2.3 Plasmid and strain construction
After codon optimization for heterologous expression in E. coli , the coding region derived from the putative SuSy mentioned above was synthesized and cloned into pRSFDuet-1 (Novagen) between the restriction endonuclease sites Nco I and Eco RI by GenScript (Nanjing, China). A 6-histidine tag was added at the C -terminus of SuSy. The generated plasmids were named as pRSF-Mc SuSy, pRSF-Cb SuSy1, and pRSF-Cb SuSy2, respectively.
The plasmid pRSF-Mc SuSy was used as the template for site-directed mutagenesis by a Mut Express® II Fast Mutagenesis Kit V2 (Vazyme Biotech Co., Ltd, Nanjing, China). The primers used in PCR to produce the plasmid mutants are listed in Table S1.
The synthesized code-optimized gene of UGT51m (ONH78233, excludingN -terminal 721 amino acids, containing seven mutations S81A/L82A/V84A/K92A/E96K/S129A/N172D) and the mutation S31D/K684T/N685D of Mc SuSy were subcloned into the restriction endonuclease sitesNde I/Xho I and Nco I/Eco RI of the pRSFDuet-1, respectively.[21] The obtained plasmid was named pRSF-UGT51m-Mc SuSym. Then, the coding region of the Mc SuSy mutant was replaced by that of At SuSy1 in pRSF-UGT51m-Mc SuSym, giving another plasmid named pRSF-UGT51m-At SuSy1.
The aforementioned plasmids were respectively transformed into E. coli BL21 (DE3) competent cells (TransGen Biotech, Beijing, China), resulting in the corresponding recombinant strain.