AUTHOR=Feng Yingjie , Qi Wenyuan , Yang Jinchu , Liu Wenzhao , Yang Zongcan , Wang Ke , Mao Duobin , Huang Shen , Zhang Tingting 

TITLE=Macrotranscriptomics analysis for decoding the role of Klebsiella variicola H8 in aroma compound biosynthesis during fermentation of reconstituted tobacco leaf concentrate

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 13 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2025.1635651

DOI=10.3389/fbioe.2025.1635651

ISSN=2296-4185

ABSTRACT=Microbial fermentation shapes the reconstituted tobacco leaf concentrate’s (RTLC) chemical composition and sensory quality. This study employed macrotranscriptomic analysis to investigate how the aroma-enhancing bacterium Klebsiella variicola H8 modulates RTLC fermentation. High-throughput second-generation 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 h, 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 h, 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.