# US 2014/0154762 A1 · Genetically Enhanced Cyanobacteria Lacking Functional Genes Conferring Biocide Resistance for the Production of Chemical Compounds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154762 A1 - Document ID: US20140154762A1 - Status: Published application - Kind: A1 - Application number: 14/132,012 - Filed: 2013-12-18 - Published: 2014-06-05 - Earliest claimed: 2011-06-24 - Assignees: Algenol Biofuels Inc. - Inventors: Ulf DUEHRING; James LEE; Kerstin BAIER - Web page: https://patentgrep.com/patent/US20140154762A1 ## CPC - C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine} - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)} - C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes} - C12P 7/065: {with microorganisms other than yeasts} - Y02E 50/10: Biofuels, e.g. bio-diesel - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract One embodiment of the invention provides a genetically enhanced cyanobacterium producing a first chemical compound comprising: a genetically enhanced genome with a first gene inactivation in a first essential or conditionally essential gene of the cyanobacterium, and a first extrachromosomal plasmid harboring the first essential or conditionally essential gene and at least one first production gene for production of the first chemical compound, wherein the cyanobacterium lacks a functional gene conferring biocide resistance. These cyanobacteria can produce a first valuable chemical compound in long-term cultures without the need to employ genes conferring biocide resistance. ## Claims ### Claim 1 A genetically enhanced cyanobacterium producing ethanol, comprising: a) a genetically enhanced genome with a first gene inactivation in a first essential or conditionally essential gene of the cyanobacterium selected from the group consisting of smtAB, leuB, ziaRA, corRT, narB and pyrF; and b) a first extrachromosomal plasmid harboring the first essential or conditionally essential gene and at least one first production gene for the production of ethanol, wherein the at least one first production gene for the production of ethanol encodes an enzyme selected from the group consisting of Adh, Pdc, AdhE and combinations thereof; wherein the cyanobacterium lacks a functional gene conferring biocide resistance, further wherein the genome harbors more than one copy of the first essential or conditionally essential gene and wherein all copies of the first essential gene carry at least one gene inactivation, and further wherein said genetically enhanced cyanobacterium produces ethanol. ### Claim 2 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first gene inactivation comprises an at least partial deletion of the first essential or conditionally essential gene. ### Claim 3 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first gene inactivation comprises a complete deletion of the first essential or conditionally essential gene. ### Claim 4 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first extrachromosomal plasmid comprises an endogenous plasmid of the cyanobacterium into which the first essential or conditionally essential gene and the at least one first production gene are integrated. ### Claim 5 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the at least one production gene comprises at least two first and second production genes coding for separate first and second production enzymes, which produce ethanol. ### Claim 6 (depends on 5) The genetically enhanced cyanobacterium of claim 5, wherein the first and second production genes are transcriptionally controlled by different promoters. ### Claim 7 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the essential or conditionally essential gene is transcriptionally controlled by a different promoter than the at least one first production gene. ### Claim 8 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first essential gene encodes one of the following: a) a first essential biocatalyst which is involved in the production of a first essential factor, which cannot promote complete genetic segregation of the cyanobacterium with regard to the first gene inactivation if present in the growth medium of the cyanobacteria; or b) the first essential factor itself, which cannot promote complete genetic segregation of the cyanobacterium with regard to the first gene inactivation if present in the growth medium of the cyanobacteria. ### Claim 9 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the at least one first production gene is under the transcriptional control of an inducible or constitutive promoter. ### Claim 10 (depends on 1) The genetically enhanced cyanobacterium of claim 1, further comprising: a) a second gene inactivation in a second essential or conditionally essential gene in the genome of the cyanobacterium, wherein the second essential or conditionally essential gene is different from the first essential or conditionally essential gene; and b) at least one second production gene different from the first production gene; wherein the second essential or conditionally essential gene and the second production gene are included on either the first extrachromosomal plasmid or on a second extrachromosomal plasmid. ### Claim 11 (depends on 10) The genetically enhanced cyanobacterium of claim 10, wherein the second production gene encodes an endogenous enzyme of the cyanobacterium, the expression of the endogenous enzyme resulting in an increased rate of production of ethanol compared to the respective cyanobacterium harboring the first production gene, but lacking the second production gene. ### Claim 12 (depends on 11) The genetically enhanced cyanobacterium of claim 11, wherein the endogenous enzyme is selected from the group consisting of enzymes of the glycolysis pathway, Calvin cycle, intermediate steps of metabolism, amino acid metabolism, the fermentation pathway and the citric acid cycle. ### Claim 13 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first gene inactivation comprises a partial deletion of the first essential or conditionally essential gene and wherein the first extrachromosomal plasmid harbors a gene which is homologous to the first essential or conditionally essential gene inactivated by the first gene inactivation. ### Claim 14 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first gene inactivation comprises a complete deletion of the first essential or conditionally essential gene and wherein the first extrachromosomal plasmid harbors the same first essential or conditionally essential gene inactivated by the first gene inactivation. ### Claim 15 (depends on 1) The genetically enhanced cyanobacterium of claim 1, wherein the first essential or conditionally essential gene included on the first extrachromosomal plasmid encodes an essential or conditionally essential biocatalyst, which is homologous or analogous to the biocatalyst encoded by the first essential or conditional essential gene in the genome of the cyanobacterium harboring the first gene inactivation. ### Claim 16 A genetically enhanced cyanobacterium producing ethanol, comprising: a) a genetically enhanced genome with a first gene inactivation in a first conditionally essential gene of the cyanobacterium; wherein the first conditionally essential gene is selected from the group consisting of narB, ziaA, smtA, corT, and combinations thereof; and b) a first extrachromosomal plasmid harboring the first essential or conditionally essential gene and at least one first production gene for the production of ethanol; wherein the cyanobacterium lacks a functional gene conferring biocide resistance. ### Claim 17 A genetically enhanced cyanobacterium producing ethanol, comprising: a) a genetically enhanced genome with a first gene inactivation in a first essential or conditionally essential gene of the cyanobacterium; and b) a first extrachromosomal plasmid harboring the first essential or conditionally essential gene and at least one first production gene coding for Pdc enzyme and a second production gene coding for Adh enzyme, wherein a transcription terminator sequence is present between the first and second production gene and wherein the first production gene is controlled by an inducible promoter and the second production gene is under the transcriptional control of a constitutive promoter; wherein the cyanobacterium lacks a functional gene conferring biocide resistance. ### Claim 18 (depends on 17) The genetically enhanced cyanobacterium of claim 17, showing an ethanol production rate from 0.024%(v/v)d−1 to 0.032% (v/v)d−1. ### Claim 19 A method for producing ethanol, comprising the method steps of: a) obtaining a genetically enhanced cyanobacterium producing ethanol, comprising i) a genetically enhanced genome with a first gene inactivation in a first essential or conditionally essential gene of the cyanobacterium selected from the group consisting of smtAB, leuB, ziaRA, corRT, narB and pyrF; and ii) a first extrachromosomal plasmid harboring the first essential or conditionally essential gene and at least one first production gene for the production of ethanol, wherein the at least one first production gene for the production of ethanol encodes an enzyme selected from the group consisting of Adh, Pdc, AdhE and combinations thereof; wherein the cyanobacterium lacks a functional gene conferring biocide resistance, further wherein the genome harbors more than one copy of the first essential or conditionally essential gene and wherein all copies of the first essential gene carry at least one gene inactivation; b) culturing said genetically enhanced cyanobacterium in the absence of a biocide, the cyanobacterium producing ethanol; and c) recovering the ethanol. ### Claim 20 (depends on 19) The method of claim 19, wherein the cyanobacterium harbors a first gene inactivation in a first conditionally essential gene and in method step a) the cyanobacteria are cultured under a condition rendering the first conditionally essential gene an essential gene. ### Claim 21 (depends on 1) A method for producing genetically enhanced cyanobacteria according to claim 1, comprising the method steps of: i) transforming the cyanobacterium by introducing a first gene inactivation into a first essential or conditionally essential gene of the cyanobacterium; and ii) introducing the first extrachromosomal plasmid harboring the first essential or conditionally essential gene and the at least one first production gene into the genetically enhanced cyanobacterium. ### Claim 22 (depends on 21) The method of claim 21, wherein method step i) comprises replacing at least a part of the first essential or conditionally essential gene of the cyanobacterium with a recombinant nucleic acid sequence, thereby creating the first gene inactivation. ### Claim 23 (depends on 21) The method of claim 21, wherein method step i) comprises the following substeps: i1) transforming the cyanobacteria with a first recombinant nucleic acid sequence, wherein the nucleic acid sequence comprises a first selectable gene conferring resistance to a selectable marker and a second counterselectable gene conferring sensitivity to a counterselectable marker; i2) selecting for transformed cyanobacteria by subjecting the cyanobacteria to the selectable marker; i3) transforming the cyanobacteria obtained from step i2) with a second recombinant nucleic acid sequence lacking the first selectable and second counterselectable gene by replacing at least a part of the first recombinant nucleic acid sequence, thereby creating transformed cyanobacteria lacking a functional first selectable and functional second counterselectable gene; i4) selecting for transformed cyanobacteria from step i3) via subjecting the cyanobacteria to the counterselectable marker; and i5) introducing the extrachromosomal plasmid into the cyanobacteria obtained from step i4). ### Claim 24 (depends on 21) The method of claim 21, wherein the genome of the cyanobacteria harbors more than one copy of the first essential gene, the essential gene encoding a biocatalyst for the production of a first essential factor, which cannot promote complete genetic segregation of the cyanobacterium with regard to the first gene inactivation if present in the growth medium and which includes the following substeps: i′1) first gene inactivations are created in not all copies of the first essential gene by replacing at least parts of the first essential gene by a first recombinant nucleic acid; i′2) selecting for the cyanobacteria obtained in substep i′1) by subjecting the cyanobacteria to the first selectable marker, followed by step ii) of introducing the first extrachromosomal plasmid harboring the first essential or conditionally essential gene and the at least one first production gene into the genetically enhanced cyanobacteria of substep I′2); i′3) subjecting the cyanobacteria from step ii) to concentrations of the first selectable marker higher than the concentration used in substep i′2); i′4) transforming the cyanobacteria obtained from step i′3) with the second recombinant nucleic acid; and i′5) selecting for cyanobacteria obtained in substep i′4) by subjecting the cyanobacteria to the counterselectable marker.