The N-terminal domain of rhamnosyltransferase EpsF influences exopolysaccharide chain length determination in Streptococcus thermophilus 05-34

Abstract

Glycosyltransferases are key enzymes involved in the assembly of repeating units of exopolysaccharides (EPS). A glycosyltransferase generally consists of the N-terminal and the C-terminal domain, however, the functional role of these domains in EPS biosynthesis remains largely unknown. In this study, homologous overexpression was employed to investigate the effects of EpsFN, a truncated form of rhamnosyltransferase EpsF with only the N-terminal domain, on EPS biosynthesis in Streptococcus thermophilus 05-34. Reverse transcription qPCR and Western blotting analysis confirmed the successful expression of epsFN in 05-34 at the transcription and translation level, respectively. Further analysis showed that the monosaccharide composition and yield of EPS were not affected by the overexpression of epsFN, whereas the molecular mass decreased by 5-fold. Accordingly, the transcription levels of genes involved in EPS biosynthesis, including chain-length determination gene epsC, were down-regulated by 5- to 6-fold. These results indicated that the N-terminal domain of EpsF alone could influence the molecular mass of EPS, probably via lowering the concentration of sugar precursors, which may lead to decreased expression of genes responsible for chain-length determination.

CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus

“…We also observed that epsF overexpression increased EPS yields by 17%. This observation was inconsistent with Wang et al (2020), who indicated that epsF overexpression did not affect EPS production in S. thermophilus. This may have been due to strain-specific differences.…”

Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34

“…S. thermophilus 05CK and 05epsCh 6 were grown under static conditions at 37°C in MRS broth supplemented with 5 μg/mL chloramphenicol for 30 h prior to protein extraction. Protein extraction and Western blot were performed as previously described ( Wang et al, 2020 ).…”

“…The HPGPC system consisted of Agilent 1260 series HPLC system (Agilent, United States) and Shodex SUGAR KS series containing KS-804 and KS-805 columns (Shodex Co., Tokyo, Japan). The purified EPS was detected using refractive index detector (RI-2000, Waters, United States) according to the method which we used previously ( Wang et al, 2020 ). Briefly, 40 μg purified EPS was eluted with 20 mM NaCl at a flow rate of 0.8 mL/min.…”

“…The molecular structure of EPS has been proved to be regulated by a specific gene cluster ( Zeidan et al, 2017 ). Generally, the eps cluster of S. thermophilus can be divided in three parts: the first region includes the regulatory genes of EPS production ( epsA, epsB ) and chain length determination genes ( epsC, epsD ); the second region is associated with biosynthesis of repeating units of EPS ( epsE, epsI , and epsJ ); the third region is responsible for polymerization and export of repeating units ( epsH, epsK ) ( Zeidan et al, 2017 ; Wang et al, 2020 ). Notably, the chain length determination gene epsC and epsD are involved in polysaccharide assembly ( Cui et al, 2017 ).…”

Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes

“…The gene products of epsCD , which are proposed to form a membrane-located complex, also bear topological and sequence similarities to ABC transport systems, suggesting that they play a dual role in EPS transport and chain length control (Stingele et al 1996 ). It is assumed that the transcription level of epsC (and likely epsD) influences chain length, while the availability of nucleotide sugars may also modulate chain length also (Wang et al 2020 ). Polymerization of the EPS repeating subunits is suggested to be a function of EpsJ, possibly through complexation with EpsC and EpsD (Stingele et al 1996 ).…”