Identification of a Key Gene Involved in Branched-Chain Short Fatty Acids Formation in Natto by Transcriptional Analysis and Enzymatic Characterization in Bacillus subtilis

Abstract

Natto as a fermented soybean product has many health benefits for human due to its rich nutritional and functional components. However, the unpleasant odor of natto, caused by the formation of branched-chain short fatty acids (BCFAs), prohibits the wide acceptance of natto products. This work is to identify the key gene of BCFAs formation and develop the guidance to reduce natto odor. Transcriptional analysis of BCFAs synthesis pathway genes was conducted in two Bacillus subtilis strains with obvious different BCFAs synthesis abilities. The transcriptional levels of bcd, bkdAA, and ptb in B. subtilis H-9 were 2.7-fold, 0.7-fold, and 8.9-fold higher than that of B. subtilis H-4, respectively. Therefore, the ptb gene with the highest transcriptional change was considered as the key gene in BCFAs synthesis. The ptb encoded enzyme Ptb was further characterized by inducible expression in Escherichia coli. The recombinant Ptb protein (about 32 kDa) was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The catalysis functions of Ptb were confirmed on substrates of isovaleryl-CoA and isobutyryl-CoA, and the higher catalysis efficiency of Ptb on isovaleryl-CoA explained the higher level of isovaleric acid in natto. The optimal activities of Ptb were observed at 50 °C and pH 8.0, and the enzymatic activity was inhibited by Ca, Zn, Ba, Mn, Cu, SDS, and EDTA. Collectively, this study reports a key gene responsible for BCFAs formation in natto fermentation and provides potential strategies to solve the odor problem.

Dioxin impacts on lipid metabolism of soil microbes: towards effective detection and bioassessment strategies

“…However, an opposite scenario, with low ratios of BCFAs/SCFAs and SFAs/USFAs, was found in TCDD-exposed bacteria (Hanano et al 2019b). This observation is consistent with earlier reports suggesting that adaptation of certain Bacilli to environmental changes, such as decreasing temperature and pH, involves a reduced level of BCFAs Table 2 Environmental and an increased level of USFAs (Silbert et al 1973;Kaneda 1977;Chen et al 2017b;Hong et al 2017). In other words, TCDD-exposed bacteria exhibit a specific lipidic "signature" due to the presence of BCUFAs.…”

Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids

“…In the cascade reaction system, an amino acid dehydrogenase is needed to recycle the amino donor by consuming ammonium and NAD(P)H. Although more than 24770 amino acid sequences of amino acid dehydrogenases are stored in the UniProt database, only a few have considerable potential as biocatalysts . Among them, five distinct AADHs, namely alanine dehydrogenase (AlaDH), leucine dehydrogenase (LeuDH), glutamate dehydrogenase (GluDH), phenylalanine dehydrogenase (PheDH), and meso ‐diaminopimelate dehydrogenase (DAPDH), were employed herein for this recycling purpose. Finally, two well‐studied ADHs (BsADH and TbADH) were chosen to regenerate the cofactor NAD(P)H. All of these enzymes were successfully cloned, overexpressed, and purified (see Figure S1), and exhibited excellent stability during purification and subsequent storage, indicating their high suitability for industrial applications.…”

Production of antibacterial compounds using Bacillus spp. isolated from thermal springs in Saudi Arabia

“…Furthermore, it has also been isolated from Bacillus spp. including B. amyloliquefaciens ( Chen et al, 2017 ), B. subtilis ( Hong et al, 2017 , Mumtaz et al, 2019 ), B. velezensis ( Calvo et al, 2020 ), B. cereus and Paenibacillus polymyxa ( Mumtaz et al, 2019 ).…”