Identification of a Novel Chitinase from Bacillus paralicheniformis: Gene Mining, Sequence Analysis, and Enzymatic Characterization.

In this study, a novel strain for degrading chitin was identified as Bacillus paralicheniformis HL37, and the key chitinase CH1 was firstly mined through recombinant expression in Bacillus amyloliquefaciens HZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes in B. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1.

Molecular Mechanism of Pasteurized Akkermansia muciniphila in Alleviating Type 2 Diabetes Symptoms.

Type 2 diabetes (T2DM) significantly diminishes people's quality of life and imposes a substantial economic burden. This pathological progression is intimately linked with specific gut microbiota, such as Akkermansia muciniphila. Pasteurized A. muciniphila (P-AKK) has been defined as a novel food by the European Food Safety Authority and exhibited significant hypoglycemic activity. However, current research on the hypoglycemic activity of P-AKK is limited to the metabolic level, neglecting systematic exploration at the pathological level. Consequently, its material basis and mechanism of action for hypoglycemia remain unclear. Drawing upon this foundation, we utilized high-temperature killed A. muciniphila (H-K-AKK) with insignificant hypoglycemic activity as the control research object. Assessments were conducted at pathological levels to evaluate the hypoglycemic functions of both P-AKK and H-K-AKK separately. Our study unveiled for the first time that P-AKK ameliorated symptoms of T2DM by enhancing the generation of glucagon-Like Peptide 1 (GLP-1), with pasteurized A. muciniphila total proteins (PP) being a pivotal component responsible for this activity. Utilizing SDS-PAGE, proteomics, and molecular docking techniques, we deeply analyzed the material foundation of PP. We scientifically screened and identified a protein weighing 77.85 kDa, designated as P5. P5 enhanced GLP-1 synthesis and secretion by activating the G protein-coupled receptor (GPCR) signaling pathway, with free fatty acid receptor 2 (FFAR-2) being identified as the pivotal target protein for P5's physiological activity. These findings further promote the widespread application of P-AKK in the food industry, laying a solid theoretical foundation for its utilization as a beneficial food ingredient or functional component.

Transcriptome analysis reveals the underlying mechanism for over-accumulation of alkaline protease in Bacillus licheniformis.

AIMS: Bacillus licheniformis AQ is an industrial strain with high production of alkaline protease (AprE), which has great industrial application value. However, how to regulate the production of AprE in the process of industrial fermentation is still not completely clear. Therefore, it is important to understand the metabolic process of AprE production in the industrial fermentation medium. METHODS AND RESULTS: In this study, transcriptome sequencing of the whole fermentation course was performed to explore the synthesis and regulation mechanism of AprE in B. licheniformis AQ. During the fermentation process, the AprE got continuously accumulated, reaching a peak of 42 020 U/mL at the fermentation endpoint (48 h). Meanwhile, the highly expressed genes were observed. Compared with the fermentation endpoint, there were 61 genes in the intersection of differentially expressed genes, functioning as catabolic processes, peptidases and inhibitors, chaperones, and folding catalysts. Furthermore, the protein-protein interactions network of AprE was constructed. CONCLUSION: This study provides important transcriptome information for B. licheniformis AQ and potential molecular targets for further improving the production of AprE.

An Efficient Prephenate Dehydrogenase Gene for the Biosynthesis of L-tyrosine: Gene Mining, Sequence Analysis, and Expression Optimization.

L-tyrosine is a key precursor for synthesis of various functional substances, but the microbial production of L-tyrosine faces huge challenges. The development of new microbial chassis cell and gene resource is especially important for the biosynthesis of L-tyrosine. In this study, the optimal host strain Bacillus amyloliquefaciens HZ-12 was firstly selected by detecting the production capacity of L-tyrosine. Subsequently, the recombinant expression of 15 prephenate dehydrogenase genes led to the discovery of the best gene, Bao-tyrA from B. amyloliquefaciens HZ-12. After the overexpression of Bao-tyrA, the L-tyrosine yield of the recombinant strain HZ/P43-Bao-tyrA reach 411 mg/L, increased by 42% compared with the control strain (HZ/pHY300PLK). Moreover, the nucleic acid sequence and deduced amino acid sequence of the gene Bao-tyrA were analyzed, and their conservative sites and catalytic mechanisms were proposed. Finally, the expression of Bao-tyrA was regulated through a promoter and 5'-UTR sequence to obtain the optimal expression elements. Thereby, the maximum L-tyrosine yield of 475 mg/L was obtained from HZ/P43-UTR3-Bao-tyrA. B. amyloliquefaciens was applied for the first time to produce L-tyrosine, and the optimal prephenate dehydrogenase gene Bao-tyrA and corresponding expression elements were obtained. This study provides new microbial host and gene resource for the construction of efficient L-tyrosine chassis cells, and also lays a solid foundation for the production of various functional tyrosine derivatives.

A comprehensive review of spermidine: Safety, health effects, absorption and metabolism, food materials evaluation, physical and chemical processing, and bioprocessing.

Spermidine, a natural autophagy inducer, has a variety of health effects, such as antitumor, antiaging, anti-inflammation, cardiovascular protection, and neuromodulation. It has been a hot topic in the field of food processing, and current research findings suggest that spermidine-rich foods may be used in intervention and prevention of age-related diseases. In this article, recent findings on the safety, health effects, absorption and metabolism of spermidine were reviewed, and advances in food processing, including the raw materials evaluation, physical and chemical processing, and biological processing of spermidine, were highlighted. In particular, the core metabolic pathways, key gene targets, and efficient metabolic engineering strategies involved in the biosynthesis of spermidine and its precursors were discussed. Moreover, limitations and future perspectives of spermidine research were proposed. The purpose of this review is to provide new insights on spermidine from its safety to its food processing, which will advance the commercial production and applications of spermidine-rich foods and nutraceuticals.

Biosynthesis of a Novel Bioactive Metabolite of Spermidine from Bacillus amyloliquefaciens: Gene Mining, Sequence Analysis, and Combined Expression.

Spermidine is a biologically active polyamine with extensive application potential in functional foods. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders its industrial application. To improve the spermidine titer, key genes affecting the spermidine production were mined to modify Bacillus amyloliquefaciens. Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Gene and amino acid sequence analysis further confirmed the function of speD and speE. Then, these two genes were co-expressed to generate a recombinant strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 105.2 mg/L, improving by 11.0-fold compared with the control (HSAM2). Through optimization of the fermentation medium, the spermidine titer was increased to 227.4 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding its synthesis pattern. In conclusion, two critical genes for spermidine synthesis were obtained, and an engineering B. amyloliquefaciens strain was constructed for enhanced spermidine production.

Biogenic amines analysis and microbial contribution in traditional fermented food of Douchi.

Biogenic amines (BAs) have been reported to threaten the Douchi safety, while the BAs formation mechanism and corresponding control method have not been clarified for Douchi. The present study aims to investigate the microbial contribution to BAs in Douchi, and to find the beneficial strain for BAs control. Firstly, the BAs profiles of 15 Douchi samples were analyzed, and common 6 kinds of BAs were detected from different samples. All the samples showed the total BAs contents within the safe dosage range, while the histamine concentrations in 2 samples and ß-phenethylamine in 6 samples were above the toxic level. Then, the bacterial and fungal communities were investigated by high-throughput sequencing analysis, and Bacillus and Candida were identified as the dominant bacteria and fungi genus, respectively. Furthermore, nineteen strains were selected from the dominant species of Douchi samples, including 14 Bacillus strains, 2 Staphylococcus strains, 1 Enterococcus strain and 2 Candida strains, and their BAs formation and degradation abilities were evaluated. B. subtilis HB-1 and S. pasteuri JX-2 showed no BAs producing ability, and B. subtilis GD-4 and Candida sp. JX-3 exhibited high BAs degradation ability. Finally, fermented soybean model analysis further verified that B. subtilis HB-1 and S. pasteuri JX-2 could significantly reduce BAs. This study not only contributed to understanding the BAs formation mechanism in Douchi, but also provided potential candidates to control the BAs in fermented soybean products.