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

Abstract

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (∆wzz). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (∆wzz) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 (ste8-) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.

A severe leakage of intermediates to shunt products in acarbose biosynthesis

“…For the expression of ValC, gene valC was amplified from the genome of S. hygroscopicus var. jinggangensis 5008 and cloned into pET-30a 56 to generate pET30a-valC. BL21(DE3) with the recombinant plasmid was cultured in LB medium containing kanamycin (50 µg/mL) at 37°C to OD 600 nm of 0.6-0.8.…”

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.…”