Auto-induction-based Rapid Evaluation of Extracellular Enzyme Expression from Lac Operator-involved Recombinant Escherichia coli

• Published in: Applied biochemistry and biotechnology 2014-12, Vol.174 (7), p.2516-2526
• Main Authors: Chen, Wen-Bo, Nie, Yao, Mu, Xiao-Qing, Yan, Wei, Xu, Yan, Xiao, Rong
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biochemistry; Biological and medical sciences; bioprocessing; Biotechnology; Cellular biology; Chemistry; Chemistry and Materials Science; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene expression; genes; Glycoside Hydrolases - biosynthesis; Glycoside Hydrolases - genetics; Gram-negative bacteria; Klebsiella; Klebsiella - enzymology; Klebsiella - genetics; Lac Operon - physiology; pullulanase; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; screening; secretion
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1201-y

Degeneration of engineered strains and decreased production of target gene products were often observed during recombinant bioprocess. Although several strategies have been developed to ensure high levels of target gene products, these methods seemed to be complex and laborious. By investigating possible factors contributing to the decreased yield, degeneration of host cells was identified as the main cause. Based on the principle of auto-induction and the fact that the interaction between colored substrates and gene products could present visible changes, a convenient and accurate screening method was developed to evaluate the production performance of engineered strains. Due to the typicality of pullulanase from genus Klebsiella, which has been a model for the research of secretion mechanism in gram-negative bacteria, an engineered E. coli producing extracellular pullulanase was employed to illustrate the method. Consequently, according to the capability to form a colorless halo, colonies could be divided into two groups, one surrounded by clear zone and the other unable to present transparent haloes. Furthermore, the high capability of these colonies was confirmed by performing pullulanase production in liquid medium. Compared with other methods for evaluating engineered strains, the visible screening technique was suggested to have the advantages of effectiveness and accuracy.