title: Effect of silent mutations in Translational Initial Region on the production of recombinant cutinase in Escherichia coli.
abstract: Translational Initial Region (TIR) is the threshold of an intracellular translation process, and tiny alterations in this region are reported to intensely influence the downstream expression. Such property provides a potential utilization in extracellular production of recombinant enzyme. As an esterase, cutinase is an essential catalyst in the process of textile scouring, and has a potential application in food and chemical industry. In the present study, a bacterial cutinase (Tfu_0883) from Thermobifida fusca was expressed in Escherichia coli with pelB as its signal peptide using trc as its promoter. A subsequent TIR degeneracy mutagenesis was then carried out in the initial sequence of pelB. A fast screening method for these mutants was developed and a series of strains with different expression strengths were accordingly obtained. Among these mutants, a high cutinase production level of 38.0 U/ml was achieved, which is three times that of the control group. This study explored the potential utilization of TIR degeneracy mutagenesis in the production of industrial enzymes.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave, Wuxi, Jiangsu 214122, China. hill_hunter@163.com
authors.forename: Zhi-guo
authors.initials: ZG
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Kong-liang
authors.initials: KL
authors.lastname: Zhu
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase