Multilevel metabolic engineering for enhanced synthesis of S-adenosylmethionine by Bacillus amyloliquefaciens.

OBJECTIVES: To enhance the de novo synthesis of SAM, the effects of several key genes on SAM synthesis were examined based on modular strategy, and the key genes were manipulated to obtain an engineered strain with high SAM production. RESULTS: In Bacillus amyloliquefaciens HSAM6, the deletion of argG gene to block aspartic acid branching degradation increased SAM titer to 254.78 ± 15.91 mg/L, up 18% from HSAM6. Subsequently, deleting the moaA gene to boost the supply of 5-methyltetrahydrofolate led to the stunted growth and the plummeting yield of SAM. Further improvement of strain growth by overexpression of the citA gene, while SAM synthesis was not significantly enhanced. Finally, the maximum SAM titer (452.89 ± 13.42 mg/L) was obtained by overexpression SAM2 gene using the multicopy plasmid. CONCLUSIONS: The deletion of argG gene and the overexpression of SAM2 gene significantly improved SAM synthesis in B. amyloliquefaciens.

Enhancement of S-adenosylmethionine production by deleting thrB gene and overexpressing SAM2 gene in Bacillus amyloliquefaciens.

OBJECTIVES: To improve the S-adenosylmethionine (SAM) production in methionine-free medium, effects of deleting genes of SAM decarboxylase (speD) and homoserine kinase (thrB) on SAM titers were investigated, and the SAM synthetase gene (SAM2) was also overexpressed. RESULTS: In B. amyloliquefaciens HSAM2, deleting speD to block the SAM utilization pathway significantly reduced the SAM titer. After knockout of thrB to block the branched pathway, the resulted mutant HSAM4 produced 143.93 mg/L SAM, increasing by 42% than HSAM2. Further plasmid-based expression of SAM2 improved the SAM titer to 226.92 mg/L, and final optimization of key fermentation parameters resulted in the maximum SAM titer of 412.01 mg/L in flasks batch fermentation. CONCLUSIONS: Deleting thrB and overexpressing SAM2 gene were efficient for enhanced SAM production in B. amyloliquefaciens. The maximum SAM titer in flasks batch fermentation was much higher than that of previous reports.

Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle.

BACKGROUND: S-Adenosylmethionine (SAM) is a critical cofactor involved in many biochemical reactions. However, the low fermentation titer of SAM in methionine-free medium hampers commercial-scale production. The SAM synthesis pathway is specially related to the tricarboxylic acid (TCA) cycle in Bacillus amyloliquefaciens. Therefore, the SAM synthesis pathway was engineered and coupled with the TCA cycle in B. amyloliquefaciens to improve SAM production in methionine-free medium. RESULTS: Four genes were found to significantly affect SAM production, including SAM2 from Saccharomyces cerevisiae, metA and metB from Escherichia coli, and native mccA. These four genes were combined to engineer the SAM pathway, resulting in a 1.42-fold increase in SAM titer using recombinant strain HSAM1. The engineered SAM pathway was subsequently coupled with the TCA cycle through deletion of succinyl-CoA synthetase gene sucC, and the resulted HSAM2 mutant produced a maximum SAM titer of 107.47 mg/L, representing a 0.59-fold increase over HSAM1. Expression of SAM2 in this strain via a recombinant plasmid resulted in strain HSAM3 that produced 648.99 mg/L SAM following semi-continuous flask batch fermentation, a much higher yield than previously reported for methionine-free medium. CONCLUSIONS: This study reports an efficient strategy for improving SAM production that can also be applied for generation of SAM cofactors supporting group transfer reactions, which could benefit metabolic engineering, chemical biology and synthetic biology.

Evaluation of the Biogenic Amines and Microbial Contribution in Traditional Chinese Sausages.

Biogenic amines (BAs) in sausages represent a health risk for consumers, and thus investigating the BAs accumulation mechanism is important to control the BAs. In this study, the BAs profiles of 16 typical Chinese sausage samples were evaluated, and 8 kinds of common BAs were detected from different samples. As a whole, the BAs contents of the majority of Chinese sausage samples were within the safe dosage range, except that the total BAs and histamine concentrations of sample HBBD were above the toxic dosage levels. Furthermore, the bacterial and fungal communities of the Chinese sausage samples were investigated by high-throughput sequencing analysis, and Staphylococcus, Bacillus, Lactococcus, Lactobacillus, Debaryomyces, and Aspergillus were identified as the predominant genera. Accordingly, 13 representative strains were selected from the dominant genera, and their BAs formation and degradation properties were evaluated. Finally, the results of fermented meats model experiment indicated that the Staphylococcus isolates including Staphylococcus pasteuri Sp, Staphylococcus epidermidis Se, Staphylococcus carnosus Sc1, Staphylococcus carnosus Sc2, and Staphylococcus simulans Ss could significantly reduce BAs, possessing the potential as the starter cultures to control the BAs in fermented meat products. The present study not only helped to explain the BAs accumulation mechanism in Chinese sausage, but also developed the candidates for potential BAs control in fermented meat products.

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.