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Background: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level., Results: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance., Conclusion: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: Streptococcus thermophilus is a major starter used in the dairy industry and it could improve the flavor of fermented products. It is necessary to improve biomass of S. thermophilus for its application and industrialization. The utilization of carbon sources directly affects the biomass of S. thermophilus. Therefore, the carbohydrate metabolism of S. thermophilus should be investigated., Results: In the present study, metabolic parameters and gene expression of S. thermophilus S-3 with different carbon sources were investigated. The physicochemical results showed that S. thermophilus S-3 had high lactose utilization. Transcriptome analysis found that approximately 104 genes were annotated onto 15 carbohydrate metabolic pathways, of which 15 unigenes were involved in the phosphotransferase system and 75 were involved in the ATP-binding cassette transporter system. In addition, 171 differentially expressed genes related to carbohydrate metabolism were identified. Expression of the galactose metabolism genes lacSZ and galKTEM increased significantly from the lag phase to the mid-exponential growth phase as a result of the global regulator protein, catabolite control protein A (CcpA). The high expression of galK in the mid- to late- phases indicated that the metabolite galactose is re-transported for intracellular utilization. CcpA regulation may also induce high expressions of glycolytic pathway regulated-genes related to lactose utilization, including ldh, fba, eno, pfkA, bglA, pgi, pgm and pyk, producing optimal glycolytic flux and S. thermophilus S-3 growth., Conclusion: The present study provides new insights into the carbon metabolism regulation and provide theoretical support for high-density fermentation of S. thermophilus S-3. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Streptococcus thermophilus is an economically prominent starter for common dairy products due to its potential health and nutritional benefits. However, lack of precise genetic manipulation approaches has greatly hampered the industrial application of S. thermophilus.. Herein, we developed an efficient genome editing toolbox (pKLH353) based on CRISPR/nCas9 (Cas9 nickase) in S. thermophilus to seamlessly edit single or multiple genes. A native constitutive promoter library was used to optimize the nCas9 and sgRNA expression with gene deletion efficiencies of 14-60%. The epsA, epsB and epsE were identified as key genes affecting exopolysaccharide (EPS) biosynthesis in S. thermophilus S-3 using the CRISPR/nCas9 toolbox. Moreover, compared to the wild-type, knockout of epsC, epsE or epsG led to a decrease of EPS titer with reducing in its molecular weight (>2.5-fold) and intrinsic viscosity (>19.8-fold). The ratio of monosaccharide composition of the mutants has also changed, suggesting that these eps genes are involved in the chain length synthesis and repeat unit assembly. Taken together, this CRISPR/nCas9 system can serve as a basic toolkit for precise genetic engineering of S. thermophilus and facilitate strain engineering to produce bio-based products., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: As a natural food additive, exopolysaccharide (EPS) produced by Streptococcus thermophilus can improve product viscosity and texture. The protein EpsA is a putative pathway-specific transcriptional regulator for EPS biosynthesis in S. thermophilus., Results: According to comparative analysis of EPS biosynthetic gene clusters, a conserved region of epsA (609 bp) was employed to design primer pair epsA-F/R as a molecular marker for the isolation of EPS-producing (EPS + ) S. thermophilus. Two EPS + S. thermophiles strains, AR333 and S-3, were band-positive, whereas Lactococcus lactis NZ9000 (non-EPS-producing, EPS - ), Lactobacillus casei LC2W (EPS + ) and L. plantarum AR113 (EPS + ) were negative by polymerase chain reaction (PCR) amplicon bands using the epsA probe. This indicated good specificity of the epsA probe to EPS + S. thermophilus. Moreover, based on PCR screening with the epsA probe, 23 positive strains were isolated and identified as S. thermophilus from our microbial library and natural fermented milk with 141.3-309.2 mg L -1 of EPS production, demonstrating the validity of our molecular marker screening method., Conclusion: The designed molecular marker of epsA can rapidly screen EPS + S. thermophilus, which has potential application in the dairy and other food industries. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Exopolysaccharides (EPSs) from lactic acid bacteria have great effect on the quality of fermented milk products. However, the mechanism for the quality improvement has not been well described. This study aimed to investigate the molecular binding kinetics and interactions between EPS obtained from Streptococcus thermophilus AR333 (EPS333) and casein of milk (CM) in a simulated acidifying process. The results indicated that EPS333 had a significant effect on the stability of casein micelles at acidic pH (6.0-4.5) according to the turbidity, ζ-potential, particle size and distribution analysis. The adsorption-desorption study by bio-layer interferometry identified the direct affinity binding between EPS333 and CM, the interactive moiety of casein was α-casein, rather than β- or κ-casein. Fluorescence quenching analysis revealed that the force types of interaction between EPS333 and CM were dynamically changeable during the acidifying process, mainly from electrostatic interaction at pH 7.0-6.5, to hydrophobic or hydrogen bonding at pH 6.5-5.5, and then transferred to electrostatic interaction again at pH 5.5-5.0. Conclusively, EPS333 could bind with CM directly via different binding forces during acidifying process to stabilize the properties of casein micelles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A lactic acid bacteria strain AR333 with high-production of exopolysaccharides (EPS) was screened out from 350 bacteria strains isolated from naturally fermented dairy products. It was identified as Streptococcus thermophilus by morphological observation, physiological and biochemical characteristics, and 16S rDNA sequence determination. The EPS from AR333 were purified through DEAE-Sepharose Fast Flow and Sepharose CL-6B, and the purified fraction was designated as EPS333. In vitro test showed that EPS333 could stimulate macrophage RAW 264.7 to release NO significantly (p<0.05). The further work tried to elucidate the structural features of EPS333 via FT-IR spectrum, high-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography (HPSEC). The results showed that EPS333 was a pure neutral polysaccharide with monosaccharide composition of galactose and glucose in a molar ratio of 6:5. A certain amount of acetyl groups might exist in EPS333 according to the FT-IR spectrum. The molecular weight (Mw) was estimated to be 332 kDa. Current study suggested that the lactic bacteria strain AR333 could be a potential source of immunoregulatory polysaccharide., (Copyright © 2016. Published by Elsevier B.V.)

This study investigates the impact of urea and β-GP on the growth of Streptococcus thermophilus S-3, a bacterium commonly used in industrial fermentation processes. Through a series of growth experiments, transcriptome, metabolome, and omics-based analyses, the research demonstrates that both urea and β-GP can enhance the biomass of S. thermophilus, with urea showing a more significant effect. The optimal urea concentration for growth was determined to be 3 g/L in M17 medium. The study also highlights the metabolic pathways influenced by urea and β-GP, particularly the galactose metabolism pathway, which is crucial for cell growth when lactose is the substrate. The integration of omics data into the genome-scale metabolic model of S. thermophilus, iCH502, allowed for a more accurate prediction of metabolic fluxes and growth rates. The study concludes that urea can serve as a viable substitute for β-GP in the cultivation of S. thermophilus, offering potential cost and efficiency benefits in industrial fermentation processes. The findings are supported by validation experiments with 11 additional strains of S. thermophilus, which showed increased biomass in UM17 medium. [ABSTRACT FROM AUTHOR]

Superoxide dismutase (SOD) plays an essential role in eliminating oxidative damage of lactic acid bacteria. Streptococcus thermophilus , an important probiotic lactic acid bacterium, often inevitably suffers from various oxidative stress during dairy fermentation. In this study, to confer high-level oxidative resistance, the sod gene from Lactobacillus casei was heterologous expressed in S. thermophilus S-3 using our previous constructed native constitutive promoter library. The enzyme activity of SOD was significantly enhanced in engineered S. thermophilus by promoter #14 (2070 U/mg). Furthermore, the strategy of multi-copy sod -expressing cassettes was employed to improve SOD activity. The maximum activity (2750 U/mg) was obtained by the two-copy sod recombinant, which was 1.5-fold higher than that of one-copy recombinant. In addition, the survival rate of multi-copy sod recombinants was increased about 97-fold with 3.5 mmol/L H2O2 treatment. To our knowledge, this is the first report of multi-copy sod gene expression in S. thermophilus , which exerts a positive effect on coping with oxidative stress to enhance the potential of industrial application. [ABSTRACT FROM AUTHOR]

An exopolysaccharide EPS333 was produced by a ropy Streptococcus thermophilus strain AR333 when grown on reconstituted skim milk. The molecular weight, radius of gyration (Rg), hydrodynamic radius (R h ) and intrinsic viscosity ([ η ]) of EPS333 were determined as 313.7 kDa, 33.96 nm, 25.98 nm, and 1.874 dL/g, respectively by high performance size exclusion chromatography (HPSEC). The conformational analysis demonstrated that EPS333 presented as compact and semi-stiff or stiff chain in aqueous solution. Monosaccharide analysis identified that EPS333 was composed of galactose, glucose and galactosamine in a molar ratio of 3: 2: 1. The backbone of EPS333 consisted of β-(1→3)-Gal p , β-(1→3,6)-Glc p , α-(1→3)-2-Gal p NAc, and α-(1→4)-Glc p . The branch chain was terminated at O -6 position of β-(1→3,6)-Glc p by α-T-Gal p . With the help of 1D & 2D NMR spectroscopy, the repeating unit of EPS333 was proposed. Further studies revealed that the in situ produced EPS333 could improve the quality of fermented milk to be close to the commercial product. [ABSTRACT FROM AUTHOR]

[Display omitted] • A high-throughput screening (HTS) method was developed based on congo red staining. • The mutants with different yields of exopolysaccharide (EPS) were obtained by HTS. • The HTS method is efficient for isolating EPS-producing Streptococcus thermophilus. As a thickening or stabilizing agent, exopolysaccharide (EPS) can significantly enhance food texture. However, the conventional analytical and screening methods of EPS have been unable to meet the current needs of industrial production, owing to low efficiency and heavy time-consumption. A simple and rapid qualitative/quantitative method is needed to accelerate the selection of high EPS-producing strains. Here, a high-throughput screening (HTS) program for EPS production was established in S. thermophilus by combining Congo red agar method (CRA, primary screening) and microplate colorimetric assay (MPC, secondary screening). The correlation coefficient (R2) between CRA/MPC and the phenol-sulfuric acid assay (a classical EPS quantification method) were 0.779 and 0.862, respectively, suggesting the feasibility of this HTS method. A mutant library (>300 colonies) of S. thermophilus was constructed using ribosome engineering strategy, and EPS-producing mutants with the titer from 50 mg/L to 200 mg/L were rapidly obtained by our developed HTS method, confirming that the HTS method is simple and efficient for the isolation of EPS-producing S. thermophilus. Taken together, this HTS method was developed for rapidly screening of EPS-producing S. thermophilus , which could be a valuable tool not only for the rapid detection of EPS production capabilities, but also for the screening of strain libraries from genetic engineering with desirable characteristics. [ABSTRACT FROM AUTHOR]