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Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon

Li, Youran, Jin, Ke, Zhang, Liang, Ding, Zhongyang, Gu, Zhenghua, Shi, Guiyang
Journal of agricultural and food chemistry 2018 v.66 no.36 pp. 9456-9464
Bacillus licheniformis, GRAS substances, alpha-amylase, catabolite repression, consensus sequence, genetically engineered microorganisms, glucan 1,4-alpha-maltohydrolase, industrial applications, mutation, nucleotides, operon, plasmids, protein engineering, xylose
The xylose operon can be an efficient biological component for regulatory expression uses in Bacillus licheniformis. However, its characteristic susceptibility to carbon catabolite repression (CCR) makes its application inconvenient. In this study, plasmids harboring the wild-type operons from three Bacillus species were constructed and introduced into B. licheniformis. These plasmids ensured secretory expression of maltogenic α-amylase (BLMA) in B. licheniformis under strict regulation. The glucose-mediated CCR was then alleviated by engineering the xylose operon of the expression system. Evidence showed that mutations in the highly conserved nucleotides of the identified catabolite responsive element (cre) consensus sequence prevented association of the regulator CcpA with DNA, thus resulting in an increase in BLMA activity of up to 12-fold. Furthermore, features of this engineered system for inducible expression were investigated. Induction in mid-log phase using 10 g/L xylose at 37 °C was found to be beneficial for promoting the accumulation of recombinant product, and the maximum yield of BlmMA reached 715.4 U/mL. This study contributes to the industrial application of the generally recognized as safe (GRAS) workhorse B. licheniformis.