Over the past 20 years, researchers have used multi-omics techniques to study microbial diversity and metabolic function on tobacco leaves. The unique metabolic function of tobacco microorganisms has attracted extensive attention from researchers, which is an important research field in tobacco industry to improve the intrinsic quality of tobacco leaf with microbial agents. The microorganisms are particularly rich on the surface of tobacco leaf, and their metabolic function is closely related to the change of tobacco leaf chemical composition. Some microorganisms have important metabolic functions, such as: degrading macromolecular and harmful substances in tobacco leaves, and they have different degradation rates and pathways for the substances. At present, many functions of tobacco leaf microorganisms have not been fully verified and analyzed. In the future, more novel culture methods are needed to screen and isolate microorganisms on the surface of tobacco leaves, deeply tap their metabolic potential, explore the application value of microorganisms in the tobacco industry, and further promote the innovation and development of the industry.

Sensory attributes strongly influence consumers’ preferences for products. The inoculation of the Klebsiella variicola H8 strain in a reconstituted tobacco leaf concentrate (RTLC) solution increased neutral aroma-enhancing compound (NAEC) production by 45%, decreased the nicotine level by 25%, decreased the water-soluble total sugar content by ~36%, and improved the sensory quality by 5.71%. The production of NAECs such as dihydrokiwi lactone (DHKL: 192.86%), 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene (THTMN: 177.77%), 2,4-di-tert-butylphenol (DTBP: 25%), 4-oxoisofolkone (OIFK: 116.66%,) 1,9-heptadecadiene-4,6-diyn-3-ol (HDD: 116.67%), β-damastrone (BDS: 116.67), and megastigmatrienone A (MSTA: 116.67%) was increased. A metagenomics analysis of the microbial community in the fermented RTLC (FRTLC) was performed to elucidate the mechanism by which NAECs were produced. As a result, 24 groups of functional genes were identified, and among them, five families of carbohydrate-active enzymes, (i) glycoside hydrolase (GH), (ii) glycosyltransferase (GT), (iii) polysaccharide lyase (PL), (iv) carbohydrate esterase (CE), and (v) auxiliary active enzyme (AA), were found to be positively correlated with the production of NAECs. However, among the GHs, the GHs annotated from the H8 strain chromosome displayed the highest relative abundance and a positive correlation with the production of NAECs. Specifically, the GH13-14, GH13-20, GH13-38, GH13-25, GH13-10, GH42, and GH28 genes of the H8 strain were relatively more abundant and were key contributors to the production of NAECs. The correlation analyses revealed that the H8 strain plays a leading role among all the microorganisms in FRTLC in the production of NAECs. Our findings support the application of Klebsiella variicola in NAEC production and a reduction in nicotine content in tobacco products.

Alpha-cembratriene-4,6-diol (α-CBT-diol) is a complex diterpenoid primarily found in Solanaceae (i.e., tobacco leaves), Pinaceae, and marine corals. Due to its intricate chemical structure, it serves as a precursor for several aroma compounds, including farnesal. Farnesal and its derivatives have applications across various fields, such as the fragrance and flavor industry, pharmaceuticals, agriculture, and cosmetics. In this study, Stenotrophomonas maltophilia H3-1, a strain capable of efficiently biodegrading α-CBT-diol into farnesal, was isolated from soil and identified through 16S rDNA sequence analysis. S. maltophilia H3-1 biodegraded 93.3% of α-CBT-diol (300 mg/L) within 36 h when grown under optimized culture conditions, including a temperature of 40 °C, pH of 8, 2 g/L maltose, and 2 g/L ammonium sulfate. Theoretically, this strain can produce 201 mg/L of farnesal during the biotransformation of α-CBT-diol. The putative α-CBT-diol bioconversion pathway expressed in S. maltophilia H3-1 is also proposed. This is the first study to report the bioconversion of α-CBT-diol into the high-value compound farnesal using a novel S. maltophilia H3-1 strain. It highlights that other compounds found in tobacco can also be bioconverted into valuable products.