The present disclosure provides the identification of tobacco Nic1 locus. Also provided are tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants. Also provided are compositions and methods for producing tobacco plants having novel Nic1 mutations or alleles to reduce nicotine levels. Further provided are sequence polymorphisms and molecular markers for breeding tobacco with reduced nicotine or nicotine free while maintaining tobacco leaf grade and tobacco product quality.

The present disclosure provides approaches for reducing tobacco-specific nitrosamines (TSNAs) in tobacco. Some of these approaches include genetically engineering tobacco plants to increase one or more antioxidants, increase oxygen radical absorbance capacity (ORAC), increase phenylalanine, or reduce nitrite. Also provided are methods and compositions for producing modified tobacco plants and tobacco products therefrom comprising reduced TSNAs.

Methods of modifying the tobacco-specific nitrosamine content of a tobacco material are described herein. One exemplary method comprises contacting a tobacco material with a composition comprising salt, sugar, enzyme, lactic acid bacteria, yeast, or a combination thereof to reduce the total bacterial content; curing the tobacco material; and fermenting the tobacco material in the presence of one or more microorganisms. The method can provide a fermented tobacco material having a tobacco-specific nitrosamine content that is reduced relative to a fermented tobacco material that has not been subjected to the disclosed method steps. In certain embodiments, the tobacco-specific nitrosamine content of the fermented tobacco material is no more than that of the cured tobacco material. Tobacco-containing products including such treated tobacco materials are also provided.

Methods of modifying the tobacco-specific nitrosamine content of a tobacco material are described herein. One exemplary method comprises contacting a tobacco material with a composition comprising salt, sugar, enzyme, lactic acid bacteria, yeast, or a combination thereof to reduce the total bacterial content; curing the tobacco material; and fermenting the tobacco material in the presence of one or more microorganisms. The method can provide a fermented tobacco material having a tobacco-specific nitrosamine content that is reduced relative to a fermented tobacco material that has not been subjected to the disclosed method steps. In certain embodiments, the tobacco-specific nitrosamine content of the fermented tobacco material is no more than that of the cured tobacco material. Tobacco-containing products including such treated tobacco materials are also provided.

Methods and compositions are provided for expressing an endolysin in a bacterium or in a tobacco plant or plant part. Expression of the endolysin can reduce the total bacterial load on the plant and in tobacco products produced from the plant. Accordingly, the level of tobacco specific nitrosamines (TSNA) in a cured tobacco product can be decreased when the tobacco leaves used in the product were harvested from a plant expressing endolysin or when bacteria modified to express endolysin is used to treat the tobacco. Thus, bacteria and tobacco plants and plant parts are provided that express an endolysin. Further provided are methods for decreasing the microbial load on a tobacco plant and in a tobacco product by applying a solution of bacteria modified to express an endolysin or by modifying a tobacco plant to express endolysins.

Smokeless tobacco products including such modified tobacco plant parts are also provided.

The present invention features tobacco nicotine demethylase nucleic acid and amino acid sequences, tobacco plants and plant components containing such sequences, including tobacco plants and plant components having reduced expression or altered enzymatic activity of nicotine demethylase, methods of use of nicotine demethylase sequences to create plants having altered levels of nornicotine or N-nitrosonornicotine (NNN) or both relative to a control plant, as well as tobacco articles having reduced levels of nornicotine or NNN.

The present invention features tobacco nicotine demethylase nucleic acid and amino acid sequences, tobacco plants and plant components containing such sequences, including tobacco plants and plant components having reduced expression or altered enzymatic activity of nicotine demethylase, methods of use of nicotine demethylase sequences to create plants having altered levels of nornicotine or N-nitrosonornicotine (NNN) or both relative to a control plant, as well as tobacco articles having reduced levels of nornicotine or NNN.

The present disclosure provides tobacco Nic1b locus and associated genes. Also provided are tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants. Further provided are compositions and methods for producing tobacco plants having novel mutations or alleles to reduce nicotine levels. Further provided are sequence polymorphisms and molecular markers for breeding tobacco with reduced nicotine or alkaloids while maintaining tobacco leaf grade and tobacco product quality.

Bacteriophage are provided, and methods of making and using the bacteriophage also are provided. The bacteriophage can be provided in a tobacco product, where the bacteriophage reduces the number of viable bacteria in the tobacco product and includes a nucleic acid sequence encoding an endolysin having at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1 or to the amino acid sequence shown in SEQ ID NO: 2.

Bacteriophage are provided, and methods of making and using the bacteriophage also are provided. The bacteriophage can be provided in a tobacco product, where the bacteriophage reduces the number of viable bacteria in the tobacco product and includes a nucleic acid sequence encoding an endolysin having at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1 or to the amino acid sequence shown in SEQ ID NO: 2.