformed into competent cells {E. coli cells {E. coli Top 10 + pRL528 + pRL443 CmCb) by electroporation for subsequent transformation into ABICyanol .

Table 5: Intergenic Sequence PCR Templates.

Table 6: Primer names and sequences

[0225] Once the plasmids were constructed, they were transformed into ABlCyanol by conjugation as described in the above examples. After transformation, candidates were repeatedly plated onto repressed medium (mBGl 1 nitrate free with 2 mM urea and 2 mM ammonium and 150 με/ml kanamycin) until the cultures were cleaned of E. coli. Once axenic, the candidates were PCR confirmed to contain the appropriate plasmid.

Example 6

Determination of ethanol production using gas chromatography

[0226] Two kinds of GC headspace measurements were performed: a) REPP A (applied for clone testing and short-term characterizations of cultures cultivated in GC vials with a duration of 24 hours, and b) single GC single measurements (applied for

measurements of EtOH concentrations in samples daily taken from PBR cultures) by measuring the ethanol content after transferring 0.5 mL of the PBR cultures into GC vials after certain points of time of cultivation in the photobioreactor.

[0227] Ethanol production potential for individual transformant clones was completed via REPPA - relative ethanol production potential assay. Transformant clone patches were cultured up in liquid medium of nitrate-free mBGl 1(10, instant ocean salt mixture) supplemented with 150 μg/ml kanamycin, 2 mM urea and 2 mM ammonium. After sufficient growth under light at 37 °C with shaking (120 rpm), cultures were induced for 20 hours in a vented 50-mL falcon tube. Cultures were induced by spinning the cultures down and resuspending the cells to 0.8 OD (ODstart) in 15-mL mBGl 1 (10). GC vial assays were then carried out the next day by

resuspending the pellets to a 0.8 OD in 1 1-mL fresh mBGl 1 (10) with 90 mM sodium

bicarbonate. GC vials were then incubated at 37°C with 24-hours continuous -90-100 μπιοΐ photons m ^"2 s ^"1 light. There were 5 vials per culture (four for GC analysis and one for OD measurement). Each vial had 4 mL of air removed from and 4 mL of pure C0 ₂ directly added to the headspace. The OD at the end of incubation (ODend) was measured from one vial; and, the average of ODstart and ODend was used to report ethanol productivity as % (v/v) per ODavg per 24 hours.

[0228] GC single measurements did not involve the cultivation of the strains in the GC vials. GC single measurements were performed in order to characterize the long-term ethanol production of strains which were already known to produce ethanol in sufficient quantities in REPPA assays. GC single measurements further differed from REPPA measurements in the volume of the culture (2 mL in REPPA and 0.5 mL aliquots taken from a photobioreactor culture in GC single measurements). In single GC measurements only the absolute amount of ethanol produced at a certain point of time was determined, whereas the REPPA measurements determines the course of ethanol production during a certain period of time up to 24 hours of growing the cells a GC vial under constant illumination.

[0229] Headspace GC measurement of ethanol was performed for both REPPA and single gc measurements using an Agilent 7890A gas chromatograph with split/splitless (SSL) inlet and flame ionization detector coupled to an Agilent 7697A headspace autosampler. An Agilent DB- ALC1 capillary column (30 m x 0.32 mm x 1.8 μπι) was maintained at 40°C at 3.5 mL/min constant flow of helium and held isothermally for 5.5 minutes for separation and quantitation of the ethanol concentration in the sample. A calibration curve was constructed before each analysis using integrated peak areas resulting from ethanol standards prepared gravimetrically in 35 psu seawater in concentrations of 0.001 % v, 0.01% v, 0.1% v, and 1% v using a 1/x weighted linear fit. Samples were introduced via automated headspace sampling after incubation at 85°C for 30 minutes with high stirring into a 1 mL loop maintained at 85°C. Timing of the headspace sample introduction was as follows: 0.2 min vial pressurization to 15.1 psi, 0.2 min of loop fill, 0.05 min of loop equilibration, and 0.5 min of inject time. The headspace sample was then transferred to the GC via a 125°C capillary transfer line into the SSL inlet at a temperature of 250°C and a split ratio of 25: 1. Detection was performed with a flame ionization detector held at 300°C with a fuel gas flow of 30 mL/min, an air flow of 300 mL/min, and a makeup gas flow of 25 mL/min. The ethanol concentration of the sample was calculated from the peak area obtained compared to the aforementioned calibration curve.

Example 7

Evaluation of nirA complementation/essential gene operon strains

[0230] AB l Cyano l :pAB 193 (SEQ ID NO: 9), ABlCyanol :pAB 194 (SEQ ID NO: 10), and ABl Cyanol :pAB 195 (SEQ ID NO: 1 1 ) were evaluated in photobioreactors (mLvPBRs) in comparison with a control strain AB l Cyanol :pAB722 (SEQ ID NO: 8). All cultures were streaked onto mBGl 1 nitrate free, 2 mM ammonium, and 2 mM urea plates with 150 μg/mL kanamycin directly from cryo preserved cultures. Six single colonies for each of the strains were patched onto the same medium, and then tested for the presence of the pdc/nirA operon. Strains that lacked a functional pdc/nirA operon were labelled as revertants. As an example, the presence or absence of the pdc/nirA operon could be detected through immunofluorescence techniques. The three candidates with the lowest revertant percentages were spread-plated onto the same repression medium described above. The plate cultures were then scraped into 150 mL bottles with natural well water (NWW) supplemented with nitrate-free (NF) BG- 1 1 nutrients containing 5.5 mM Urea and 150 μg/mL kanamycin. The bottles were grown for 5 days with constant 1% C0 ₂ bubbling before being used to inoculate the mLvPBRs.

[0231] The following procedure describes the standard lab conditions under which a 0.4 L "mini lab vertical photobioreactor" (mLvPBR) was operated as well as the necessary parts, ports, etc. to construct this 0.4 L vPBR. mLvPBRs were made from the fusion of 5 cm inner diameter, 5.7 cm outer diameter Pyrex tubing (National Scientific Company, Inc.) and Corning Pyrex 100-mL screw cap media storage bottles (Fisher Scientific 16157-103). A 20.3 cm section of Pyrex tubing was welded between the bottom 1.3 cm and top 6.4 cm of the bottle (including the screw cap threads). Eagle Laboratory Glass Company (Painesville, OH) performed all glassware fabrication. The filling volume was about 0.4 L leading to a liquid height of about 21.5 cm and a headspace of about 4.5 cm. Caps were fitted with straight tapered 1/8" to 7/32" barbed polypropylene couplings (Fisher Scientific 15-315-28A), one each for gas inflow, gas outflow, and sampling. The diffuser consisted of a 1.5" section of EPDM 3/16" inner diameter tubing, which was perforated 12 times per inch by a standard sewing machine with a 60 gauge needle and attached to a polypropylene elbow fitting at the end of a section of tubing extending to the bottom of the reactor. On one side of the mLvPBR, a uniform 230 μιηοΐ m ^"2 s ^"1 light surface generated by a light panel of 4 x T5HO 54W 6500K fluorescent bulbs operating in a 12/12hour day/night cycle. The temperature was set to 30°C ± 4 °C during day/night from light source heating/cooling. Culture mixing and introduction of gasses was attained by constant air bubbling through perforated EPDM tubing. Gas flow was delivered at -22 mL min ^"1 to each reactor via a set of 30μπι machined restriction orifice with a head pressure set at 27 psiG. Gas flow operated in constant sparging mode (day and night) with a fixed C0 ₂ supply of 0.25% at night and 0.5-6.0% supply during the day depending on growth and pH. The standard cell density for starting a cultivation experiment OD7 ₅₀ nm = 0.2 in 35psu natural well water amended with BG1 1 nutrients, 17 mM Nitrate and 0.75 mM Urea.

[0232] Each culture was inoculated into one mLvPBR, so each strain was evaluated using three biological replicates. Strains were inoculated by dilution of cultures into mLvPBRs to an initial Optical Density (OD750) of 0.3 with an initial culture volume of ~400mL. Cultures were maintained at ambient air with a range of 25 to 28 °C during the night with an increase to ~ 33 °C during the photoperiod (due to heating from the light source). Light was supplied from one side of the reactor with a homogeneous light field set to 230 μπιοΐ photons/m ^"2 s ^"1 to approximate the average annual irradiance observed at a 4: 1 height to space ratio from outdoor vPBR cultures. Introduction of gasses was done via the same type of tubing currently in use in large-scale vPBRs (smooth EPDM). The length of gas tubing (ca. 4 cm) in each reactor was representative of the length per chamber in a 17L-vPBR. Gas flow was set 30 mL min ^'1 (scaled to match 0.5 L min- 1 for a 17L-vPBR) and delivered through machined restriction orifices to eliminate the need for mass flow controllers. C0 ₂ was delivered constantly from 0.5-5%; and, the percentage was controlled based on OD and pH over time. The standard cell density for starting a cultivation experiment OD7 ₅ 0nm = 0.2 in 35psu natural well water amended with BG1 1 nutrients, 17 mM Nitrate and 0.75 mM Urea.

[0233] After initial inoculation, cultures were grown in a 21 -day batch. At day 21 , cultures were diluted 20: 1 into new mLvPBRs. The second batch was also grown for 21 days, after which a second 20: 1 dilution step was performed. The third batch was 21 days in length as well, for a total of 9 weeks of cultivation. The effect of these several dilutions on various measurements, such as OD, ethanol levels, and the percentage of cells that were no longer able to produce ethanol, can be found in FIGs. 4A, 4B, 4C, and 4D.

[0234] The same strains were evaluated twice in mLvPBRs. For the first round of evaluation, 17 mM nitrate was used. The second round of evaluations used 23 mM nitrate. For both

evaluations, OD and ethanol measurements were taken three times per week.

[0235] Samples for PDC activity (FIG. 6A) and ADH activity (FIG. 6B) were taken weekly for the first three weeks of the evaluation.

[0236] Ethanol in the culture (FIG. 4B) was quantified by headspace analysis via gas

chromatography as described in the examples above. To correct for ethanol loss due to venting, ethanol concentration in the headspace was assumed to be in equilibrium with ethanol or acetaldehyde in the culture. Ethanol losses to the vapor phase were estimated using a vapor- liquid equilibrium (VLE) model. The venting-loss corrected ethanol data was calculated by adding the ethanol venting loss and the culture ethanol concentration. Data generated were then presented as VLE-corrected ethanol (%, v/v) (FIG. 4C).

[0237] Cell population analysis (FIG. 4D) was performed every week, utilizing either colony counting on a solid medium plate (for the first two weeks) or by immunofluorescence techniques. For the plate counting method, cultures were diluted to an OD750 of 1.0, then serially diluted to appropriate levels for obtaining countable numbers of revertant cells. The diluted samples were plated onto mBGl 1 plates containing Instant Ocean sea salt (Spectrum Brands, Blacksburg, VA, USA). Revertant cells were defined as colony forming units (CFUs) that appeared larger than other colonies. The larger colonies indicate the loss of ethanol production and thus were able to grow faster and produce larger colonies (as found by previous research, data not shown). The total number of cells plated was obtained by counting the lowest dilution, usually 10 ^"4 or 10 ^"5 .

[0238] An immunofluorescence method was used after the first two weeks of culture growth. This method used flow cytometry to determine the percentage of cells still producing the PDC enzyme. For this method, samples were fixed, immunostained to recognize the PDC protein, and then analyzed by use of flow cytometry.

Results:

[0239] Host cell growth rate: As shown in FIG. 4A, the control strain had a higher growth rate over time (as measured by OD75o), particularly during the later stages of the culture. By the third dilution, the effect was particularly pronounced.

[0240] Ethanol production: As shown in FIG. 4B, however, ethanol production was higher in the nirA complementation strain than in the control strain. Again, the difference became more pronounced during the later stages of the culture. A similar result was seen when the ethanol was corrected for vapor loss ("VLE corrected"). Thus, the nirA complementation strain was capable of producing much more ethanol over a longer time than the control strain. In fact, the control strain produced very little ethanol by the end of the run. This data clearly shows that the nirA complementation method, as an essential gene system, does indeed result in an increased stability of ethanol production in the cyanobacterial cells. The increased ethanol level in comparison to the control was particularly strong after several dilutions of the culture, as can be seen in FIG. 4B and FIG. 4C.

[0241] Genetic stability of production gene(s): A significant reason for this difference in ethanol production can be seen in FIG. 4D, showing that the control strain had a higher percentage of cells that were no longer making ethanol, which became more pronounced throughout the run. These cells had reverted by mutations (often in or upstream of the pdc gene) that inactivated the ethanol producing cassette. While the nirA complementation strains were likely to have a similar mutation rate as the control strains, the cells (in the nirA complementation strains) that mutated to inactivate ethanol production were also unable to express nirA, so those cells died, rather than being able to grow faster, as in the control strains. Because these cells could grow faster than the ethanol producing cells, they were capable of taking over the entire control culture by the end of the run: by week 9, the reversion rate of the control strain was almost 100%. Again, the difference between the induced culture having the essential operon system and the control became more pronounced over time, particularly after several culture dilutions occurred (FIG. 4D). The data shows that the nirA gene works well as an essential gene, and that the essential gene operon method is capable of increasing the stability of the ethanol producing cells over time. This is particularly useful for outdoor, long-term commercial growth of the cyanobacterial cultures to produce a product, such as ethanol. Example 8

Three gene nirA complementation/essential gene operon strains

[0242] The use of the essential operon system, with nirA as the essential gene, was also demonstrated with both ethanol production genes pdc and adh located immediately upstream of the nirA gene on the same operon. This gene arrangement generally necessitates that both pdc and adh be adequately expressed in order for the nirA gene to be expressed.

[0243] Respective pdc, adh, and nirA genes were cloned into an ethanologenic plasmid backbone. The ethanologenic plasmid backbone used was a pABICYAN01-6.8-based vector containing a kanamycin resistance marker. The promoter PnirA*2, pdc, intergenic sequence IScpcBA*l , adh, and nirA genes were PCR-amplified using plasmid vectors that contain the elements as templates. All of the PCR products were cloned into the pABICYAN01-6.8-based vector containing a kanamycin resistance marker to create plasmid #2286.

[0244] The nirA complement strain ABlCyanol :#2286 (SEQ ID NO: 68) was evaluated in photobioreactors (LvPBRs). All cultures were grown in 20mL mBGl 1 nitrate free (NF), 2 mM ammonium, and 2 mM urea directly from cryo preserved cultures. The cultures were then inoculated into 50 mL of the same medium. 50mL cultures were then inoculated into 700 mL bottles with natural well water supplemented with BG-1 1 nutrients containing 5.5 mM Urea to a starting density of OD7so of 0.2. The bottles were grown for 5 days with constant 1% CO2 bubbling before being used to inoculate the LvPBRs.

[0245] The following procedure describes the standard lab conditions under which a 1.2L "vertical photobioreactor" (LvPBR) was operated as well as the necessary parts, ports, etc. to construct this 1.2L vPBR. LvPBRs were constructed via fusion of 5 cm inner diameter, 5.7 cm outer diameter Pyrex tubing (National Scientific Company, Inc.) and Corning Pyrex 100-mL screw cap media storage bottles (Fisher Scientific 16157-103). A 28" section of Pyrex tubing was fused between the two top halves of a 100 mL glass media bottle including the screw cap threads so that port caps could be constructed on the bottom and top of the reator. An additional glass pH probe port was fused into the middle of the glass reactor at an angle. Eagle Laboratory Glass Company (Painesville, OH) performed all glassware fabrication. The filling volume was about 1.2 L. The LvPBR was equipped with several ports for operation: a sampling port (on the top of the LvPBR, [0246] a gasin port (on the bottom of the LvPBR), a pH probe port, a mediumin port (on the top of the LvPBR) ), a mediumout port (on the bottom of the LvPBR) and a gasout port (on the top of the LvPBR). The standard light conditions was a uniform light field from one side with 350 μπιοΐ m ^"2 s ^"1 at the LvPBR surface generated by a light panel which consists of 8 T5 54W 6500K fluorescent bulbs operating in a 12/12 hour day/night cycle. Introduction of gasses was done via a diffuser on the bottom reactor cap, which consisted of a 1.5" section of EPDM 3/16" inner diameter tubing, which was perforated 12 times in a concentric circle around the diffuser then attached to the reactor cap via a polypropylene luer fitting.

[0247] The mixing was realized via the ascending air bubbles through the liquid culture. The gas flow was operating in a constant sparging mode (day and night) with air enriched with max 4 % C0 ₂ introduced on demand via pH control (pH setpoint = 7.3, day and night) and a flow rate of 38 mL min ^"1 . The standard cell density for starting a cultivation experiment OD750nm = 0.2 in 35 psu natural well water amended with BG1 1 nutrients, 17 mM Nitrate and 0.75 mM Urea.

[0248] Each culture was inoculated into three LvPBR. Strains were inoculated by dilution of cultures into LvPBRs to an initial OD750 of 0.2 with an initial culture volume of ~1.2 L. Cultures were maintained at ambient air with a range of 25 to 28 °C during the night with an increase to ~ 33 °C during the photoperiod (due to heating from the light source). Light was supplied from one side of the reactor with a homogeneous light field set to 350 μπιοΐ photons m ^"2 s ^"1 . Introduction of gasses was done via medium walled EPDM diffusers. Gas flow was set 30 mL min ^"1 . C0 ₂ was delivered constantly from 0.5-5%; and, the percentage was controlled based on pH over time. After initial inoculation, cultures were grown in a 31 -day batch.

Example 9

The essential gene operon can be located on the chromosomal DNA

[0249] The production gene can also be inserted into the host cell chromosome, so that it is positioned directly upstream of the chosen essential gene. Using nirA as an example of the essential gene, a construct for homologous recombination is prepared based on the known sequence surrounding the chosen essential gene. The construct is designed so that once it is positioned in the cell, it will replace the promoter of the endogenous nirA gene. An inducible promoter is present upstream of the production gene. A kanamycin resistance gene is placed in the construct. After transformation, selection for the transformed sequence is performed by adding kanamycin, and selection for full segregation is performed by a sequential series of plating onto agar plates with increasing amounts of kanamycin. Full segregation is verified by PCR and agarose gel electrophoresis. The modified host cells are tested for their ability to make the product, as well as long term stability of the gene insertion in the host cell.

Example 10

RNA extraction and qRT-PCR

[0250] Cultures were sampled into 15 mL or 50 mL centrifuge tubes cooled on ice. When sampling respective culture, a total of approximately 15.2 OD750 was collected. Samples were centrifuged at 12,000 x g for 10 minutes at 4°C and placed back on ice once centrifugation was complete. The supernatant was pipetted off and discarded. Pellets were resuspended in 300 μί of RNA Resuspension Buffer (lOmM sodium acetate, 200mM sucrose, 5mM EDTA) and placed back on ice. Once all pellets were resuspended, samples were transferred off of ice. A volume of 900 μΙ_. of Z6 Extraction Buffer (8M guanidine hydrochloride, 50mM β-mercaptoethanol, 20mM EDTA, 20mM MES, pH7.0) was added and samples were incubated for 5 minutes at room temperature. During the incubation, samples were mixed by inverting (15 mL tubes) or swirling (50 mL tubes) the tubes three times every 1 minute. Each culture suspension was transferred to a 1.5 mL screw cap microcentrifuge tube, flash frozen using liquid nitrogen, and then stored at - 80°C until extraction.

[0251] Total RNA was extracted using known RNA extraction protocols. For this method, the previously processed frozen culture suspension was thawed on ice. Once thawed, each culture suspension was mixed well by pipetting then divided and transferred into 2 x 1.5 mL screw cap microcentrifuge tubes. To each tube, 300 of hot (65°C) Acidic Phenol was added and incubated for 15 minutes at 65°C in an Eppendorf Thermomixer at 1 ,400 rpm. Following the incubation, tubes were centrifuged for 2 minutes at 17,000 x g at 4°C. Each supernatant was transferred into a fresh 2.0 mL screw cap microcentrifuge tube. To each supernatant transferred, 0.5 volume of chloroform risoamyl alcohol (24:1) was added for phase extraction. Tubes were shaken vigorously by hand for 15 seconds to mix, then centrifuged for 5 minutes at 17,000 x g at 4°C. The aqueous phase was transferred to a new 2.0 mL screw cap microcentrifuge tube and 1 volume of phenol:chloroform:isoamyl alcohol (25:24: 1) was added. Tubes were shaken vigorously by hand for 15 seconds to mix, then centrifuged for 5 minutes at 17,000 x g at 4°C. The aqueous phase was transferred to a new 2.0 mL screw cap microcentrifuge tube and 1 volume of chloroform:isoamyl alcohol (24: 1) was added. Tubes were shaken vigorously by hand for 15 seconds to mix, then centrifuged for 5 minutes at 17,000 x g at 4°C. The aqueous phase was transferred to a new 2.0 mL screw cap microcentrifuge tube and 1 volume of chloroform:isoamyl alcohol (24:1) was added. Tubes were shaken vigorously by hand for 15 seconds to mix, then centrifuged for 5 minutes at 17,000 x g at 4°C. The replicate aqueous phases were transferred and combined at this step into one fresh 2.0 mL screw cap

microcentrifuge tube and 1 volume of chloroform:isoamyl alcohol (24:1) was added. Tubes were shaken vigorously by hand for 15 seconds to mix. Tubes were centrifuged for 5 minutes at 17,000 x g at 4°C. The aqueous phase was transferred to a fresh 2.0 mL round bottom flip snap- cap microcentrifuge tube and 1 volume of 100% isopropanol was added and pipetted up and down to mix. In order to precipitate the RNA, tubes were incubated at -20°C for a minimum of 2 hours.

[0252] Following the precipitation incubation, tubes were centrifuged for 30 minutes at 17,000 x g at 4°C. The supernatant was removed and the pellet was washed with 1 mL of 75% Ethanol. Tubes were centrifuged for 5 minutes at 13,000 x g at 4°C. The ethanol wash step was repeated for a total of 3 times. Following the removal of the ethanol after the last wash step, tubes were left open to air dry the pellet at room temperature for approximately 5 minutes. The pellet was resuspended in 75 μΐ _^ nuclease-free water and transferred to a fresh 1.5 mL V-bottom flip snap- cap microcentrifuge tube. RNA samples were kept on ice. The concentration (ng^L), 260/280 and 260/230 ratios were recorded using the NanoDrop. 260/280 ratio values were expected to be between 1.8 and 2.2, whereas 260/230 ratios were expected to be greater than 1.

[0253] RNA samples were treated with DNase and cleaned using the Invitrogen TURBO DNA- free kit from Life Technologies. qPCR using Applied Biosystems Power SYBR Green PCR Master Mix from Life Technologies was performed with primers specific to the ABICyanol enolase gene (refer to Table 5) to check for any residual genomic DNA contamination in the RNA samples. All RNA samples resulted in no amplification above threshold level and were deemed to be clear of DNA. Synthesis of cDNA was performed using the Invitrogen Superscript VILO cDNA Synthesis Kit from Life Technologies that utilizes random hexamer primers.

[0254] The expression of pdc and nirA was quantified using SYBR green based qRT-PCR. qRT- PCR reactions were carried out using Applied Biosystems Power SYBR Green PCR Master Mix from Life Technologies. Primers and sequences used for pdc and nirA can be referred to in Table 7. A single-stranded oligo (ssDNA equivalent to each respective cDNA) of pdc and nirA was chemically synthesized by IDT DNA Technologies. Respective oligo sequences can be referred to in Table 8. Each respective ssDNA oligo was dissolved and the stock was used to prepare solutions at 12 concentrations. A volume of 2.5 of the standard at each concentration was used in qRT-PCR per reaction in duplicate to generate the standard curve. The final standard curve concentration range for each oligo was 5x10 ⁷ copies to lxl 0 ² copies. A volume of 2.5 \iL ' of each sample was used as template such that cDNA was tested in duplicate and controls were tested singly. Transcript levels were expressed as copies per nanogram of total RNA, which was based on the respective standard curve generated using qRT-PCR from the known quantity of ssDNA oligo of the target gene as described above (FIG. 5).

Table 7: Primer names, respective targets and sequences

Table 8: ssDNA Oligo names and sequences

AAG AAG GTT TGA GGG AAA ATA GAG CAA AAG CCC GTT TAA TGT GGT TAA TTG ATA AAT GGG GT