Advances in Genetic Tools and Their Application in Streptococcus thermophilus

Abstract

Streptococcus thermophilus is a traditional starter. Nowadays, key aspects of S. thermophilus physiology have been revealed concerning the phenotypic traits relevant for industrial applications, including sugar metabolism, protein hydrolysis, and the production of important metabolites that affect the sensory properties of fermented foods as well as the original cooperation with Lactobacillus delbrueckii subsp. bulgaricus. Moreover, significant advances have been made in the synthetic biology toolbox of S. thermophilus based on technological advances in the genome and its sequencing and synthesis. In this review, we discuss the recently developed toolbox for S. thermophilus, including gene expression toolsets (promoters, terminators, plasmids, etc.) and genome editing tools. It can be used for both functionalized foods and therapeutic molecules for consumers. The availability of new molecular tools, including the genome editing toolbox, has facilitated the engineering of physiological studies of S. thermophilus and the generation of strains with improved technical and functional characteristics.

Keywords: Streptococcus thermophilus; genetic tools; plasmid; production characteristics; promoters.

Figure 1

Pathways of sugar metabolism in S.thermophilus. Fructose-1P, fructose-1-phosphate; Fructose-6P, fructose-6-phosphate; FBP, fructose-1,6-biphosphate; Suc-6P, sucrose-6-phosphate; Glucose-6P, glucose-6-phosphate; Glucose-1P, glucose-1-phosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Acetyl-P, acetyl-phosphate; Xylulose-5P, xylulose-5-phosphate; Ribulose-5P, ribulose-5-phosphate; Ribose-5P, ribose-5-phosphate; PRPP, 5-phosphoribosyl diphosphate; UDP-GlcNAc, UDP-N-acetyl-glucosamine; Pyr, pyrimidines; Pur, purines; EPS, exopolysaccharides; PG, peptidoglycan; RPS, rhamnose polysaccharides; TA, teichoic acids; PTS, sugar phosphotransferase system. Arrow indicates a reaction, multiple enzymatic processes are shown by dashed arrows, double-headed arrow indicates a bidirectional reaction. The figure is original.

Figure 2

S. thermophilus proteolytic system. There are three steps in the system. Proteins are broken down in the first stage by a proteinase called PrtS, which Sortase A (SrtA) anchors to the cell wall. The second step is transport of peptides and oligopeptides. The transport of peptides is integrated by the Dpp system with five proteins (DppA, DppB, DppC, DppD, and DppE) regulated by ATP and proton formation (NAD+); the oligopeptides transport is carried out by the Ami system integrated in an operon system with seven proteins (Ami1, Ami2, Ami3, AmiC, AmiD, AmiE, and AmiF) regulated by ATP and activated by sulfur amino acid presence. The peptide ComS controls the transcriptional regulatory protein ComR linked to the Ami system. The third and final stage is the cutting of peptides by fourteen internal peptidases, five of which (PepO, PepS, PepX, PepN, and PepC) differ from those of other LAB in terms of their properties and specificity. The figure is original.

Figure 3

Overview of genome engineering tools for S. thermophilus. (A) Integration techniques based on plasmids. (B) DNA-based linear integration techniques. (C) Process flow diagram for gene editing using the CRISPR/Cas9 system, CRISPR-based methods. The description of the details in this figure has been added as follows. Arrows represent target genes. The green rectangle (_▀) represents left repair arm. The blue rectangle (_▀) represents right repair arm. S⁺, positive antibiotic selection marker. S⁻, negative selection marker. The black rectangle (^█) represents modification. gRNA-Cas9, active complex of a guide RNA and the RNA-guided endonuclease Cas9. The figure is original.