Macrotranscriptomics Analysis for Decoding the Role of Klebsiella variicola H8 in Aroma Compound Biosynthesis During Fermentation of Reconstituted Tobacco Leaf Concentrate

Feng Yingjie¹Qi Wenyuan²Aamir Rasool^3*Jinchu Yang¹Wenzhao Liu¹Zongcan Yang¹Shen Huang²Tingting Zhang¹

• ¹Technical Centre, China Tobacco Henan Industrial Limited Company, Zhengzhou, Zhengzhou, China
• ²Zhengzhou University of Light Industry, Zhengzhou, China
• ³University of Balochistan, Quetta, Pakistan

Microbial fermentation shapes the reconstituted tobacco leaf concentrate's (RTLC) chemical composition and sensory quality. This study employed macrotranscriptomic analysis to investigate how the aromaenhancing bacterium Klebsiella variicola H8 modulates RTLC fermentation. High-throughput secondgeneration RNA sequencing revealed that the transcript abundance of K. variicola H8 increased from 5.92% at the start of fermentation to 14.78% at 16 hours, accompanied by the enrichment of other key genera such as Lactobacillus and Citrobacter. Differential gene expression analysis showed that K. variicola H8 transcription correlated strongly (R² = 0.85) with water-soluble sugar degradation, while nitrogen and potassium correlations were weaker (R² = 0.47 and 0.41, respectively). Notably, the upregulation of glycoside hydrolases-particularly GH78, GH13_25, GH31, and GH28-was associated with the release of key non-volatile aroma-enhancing compounds (NAECs), such as β-damascenone (13.24 µg/g), phenylethanol (7.12 µg/g), solanone (5.89 µg/g), dihydrokiwi lactone (6.03 µg/g), and benzyl alcohol (5.15 µg/g). Furthermore, expression levels of apoptosis-related genes increased at 36 hours, coinciding with a decline in sensory quality and aroma compound accumulation.These findings reveal the dynamic microbial and enzymatic processes underpinning NAEC production and provide a mechanistic basis for optimizing microbial fermentation in tobacco processing.Keyword Metatranscriptome; Klebsiella variicola H8; reconstituted tobacco leaf concentrate; glycoside hydrolases; aroma-enhancing compounds; microbial fermentation; gene expression profiling 1 Introduction Tobacco (Nicotiana tabacum) contains ~5,000 compounds, many of which have been reported to have various pharmacological activities (Banožić et al., 2020). Recently, the extraction of bioactive compounds from plants has attracted significant interest (Suleria et al., 2016;X. Zou et al., 2021).Microbial fermentation offers several advantages, such as a high conversion rate, high specificity, and the production of high-quality aroma compounds, and is therefore applied in a wide range of food industries (Hadj Saadoun et al., 2021;Ma et al., 2024;Zara & Fan, 2023). For example, the fermentation of reconstituted tobacco leaves (RTLCs) with K. variicola H8 resulted in a ~25% reduction in nicotine