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

Zhengyuan Zhai,Shuxin Xie,Yanling Hao,Huaxi Yi,Hongxing Zhang

doi:10.3389/fmicb.2021.696222

Abstract

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

Highlights

Streptococcus thermophilus is a homofermentative facultative anaerobe, which has been widely used as a starter culture for the manufacture of dairy products, e.g., yogurt and cheese (Pega et al, 2017; Vieira et al, 2019)
Western blotting assay with anti His-Tag mouse monoclonal antibody further confirmed the production of a 26-kDa protein in S. thermophilus 05epsCh6, while no corresponding band was observed in S. thermophilus 05CK (Figure 1B)
Topological analysis with TMHMM program revealed that EpsC from S. thermophilus 05-34 is a cytoplasmic membrane protein and contains two transmembrane helices located in amino acid units 25–44 and 181–198 (Supplementary Figure 3A)

Summary

Introduction

Streptococcus thermophilus is a homofermentative facultative anaerobe, which has been widely used as a starter culture for the manufacture of dairy products, e.g., yogurt and cheese (Pega et al, 2017; Vieira et al, 2019). The presence of exopolysaccharides (EPS) produced by S. thermophilus has been reported to improve viscosity and texture, increase resistance to Homologous Over-Expression of EpsC mechanical handling and prevent syneresis (Broadbent et al, 2003; Cui et al, 2017). The in situ production of EPS by S. thermophilus has the potential to decrease the amount of added ingredients and stabilizers, such as dairy proteins, starch, pectin, and hydrocolloids (Zeidan et al, 2017). The replacement of these additives would lead to a clean label and a reduced production cost. EPS production is one of the most important and attractive properties of S. thermophilus strains

Methods

Results

Conclusion