title: 3'-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli.
abstract: 3'-Untranslated region (3'UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3'UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3'UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3'UTR. In summary, 3'UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells.
authors.affiliation: Department of Food Science &Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea.
authors.forename: Ji-Won
authors.initials: JW
authors.lastname: Song
authors.affiliation: Department of Food Science &Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea.
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Woo
authors.affiliation: Department of Chemical Engineering and School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Gyeongbuk 790-784, Republic of Korea.
authors.forename: Gyoo Yeol
authors.initials: GY
authors.lastname: Jung
authors.affiliation: Institute of Biochemistry, Department of Biotechnology &Enzyme Catalysis, Greifswald University, 17487 Greifswald, Germany.
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
authors.affiliation: Department of Food Science &Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea.
authors.forename: Jin-Byung
authors.initials: JB
authors.lastname: Park
mesh_terms: D020413:3' Untranslated Regions; D055162:Biocatalysis; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D020413:3' Untranslated Regions; D006899:Mixed Function Oxygenases; D004722:Endoribonucleases; C022537:ribonuclease E