# US 10,368,573 B2 · Reducing levels of nicotinic alkaloids in plants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,368,573 B2 - Document ID: US10368573B2 - Status: Granted - Kind: B2 - Application number: 15/858,599 - Filed: 2017-12-29 - Published: 2018-08-02 - Granted: 2019-08-06 - Earliest claimed: 2005-02-28 - Assignees: 22nd Century Limited, LLC - Applicants: 22nd Century Limited, LLC - Inventors: Takashi Hashimoto; Akira Kato - Web page: https://patentgrep.com/patent/US10368573B2 ## CPC - A24B 15/18: Treatment of tobacco products or tobacco substitutes - A24B 13/00: Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff (reconstituted tobacco products A24B3/14) - A24B 13/02: Flakes or shreds of tobacco - A24B 15/20: Biochemical treatment - A24B 15/243: {Nicotine} - A24D 1/00: Cigars; Cigarettes - A24F 47/00: Smokers' requisites not otherwise provided for - C07K 14/415: from plants - C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]} - C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine} - Y02E 50/10: Biofuels, e.g. bio-diesel - A24B: MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF - A24D: CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES OF CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES - A24F: SMOKERS' REQUISITES; MATCH BOXES; SIMULATED SMOKING DEVICES (inhaling appliances for medical purposes, shaped like cigars, cigarettes or pipes A61M15/06) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract Two genes, A622 and NBB1, can be influenced to achieve a decrease of nicotinic alkaloid levels in plants. In particular, suppression of one or both of A622 and NBB1 may be used to decrease nicotine in tobacco plants. ## Claims ### Claim 1 A method for reducing a nicotinic alkaloid in a plant comprising down-regulating A622 and NBB1 expression relative to a non-transformed control plant, wherein A622 is encoded by a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and (c) a nucleotide sequence that encodes a polypeptide with one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 and encoding a polypeptide with A622 expression; and wherein NBB1 is encoded by a nucleotide sequence selected from the group consisting of: (i) the nucleotide sequence set forth in SEQ ID NO: 3; (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; and (iii) a nucleotide sequence that encodes a polypeptide with one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ NO: 4 and encoding a polypeptide with NBB1 expression. ### Claim 2 (depends on 1) The method of claim 1, wherein the nicotinic alkaloid is nicotine. ### Claim 3 (depends on 1) The method of claim 1, wherein the nicotinic alkaloid is anatabine. ### Claim 4 A genetically engineered plant having reduced A622 and NBB1 expression relative to a non-transformed control plant, wherein A622 is encoded by a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and (c) a nucleotide sequence that encodes a polypeptide with one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 and encoding a polypeptide with A622 expression; and wherein NBB1 is encoded by a nucleotide sequence selected from the group consisting of: (i) the nucleotide sequence set forth in SEQ ID NO: 3; (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; and (iii) a nucleotide sequence that encodes a polypeptide with one or more conservative amino acid substitutions as compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 and encoding a polypeptide with NBB1 expression. ### Claim 5 (depends on 4) The plant of claim 4, wherein the plant is a tobacco plant. ### Claim 6 (depends on 5) The tobacco plant of claim 5, wherein the tobacco plant has reduced nicotinic alkaloid content relative to a non-transformed control plant. ### Claim 7 (depends on 6) The method of claim 6, wherein the nicotinic alkaloid is nicotine. ### Claim 8 (depends on 6) The method of claim 6, wherein the nicotinic alkaloid is anatabine. ### Claim 9 (depends on 6) A reduced-nicotine tobacco product comprising the tobacco plant of claim 6. ### Claim 10 (depends on 9) The reduced-nicotine tobacco product of claim 9, wherein the tobacco product is selected from the group consisting of a cigarette, cigarette tobacco, a cigar, cigar tobacco, pipe tobacco, chewing tobacco, snuff, leaf tobacco, shredded tobacco, and cut tobacco. ### Claim 11 A tobacco plant produced by a method comprising: (a) mutagenizing a plurality of tobacco plant cells thereby producing a plurality of mutagenized cells; screening plants generated from the mutagenized cells, or progeny produced therefrom, for reduced expression of A622 and NBB1; and (c) selecting one or more of the plants or progeny comprising a mutation in the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 3, wherein the plant or progeny has reduced nicotine content as compared to a control tobacco plant. ### Claim 12 (depends on 11) A tobacco product comprising the tobacco plant of claim 11, having a reduced expression of A622 and NBB1 relative to a control tobacco plant, wherein the tobacco product is selected from the group consisting of smoking cessation products, cigarettes, cigarette tobacco, cigars, cigar tobacco, snus, pipe tobacco, and chewing tobacco. ### Claim 13 (depends on 11) A product comprising the tobacco plant of claim 11, having a reduced expression of A622 and NBB1 relative to a control tobacco plant, wherein the product is selected from the group consisting of a food product, food ingredient, feed product, feed ingredient, nutritional supplement, and biofuel. ### Claim 14 A tobacco plant comprising plant cells into which one or more mutations are introduced into: (a) the region of SEQ ID NO: 1 that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and (b) the region of SEQ ID NO: 3 that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; wherein the plant exhibits reduced expression of a gene product encoded by SEQ ID NO: 1 and SEQ ID NO: 3 as compared to a control tobacco plant. ### Claim 15 (depends on 14) The tobacco plant of claim 14, wherein the tobacco plant exhibits a reduced nicotine level as compared to a control plant. ### Claim 16 (depends on 14) Seeds from the tobacco plant of claim 14, or progeny plant thereof, wherein the seeds comprise in their genomes the one or more mutations. ### Claim 17 (depends on 15) A product comprising the tobacco plant of claim 15, having reduced expression of a gene product encoded by SEQ ID NO: 1 and SEQ ID NO: 3 as compared to a control tobacco plant, and having a reduced level of nicotine as compared to a product produced from a control tobacco plant, wherein the product is selected from the group consisting of a tobacco product, food product, food ingredient, feed product, feed ingredient, nutritional supplement, and biofuel. ### Claim 18 (depends on 17) The tobacco product of claim 17, wherein the product is selected from the group consisting of smoking cessation products, cigarettes, cigarette tobacco, cigars, cigar tobacco, snus, pipe tobacco, and chewing tobacco.