To prove that the phospholipase A₁ accessory protein PlaS of Serratia marcescens has a significantly disruptive effect on the cell membrane of Escherichia coli BL21 (DE3) during heterologous expression, changes in cell membrane properties were explored, their effect on cell membrane fatty acids was identified by gas chromatography-mass spectrometry (GC-MS), and subcellular localization was observed by laser confocal microscopy. The results showed that after the heterologous expression of PlaS, the inner and outer membrane permeability of the host bacterium was significantly increased, and cell membrane fluidity and surface hydrophobicity were severely decreased, indicating loss of cell membrane functions; the saturation degree of fatty acids was decreased, that is, the relative contents of straight-chain saturated fatty acids and trans-monounsaturated fatty acids in fatty acids were reduced, and the relative contents of cis-monounsaturated fatty acids were increased. Moreover, membrane rigidity was enhanced, membrane fluidity was reduced, and the cell membrane components and structure were abnormal, so that the cells could be susceptible to death. Laser confocal microscopy showed that significant fluorescence was observed in the host cell membrane. In summary, the heterologous expression of the membrane protein PlaS in E. coli disrupts the structure and function of the host cell membrane and exhibits growth inhibition, which will provide support for further research on the mechanism of PlaS inhibition and its functional development.

In order to improve the ability of a wild-type strain (BY-1) of Pichia pastoris to produce glutathione (GSH), a mutant strain designated BY-1-26 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis. To further improve the production of glutathione by the mutant during shake flask culture, 1,2,4-triazole was added to the culture medium. Finally, adaptive evolution of the mutant strain was carried out on a microbial microdroplet culture system (MMC) using 1,2,4-triazole as a screening factor. As a result, a high-yield glutathione-producing mutant strain BY-2-24 was obtained, and its genetic stability was investigated. The results showed that a high-yield glutathione-producing mutant strain could be obtained using the wild-type strain BY-1 by ARTP mutagenesis, primary resistance screening on gradient plates, adaptive evolution on MMC and shake-flask secondary screening. The yield of glutathione produced by BY-2-24 in shake flask culture was (312.13 ± 2.62) mg/L, which was 134.26% higher than that produced by the original strain, and this mutant still had good genetic stability after seven passages. Meanwhile, the biomass was increased by 118.33%, indicating that the growth ability of the mutant strain was improved compared to the original strain. The result of this study show that the combination of ARTP and MMC can be used as a simple and effective screening method for excellent mutant strains, which provides a reference for high throughput selection of target strains.

In order to further investigate the structural properties and catalytic function of acarviosyltransferase (ATase), a key enzyme in the biosynthesis of acarbose, its gene (acbD) was cloned from Actinoplanes sp. SE50 genome and heterologously expressed in Escherichia coli. Bioinformatics analysis showed that the conserved domains of ATase, the expression product of acbD, were highly similar to those of cyclodextrin glycosyltransferase, which belongs to the glycoside hydrolase 13 (GH13) family, and ATase possessed a signal peptide and a transmembrane domain. After removal of the coding sequences in the signal peptide, the soluble expression level of acbD increased by 23.4 times as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal catalytic temperature and pH for the recombinant ATase were 30 ℃ and 7.0, respectively. The substrate spectrum showed that the recombinant ATase had the highest catalytic activity toward D-salicin (82.85 U/mL), followed by that (63.75 U/mL) toward L-sorbose. This is the first finding that L-sorbose can serve as an excellent glycosyl donor for ATase. The above results lay the foundation for further clarifying the catalytic mechanism of ATase.

In the Chinese strong-flavor Baijiu (CSFB) fermentation ecosystem, the caproic acid-anabolism of caproic acidproducing bacteria (CPBs) is very important for improving the fermentation quality of CSFB. Therefore, it is necessary to thoroughly understand the types of CPBs and their caproic acid-anabolism characteristics. This minireview introduces readers to the diversity, phylogenetic relationship, physiological and metabolic characteristics, and caproic acid synthesis mechanism of CPBs isolated from the CSFB fermentation ecosystem as well as their synergistic metabolic relationships with other CPBs or non-CPBs. This paper provides a reference for understanding the in-situ caproic acid-anabolism pattern of CPBs from the CSFB fermentation ecosystem, and further provides a theoretical basis for the future targeted application of CPBs in CSFB fermentation and for CPBs culture engineering for the synthesis of high value-added caproic acid.