Construction of Bacillus subtilis chassis strain with enhanced α-amylase expression capability based on CRISPRi screening.

Bacillus subtilis has been widely used in the expression of recombinant proteins due to its food safe and powerful secretion characteristic, but the current production level cannot meet the increasing industrial needs. To enhance the production of recombinant protein, we first screened target key genes that are directly or indirectly involved in protein synthesis, using CRISPRi technology targeting the whole genome, with industrial valuable Bacillus stearothermophilus α-amylase as the model protein. Then the screened key genes were combined, yielding a chassis strain that owning enhanced protein expression capability.

Molecular Mechanism of Double-Displacement Retaining β-Kdo Glycosyltransferase WbbB.

Glycosyltransferases (GTs) are pivotal enzymes involved in glycosidic bond synthesis, which can lead to either retention or inversion of the glycosyl moiety's anomeric configuration. However, the catalytic mechanism for retaining GTs remains a subject of controversy. In this study, we employ MD and QM/MM metadynamics to investigate the double-displacement catalytic mechanism of the retaining β-Kdo transferase WbbB.

Conformational Switch of the 250s Loop Enables the Efficient Transglycosylation in GH Family 77.

Amylomaltases (AMs) play important roles in glycogen and maltose metabolism. However, the molecular mechanism is elusive. Here, we investigated the conformational dynamics of the 250s loop and catalytic mechanism of AM using path-metadynamics and QM/MM MD simulations.

Mechanistic Insights into How the Protonation State of D234 Dictates the Reactivity in β--Acetylhexosaminidase.

β--Acetylhexosaminidases (HEXs) play important roles in human diseases and the biosynthesis of human milk oligosaccharides. Despite extensive research, the catalytic mechanism of these enzymes remains largely unexplored. In this study, we employed quantum mechanics/molecular mechanics metadynamics to investigate the molecular mechanism of HEX (HEX), which has shed light on the transition state structures and conformational pathways of this enzyme.

Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design.