Boosting the Heterologous Expression of d-Allulose 3-Epimerase in Bacillus subtilis through Protein Engineering and Catabolite-Responsive Element Box Engineering.

As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of Clostridium cellulolyticum H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the T1/2 of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, PamyE was determined as the optimal promoter for the recombinant expression of CcDAEase in Bacillus subtilis, and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL-1, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.

[Genetic polymorphism and relationship of 24 Y-STR loci among three ethnic minorities from Guizhou].

OBJECTIVE: To study the distribution of haplotypes of Y-chromosomal short tandem repeats (Y-STR) loci among three ethnic minorities from Guizhou, China. METHODS: Twenty four Y-STR loci of 174 unrelated males were amplified with a Microreader(TM)24Y Direct ID System kit. Capillary electrophoresis was carried out on an ABI 3100 Genetic Analyzer, and the data was analyzed with GeneMapper software. RESULTS: Seventy six haplotypes were identified for the 24 Y-STR loci among the three ethnic minorities, including 13 from the Qiangs, 35 from the Manchurians, and 28 from the Shes, with the corresponding Haplotype Diversity (HD) being 0.7327, 0.9578, and 0.9344. Genetic distance between the Shes and Qiangs was relatively close, whilst that for Manchurians was relatively far. CONCLUSION: Analysis of the genetic characteristics and relationship of the three ethnic minorities from Guizhou can provide a reference for the study of their origin, evolution and patrilineal fusion.