Overexpression of Escherichia coli Acetyl Esterase Using a Strategy of Multi-copy Promoters

Abstract

Acetyl esterase (AE) plays an important role in bioconversion of lignocellulosic biomass. However, the expression level of AE from wild type strains is relatively low. The aim of the study was to enhance the activity of AE using the strategy of multi-copy promoters. A commercial strain E. coli JM110 was used as the host of expression plasmid. pKK223-3-ybaC (pK-ybaC) is a recombinant plasmid, and it carries ybaC gene (encoding AE) that was derived from E. coli RB3. E. coli JM110 (pK-ybaC; EcKy) was a recombinant strain that contains pK-ybaC. Fragment P3 that contains three copies of the core-tac-promoter and fragment P5 that contains five copies of the core-tac-promoter were synthesized. Plasmid pK-ybaC was extracted from EcKy and was linearized by EcoR I and BamH I to remove its wild promoter. Then fragment P3 was subcloned into linearized pK-ybaC to generate pKTH-ybaC and fragment P5 was inserted into pK-ybaC to generate pKFI-ybaC. In-Fusion kit (Takara) was used to construct recombinant plasmids according to the instruction of the manufacturer. The two plasmids were then transformed into E. coli JM110 to get the recombinant strain E. coli JM110 (pKTH-ybaC; EcTH) and E. coli JM110 (pKFI-ybaC; EcFI). The highest activity of AE from EcTH was 3.04 U mL−1. It was 7.8 times of that from the parental strain E. coli RB3. The peak value of AE activity from EcFI was 35.66 U mL−1, which was 91.4 times of that from E. coli RB3. EcFI showed the highest transcription level of ybaC gene, and its value was 4239 times of that in E. coli RB3. The K m value was 3.49 mmol L−1 and the V max value was 43.29 µmol s−1 mg−1 for AE from EcFI. The K cat/K m value was around fivefold of that from the parental strain E.coli RB3. Two engineered bacterial strains that contain three copies and five copies of the core-tac-promoter were constructed. The activity of AE from the two engineered strains was enhanced significantly compared to that from the parental strain. The results indicated the feasibility of enhancing AE activity using the strategy of multi-copy promoters.