Tetramethylpyrazine (TTMP) is a vital bioactive alkaloid and characteristic flavor compound in fermented foods. Our previous study found that fermentation of adlay by Bacillus subtilis BJ3-2 efficiently accumulates TTMP, whereas the underlying high-yield mechanism remains unclear. This study investigated the fermentation characteristics, gene transcription and protein expression of B. subtilis BJ3-2 in dehulled adlay (BDA) and soybean (BSB), respectively, and elucidated the mechanism responsible for high-yield TTMP production. The results showed that glutamate, leucine and phenylalanine were major free amino acids in BDA. The TTMP yield in BDA at 48 h (6.11 mg/g dry weight) was 360-fold higher than that in BSB. Transcriptomic and proteomic analysis demonstrated that compared with the soybean substrate, dehulled adlay substrate significantly up-regulated the expression of alsSD and ilvBH genes and their encoding proteins in B. subtilis BJ3-2, which were involved in C5-branched dibasic acid metabolism, 2-oxocarboxylic acid metabolism, and valine, leucine and isoleucine biosynthesis. Meanwhile, acetoin degradation was inhibited by down-regulating acetoin dehydrogenase complex (acoABCL) in citrate cycle, glycolysis/gluconeogenesis and carbon metabolism. Additionally, nitrogen metabolism pathway was transcriptionally enhanced to guarantee sufficient ammonium supply. Notably, protein-protein interaction and molecular docking analyses revealed that acetohydroxyacid synthase (ilvBH) interacted tightly with α-acetolactate decarboxylase (alsD), potentially forming a metabolic channel for acetoin synthesis. In conclusion, the efficient synthesis of TTMP in BDA was primarily attributed to the high synthesis and low degradation of acetoin, and the moderate synthesis of ammonium/ammonia. This study provided a theoretical basis for the targeted and efficient biosynthesis of TTMP.
Yong-Xue Ma, Hong Zhang, Rongrong Zhang et al.· Food Research International· 1 citation
The sustainable valorization of agricultural waste, such as chili pepper straw, is often challenged by the absence of effective microbes that can degrade cell wall components. In this study, metagenomic analysis found that Pseudomonadota was the dominant phylum in the carboxymethyl cellulose (CMC)-enriched microbial communities. In addition, a cellulolytic bacterial strain, designated as Cellulomonas iranensis 7–12, was isolated from naturally decayed chili pepper straw and identified by colony morphology, Gram staining, 16 S rRNA gene sequencing, and genome-based average nucleotide identity (ANI) analysis. Within 30 h, C. iranensis 7–12 displayed robust cellulolytic activity, causing nearly complete disintegration of filter paper, a cellulose model substrate. In contrast, chili pepper straw, a structurally more complex lignocellulosic substrate, was only partially degraded, with dry-weight loss increasing from 11.98% in the uninoculated control to 32.63% after 4 d of fermentation with C. iranensis 7–12. C. iranensis 7–12 exhibited a predominantly extracellular cellulase–xylanase activity profile, with extracellular xylanase activity reaching 3.41 U/mL and exceeding the measured cellulase activities. Whole-genome sequencing of C. iranensis 7–12 identified a complete 3.79-Mb circular chromosome and a diverse CAZyme repertoire, including glycoside hydrolase families related to cellulose and hemicellulose degradation, carbohydrate-binding modules, carbohydrate esterases, and secretion-associated proteins. Moreover, scanning electron microscopy (SEM) examination revealed that the surface and internal microstructure of chili pepper straw were disrupted. Similarly, Fourier-transform infrared (FTIR) spectroscopy analysis showed marked changes in the characteristic absorption bands associated with cellulose, hemicellulose, and lignin-containing structures, indicating partial degradation of polysaccharide components and lignin-associated structural alteration. Collectively, C. iranensis 7–12 shows great potential for the bioconversion of chili pepper straw and the high-performance microbes will be further developed for the effective use of biomass resources.
