US 12,270,038 B2
Targeted mutagenesis of tobacco berberine bridge enzyme-like nucleic acids
Ralph E. Dewey, Apex, NC (US); and Ramsey S. Lewis, Apex, NC (US)
Assigned to North Carolina State University, Raleigh, NC (US)
Filed by North Carolina State University, Raleigh, NC (US)
Filed on Jun. 27, 2023, as Appl. No. 18/341,799.
Application 18/341,799 is a continuation of application No. 17/532,438, filed on Nov. 22, 2021, granted, now 11,753,649.
Application 17/532,438 is a continuation of application No. 16/778,780, filed on Jan. 31, 2020, granted, now 11,214,813, issued on Jan. 4, 2022.
Application 16/778,780 is a continuation of application No. 15/780,354, granted, now 10,590,429, issued on Mar. 17, 2020, previously published as PCT/US2016/064758, filed on Dec. 2, 2016.
Claims priority of provisional application 62/263,151, filed on Dec. 4, 2015.
Prior Publication US 2023/0323377 A1, Oct. 12, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/82 (2006.01); A01H 5/12 (2018.01); C12N 15/52 (2006.01); A24D 1/00 (2020.01)
CPC C12N 15/8243 (2013.01) [A01H 5/12 (2013.01); C12N 15/52 (2013.01); C12N 15/8213 (2013.01); A24D 1/00 (2013.01)] 14 Claims
 
1. A method of producing a tobacco product comprising tobacco from a Nicotiana plant, the method comprising:
(1) combining in a Nicotiana plant:
(A) a modification that reduces the activity of at least one of BBLa, BBLb, and BBLc or reduces the expression of a nucleic acid encoding at least one of BBLa, BBLb, and BBLc; and
(B) a mutation in at least one endogenous polynucleotide or combination of polynucleotides selected from the group consisting of:
(i) a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO:1 and a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO: 2;
(ii) a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO: 1 and a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO: 3;
(iii) a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO:2; and
(iv) a polynucleotide having about 97% to about 100% sequence identity to SEQ ID NO:3,
such that the Nicotiana plant has a nicotinic alkaloid content that is reduced as compared to a plant that is not modified per (A) and does not comprise the mutations of (B); and
(2) producing a tobacco product from the Nicotiana plant.