Part:BBa_K4395011

SpyTag-UGT73B6-SpyCatcher is a fusion protein whose thermostability is increased by the SpyTag-SpyCatcher tagging system while the catalytic activity not affected.

UGT73B6 is a member of the UGT superfamily, coding a UDP-glucosyltransferase. Its cDNA was first isolated from Rhodiola sachalinensis in 2007, which was 1,598 bp in length encoding 480 deduced amino acid residues with a conserved UDP-glucose-binding domain [1]. It is the first cloned glucosyltransferase gene involved in salidroside biosynthesis [1]. UGT73B6 can catalyzes the biosynthesis of salidroside from tyrosine by transferring glucose to the side chain of tyrosol. By using E. coli introduced with UGT73B6, salidroside was produced with a titer of 56.9 mg/L [2]. This relatively high yield attracts us to use it this year to convert tyrosol produced in second step to salidroside.

Besides salidroside, UGT73B6 could also catalyze the formation of other transglycosylation products. For example, producing icariside D2 by attaching glucose to the phenolic position of tyrosol instead of carbonyl [2]. It can also transfer glucosides to other phenolic compounds, including simple phenolic compounds, coumarin 4-methylumbelliferone, RA analogs, xanthones, and honokiol [3].

The SpyTag/SpyCatcher system is a split-in-two of the immunoglobulin-like collagen adhesion domain of Streptococcus pyogenes denoted CnaB2. CnaB2 is characterized by an internal isopeptide bond between residue Lys31 and residue Asp117. When split in two, one of which containing Lys31 and another containing Asp117, they associate and spontaneously form the isopeptide bond, thus join together [4] The composite part SpyTag-UGT73B6-SpyCatcher is a fusion protein cyclized by the SpyTag/SpyCatcher system, whose two components are fused on both ends. This cyclization process increases the thermostability of the enzyme, enabling catalysis in an unfavored heated system [5].

References: [1] Ma, LQ., Liu, BY., Gao, DY. et al. Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis . Plant Cell Rep 26, 989–999 (2007). [2] Bai, Y., Bi, H., Zhuang, Y. et al. Production of salidroside in metabolically engineered Escherichia coli. Sci Rep 4, 6640 (2014). [3] He, Qinglin et al. “Fermentative Production of Phenolic Glucosides by Escherichia coli with an Engineered Glucosyltransferase from Rhodiola sachalinensis.” Journal of agricultural and food chemistry vol. 65,23 (2017): 4691-4697. [4] Long Li, Jacob O. Fierer, et al. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag J Mol Biol. 2014 January 23; 426(2): 309–317 [5] Xiao-Bao Suna, Jia-Wen Caoa,b, et al. SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation