Cloning and characterization of a glucosyltransferase and a rhamnosyltransferase fromStreptomycessp. 139

Abstract

Aims: Ste15 and ste22 present in the Ebosin biosynthesis gene cluster (ste) were previously shown to function in Ebosin biosynthesis and both of the protein products are predicted to be glycosyltransferases. In this study, their biochemical activities were confirmed. Methods and Results: ste15 and ste22 were cloned and expressed in Escherichia coli. With a continuous coupled spectrophotometric assay and using the purified proteins, we now demonstrated that the protein Ste15 has the ability of catalysing the transfer of glucose specifically from UDP‐glucose to an Ebosin precursor that lacks glucose, the lipid carrier located in the cytoplasmic membrane of the gene ste15 disrupt mutant Streptomyces sp. 139 (ste15−). The protein Ste22 can catalyse the transfer of rhamnose specifically from TDP‐rhamnose to an Ebosin precursor that lacks rhamnose, a lipophilic carrier in the cytoplasmic membrane of the gene ste22 disrupt mutant Streptomyces sp. 139 (ste22−). Conclusions: The gene product of ste15 was identified to be a glucosyltransferase, and the protein encoded by ste22 was found to be a rhamnosyltransferase. Significance and Impact of the Study: Both of two enzymes play essential roles in the formation of repeating units of sugars during Ebosin biosynthesis. These are the first glucosyltransferase and rhamnosyltransferase in the biosynthesis of a Streptomyces exopolysaccharide to be characterized.

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…The remaining 12% utilized K12 derivates. Commercially available K12 strains used include JM109 [ 25 , 51 , 55 ], DH5α [ 30 , 56 ], NovaBlue [ 57 ], XL1 Blue [ 58 ], M15 [ 31 , 59 , 60 ], and Top10 [ 61 , 62 ]; alternatively non-commercial K12 strains W3110 [ 63 ] and MG1655 [ 62 , 64 ] were used in a limited number of cases. K12 strains serve as useful tools when plasmid instability is encountered resulting in plasmid loss from the host [ 54 ]; this may explain its use in limited cases for expression.…”

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

“…Various ste genes had been functionally characterized by our lab: ste1 and ste2 for regulating exopolysaccharides production and phenotype of S sp. 139 (Bai et al 2013 ; Geng et al 2022 ); ste5 , ste7 , ste15 , and ste22 for bifunctional glycosyl-1-phosphate transferase, fucosyltransferase, glucosyltransferase, and rhamnosyltransferase, respectively (Chang et al 2009 ; Li et al 2010 ; Qi et al 2015 ; Zhang et al 2006 ); and ste8 and ste9 for polymerization and export of exopolysaccharides (Yang et al 2013 ; Zhang et al 2014 ). However, the function of the Orf2 protein encoded by the orf2 gene, situated between ste1 and ste5 (Fig.…”

Identification and Functional Analysis of the Chain Length Determinant Gene ste8 Involved in the Biosynthesis of Ebosin by Streptomyces sp. 139

“…139, consists of 27 ORFs [27]. Some of the genes involved in the cluster have been studied separately [5,16,29]. In this paper, a functional study of ste8 is presented.…”